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1. Felske  AD, Fehr  W, Pauling  BV, von Canstein  H, Wagner-Döbler  I,     ( 2003 )

Functional profiling of mercuric reductase (mer A) genes in biofilm communities of a technical scale biocatalyzer.

BMC microbiology 3 (N/A)
PMID : 14577839  :   DOI  :   10.1186/1471-2180-3-22     PMC  :   PMC270059    
Abstract >>
Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase. We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation. Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences. The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing. The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducing Ps. aeruginosa strain was identified by its unique mercuric reductase gene.
KeywordMeSH Terms
Biofilms
2. Sevrioukova  IF, Garcia  C, Li  H, Bhaskar  B, Poulos  TL,     ( 2003 )

Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida.

Journal of molecular biology 333 (2)
PMID : 14529624  :   DOI  :   10.1016/j.jmb.2003.08.028    
Abstract >>
Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.
KeywordMeSH Terms
3. Shimizu  S, Ogawa  J, Chung  MC, Yamada  H,     ( 1992 )

Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2.

European journal of biochemistry 209 (1)
PMID : 1396711  :   DOI  :   10.1111/j.1432-1033.1992.tb17299.x    
Abstract >>
A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.
KeywordMeSH Terms
4. van Beilen  JB, Eggink  G, Enequist  H, Bos  R, Witholt  B,     ( 1992 )

DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans.

Molecular microbiology 6 (21)
PMID : 1453953  :   DOI  :   10.1111/j.1365-2958.1992.tb01769.x    
Abstract >>
The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.
KeywordMeSH Terms
5. Eaton  RW, Chapman  PJ,     ( 1992 )

Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions.

Journal of bacteriology 174 (23)
PMID : 1447127  :   DOI  :   10.1128/jb.174.23.7542-7554.1992     PMC  :   PMC207464    
Abstract >>
The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.
KeywordMeSH Terms
Intramolecular Oxidoreductases
6. Fujita  M, Amemura  A,     ( 1992 )

Purification and characterization of a DNA-dependent RNA polymerase from Pseudomonas putida.

Bioscience, biotechnology, and biochemistry 56 (11)
PMID : 1369075  :  
Abstract >>
DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida. The enzyme had the typical composition of beta',beta,alpha, and sigma subunits of eubacterial RNA polymerases. The molecular masses of the subunits were 156,000 Da, 151,000 Da, 87,000 Da, and 42,000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The NH2-terminal amino acid residues of the alpha subunit had a marked homology with those of the alpha subunit of Escherichia coli RNA polymerase. The enzyme activity was dependent on ribonucleoside triphosphates, Mg2+, and a DNA template, and was inhibited in vitro by rifampicin. The enzyme activity was maximal in the presence of 10 mM MgCl2. In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P. putida initiated transcription at the same site as that of E. coli.
KeywordMeSH Terms
7. Williams  SE, Woolridge  EM, Ransom  SC, Landro  JA, Babbitt  PC, Kozarich  JW,     ( 1992 )

3-Carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida is homologous to the class II fumarase family: a new reaction in the evolution of a mechanistic motif.

Biochemistry 31 (40)
PMID : 1390752  :   DOI  :   10.1021/bi00155a033    
Abstract >>
The gene (pcaB) for 3-carboxymuconate lactonizing enzyme (CMLE; 3-carboxymuconate cycloisomerase; EC 5.5.1.2) from Pseudomonas putida has been cloned into pMG27NS, a temperature-sensitive expression vector, and expressed in Escherichia coli N4830. The specific activity and kinetic parameters of the recombinant CMLE were comparable to those previously reported. A comparison of the deduced amino acid sequence of CMLE with sequences available in the PIR and Genbank databases revealed that CMLE has highly significant sequence homology to the class II fumarase family, particularly to adenylosuccinate lyase from Bacillus subtilis. CMLE has no significant homology to muconate lactonizing enzyme (MLE) from P. putida, its sister enzyme in the beta-ketoadipate pathway. These findings fully corroborate a prediction made by us on the basis of mechanistic and stereochemical analyses of CMLE and MLE [Chari, R. V. J., Whitman, C. P., Kozarich, J. W., Ngai, K.-L., & Ornston, L. N. (1987) J. Am. Chem. Soc. 109, 5514-5519] and suggest that CMLE and MLE were recruited into this specialized pathway from two different enzyme families.
KeywordMeSH Terms
8. Shaw  JP, Harayama  S,     ( 1992 )

Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

European journal of biochemistry 209 (1)
PMID : 1327782  :   DOI  :   10.1111/j.1432-1033.1992.tb17260.x    
Abstract >>
The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.
KeywordMeSH Terms
Plasmids
9. Voss  JA, Khedairy  H, Baker  RF, Benjamin  RC,     ( 1990 )

Molecular cloning of the xylL-xylE region from the P. putida TOL plasmid, pDK1.

SAAS bulletin, biochemistry and biotechnology 3 (N/A)
PMID : 1366507  :  
Abstract >>
A 5.2 kilobase EcoRI restriction fragment from the Pseudomonas putida HS1 TOL plasmid pDK1, encoding a portion of the lower toluene degradation pathway, was cloned into the E. coli plasmid pBR325. A detailed map of the restriction endonuclease sites was constructed and the nucleotide sequence of three contiguous XhoI fragments, with a combined total length of approximately 3.9 kilobases, has been investigated. This region was determined to contain a total of four separate open reading frames, each preceded by an identical putative ribosome-binding site (nucleotide sequence of 5'-GAGGTG-3'). These open reading frames have been tentatively identified as encoding the lower pathway enzymes catechol 2,3-dioxygenase (C23O) and 1,2-dihydroxycyclohexa-3,5-diene carboxylate dehydrogenase (DHCDH) and a subunit of the toluate 1,2-dioxygenase complex (TO).
KeywordMeSH Terms
Dioxygenases
10. Chen  LH, Kenyon  GL, Curtin  F, Harayama  S, Bembenek  ME, Hajipour  G, Whitman  CP,     ( 1992 )

4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer.

The Journal of biological chemistry 267 (25)
PMID : 1339435  :  
Abstract >>
The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL plasmid pWW0 and inserted into an Escherichia coli expression vector. Several of the genes of the metafission pathway encoded by pWW0 have been cloned in E. coli, but the overexpression of their gene products has met with limited success. By utilizing the E. coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture. 4-OT has been previously characterized and shown to be an extremely efficient catalyst (Whitman, C. P., Aird, B. A., Gillespie, W. R., and Stolowich, N. J. (1991) J. Am. Chem. Soc. 113, 3154-3162). Kinetic and physical characterization of the E. coli-expressed protein show that it is identical with that of the 4-OT isolated from P. putida. The functional unit is apparently a pentamer of identical subunits, each consisting of only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The amino acid sequence, determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions. We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues belonging to several, if not all, of the subunits.
KeywordMeSH Terms
Genes, Bacterial
11. Assinder  SJ, de Marco  P, Sayers  JR, Shaw  LE, Winson  MK, Williams  PA,     ( 1992 )

Identical resolvases are encoded by Pseudomonas TOL plasmids pWW53 and pDK1.

Nucleic acids research 20 (20)
PMID : 1331988  :   DOI  :   10.1093/nar/20.20.5476     PMC  :   PMC334361    
Abstract >>
N/A
KeywordMeSH Terms
12. Steele  MI, Lorenz  D, Hatter  K, Park  A, Sokatch  JR,     ( 1992 )

Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase.

The Journal of biological chemistry 267 (19)
PMID : 1339433  :  
Abstract >>
A 5417-base pair (bp) region of Pseudomonas aeruginosa PAO chromosomal DNA containing the mmsAB operon and an upstream regulatory gene (mmsR) has been cloned and characterized. The operon contains two structural genes involved in valine metabolism: mmsA, which encodes methylmalonate-semialdehyde dehydrogenase; and mmsB, which encodes 3-hydroxyisobutyrate dehydrogenase. mmsA and mmsB share the same orientation and are separated by a 16-bp noncoding region. The transcriptional start site for the operon has been pinpointed to a cytidine residue located 77 bp from the translational start site of the operon. mmsR is located on the opposite strand and begins 134 bp from the translational start site of mmsA. MmsR has been identified as a member of the XylS/AraC family of transcriptional regulators and appears to act as a positive regulator of the mmsAB operon. Sequence comparison of MmsA to other proteins in the data bases revealed that MmsA belongs to the aldehyde dehydrogenase (NAD+) superfamily. MmsB shares a 44% amino acid identity with 3-hydroxyisobutyrate dehydrogenase from rat liver. Mutants with insertionally inactivated mmsR, mmsA, and mmsB grow slowly on valine/isoleucine medium and exhibit reduced enzyme activity in cell-free extracts compared to P. aeruginosa PAO.
KeywordMeSH Terms
Operon
13. Fowell  SL, Lilley  KS, Jones  D, Maxwell  A,     ( 1992 )

GroEL proteins from three Pseudomonas species.

Molecular microbiology 6 (11)
PMID : 1352616  :   DOI  :   10.1111/j.1365-2958.1992.tb00880.x    
Abstract >>
N/A
KeywordMeSH Terms
14. Park  W, Jeon  CO, Hohnstock-Ashe  AM, Winans  SC, Zylstra  GJ, Madsen  EL,     ( 2003 )

Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1.

Applied and environmental microbiology 69 (6)
PMID : 12788725  :   DOI  :   10.1128/aem.69.6.3263-3271.2003     PMC  :   PMC161498    
Abstract >>
Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.
KeywordMeSH Terms
Conjugation, Genetic
Gene Transfer, Horizontal
15. Mattevi  A, Obmolova  G, Sokatch  JR, Betzel  C, Hol  WG,     ( 1992 )

The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.

Proteins 13 (4)
PMID : 1325638  :   DOI  :   10.1002/prot.340130406    
Abstract >>
The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.
KeywordMeSH Terms
16. Chowdhury  EK, Akaishi  Y, Nagata  S, Misono  H,     ( 2003 )

Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23.

Bioscience, biotechnology, and biochemistry 67 (2)
PMID : 12729017  :  
Abstract >>
The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.
KeywordMeSH Terms
17. Docquier  JD, Riccio  ML, Mugnaioli  C, Luzzaro  F, Endimiani  A, Toniolo  A, Amicosante  G, Rossolini  GM,     ( 2003 )

IMP-12, a new plasmid-encoded metallo-beta-lactamase from a Pseudomonas putida clinical isolate.

Antimicrobial agents and chemotherapy 47 (5)
PMID : 12709317  :   DOI  :   10.1128/aac.47.5.1522-1528.2003     PMC  :   PMC153319    
Abstract >>
A Pseudomonas putida strain showing broad-spectrum resistance to beta-lactams, including expanded-spectrum cephalosporins and carbapenems, was isolated from a patient with a urinary tract infection at the University Hospital of Varese in northern Italy. The isolate was found to produce metallo-beta-lactamase activity and to harbor a 50-kb plasmid, named pVA758, carrying a new bla(IMP) determinant, named bla(IMP-12). Plasmid pVA758 was not self-transferable by conjugation to either Escherichia coli or Pseudomonas aeruginosa but could be introduced by electroporation and maintained in the latter host, where it conferred resistance or decreased susceptibility to various beta-lactams. The IMP-12 enzyme is quite divergent from other IMP variants: its closest relatives are IMP-8 and IMP-2 (89 and 88% sequence identity, respectively), and IMP-1 is 85% identical to IMP-12. The bla(IMP-12) determinant is carried on an integron-borne gene cassette whose attC recombination site is related to those present in cassettes containing bla(IMP-1), bla(IMP-6), bla(IMP-7), bla(IMP-10), and bla(IMP-11) and unrelated to that present in cassettes containing bla(IMP-2) and bla(IMP-8). IMP-12 was overproduced in E. coli by using a T7-based expression system and was purified by cation-exchange chromatography followed by gel filtration. Kinetic analysis revealed that, like other IMP variants, IMP-12 exhibits an overall preference for cephalosporins and carbapenems rather than for penicillins and does not hydrolyze temocillin and aztreonam. However, IMP-12 also exhibits some notable functional differences from other IMP variants, including uniformly poor activity toward penicillins (k(cat)/K(m) values, around 10(4) M(-1). s(-1)) and a remarkably high K(m) (around 900 micro M) for imipenem.
KeywordMeSH Terms
18. Beuth  B, Niefind  K, Schomburg  D,     ( 2003 )

Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution.

Journal of molecular biology 332 (1)
PMID : 12946365  :   DOI  :   10.1016/s0022-2836(03)00860-x    
Abstract >>
Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida, a homohexameric enzyme with a molecular mass of 28.4 kDa per subunit, is a cyclic amidohydrolase catalysing the reversible conversion of creatinine to creatine. The enzyme plays a key role in the bacterial degradation of creatinine. The three-dimensional structure of creatininase from P.putida was determined and refined to 2.1A. The structure shows the six subunits arranged as a trimer of dimers and definitely disproves previous reports that the enzyme has an octameric quaternary structure. Each monomer consists of a central, four-stranded, parallel beta-sheet flanked by two alpha-helices on both sides of the beta-sheet. This topology is unique within the superfamily of amidohydrolases. Moreover, creatininase possesses a novel fold with no close structural relatives within the Protein Data Bank. Each creatininase monomer contains a binuclear zinc centre near the C termini of the beta-strands and the N termini of the main alpha-helices. These zinc ions indicate the location of the active site unambiguously. The active site is entirely buried and is not accessible from the solution without movement of parts of the protein. The two zinc ions are bridged by a water molecule and by an aspartate residue, which acts as a bidentate ligand. They differ from each other in the number and the spatial arrangement of their ligands. One of them is tetrahedrally and the other trigonal-bipyramidally ligated. Using two water molecules of the first coordination sphere as anchor points, a creatinine-water adduct resembling the transition state of the hydrolysation reaction was modelled into the active site. The resulting complex in combination with structural comparisons with other amidohydrolases enabled us to identify the most probable candidate for the catalytic base and to suggest a putative reaction mechanism. Surprisingly these structural comparisons revealed a similarity in the active-site arrangement between creatininase and the hydantoinase-like cyclic amidohydrolases that was unexpected, given the completely unrelated primary and tertiary structures. In particular, the zinc-bridging aspartate residue of creatininase is a spatially and functionally analogue to a carboxylated lysine residue found in dihydroorotase and the hydantoinases. Hence, creatininase and the hydantoinase-like cyclic amidohydrolases represent a further example of convergent evolution within the enzyme class of hydrolases.
KeywordMeSH Terms
Evolution, Molecular
Protein Folding
Protein Structure, Tertiary
19. Endoh  T, Habe  H, Yoshida  T, Nojiri  H, Omori  T,     ( 2003 )

A CysB-regulated and sigma54-dependent regulator, SfnR, is essential for dimethyl sulfone metabolism of Pseudomonas putida strain DS1.

Microbiology (Reading, England) 149 (Pt 4)
PMID : 12686641  :   DOI  :   10.1099/mic.0.26031-0    
Abstract >>
Pseudomonas putida strain DS1 utilizes dimethyl sulfide (DMS) as a sulfur source, and desulfurizes it via dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO(2)) and methanesulfonate (MSA). Its Tn5 mutant, Dfi74J, no longer utilized DMS, DMSO and DMSO(2), but could oxidize DMS to DMSO(2), suggesting that the conversion of DMSO(2) to MSA was interrupted in the mutant. Sequencing of the Tn5 flanking region of Dfi74J demonstrated that a gene, sfnR (designated for dimethyl sulfone utilization), encoding a transcriptional regulator containing an ATP-dependent sigma(54)-association domain and a DNA-binding domain, was disrupted. sfnR is part of an operon with two other genes, sfnE and sfnC, located immediately upstream of sfnR and in the same orientation. The genes encode NADH-dependent FMN reductase (SfnE) and FMNH(2)-dependent monooxygenase (SfnC). Complementation of Dfi74J with an sfnR-expressing plasmid led to restoration of its growth on DMS, DMSO and DMSO(2). An rpoN-defective mutant of strain DS1, which lacks the sigma(54) factor, grew on MSA, but not on DMS, DMSO and DMSO(2), indicating that SfnR controls expression of gene(s) involved in DMSO(2) metabolism by interaction with sigma(54)-RNA polymerase. Northern hybridization and a reporter gene assay with an sfn-lacZ transcriptional fusion elucidated that expression of the sfnECR operon was induced under sulfate limitation and was dependent on a LysR-type transcriptional regulator, CysB. This is believed to be the first report that a sigma(54)-dependent transcriptional regulator induced under sulfate limitation is involved in sulfur assimilation.
KeywordMeSH Terms
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
20. Kaderbhai  MA, Hopper  DJ, Akhtar  KM, Abbas  SK, Kaderbhai  NN,     ( 2003 )

A cytochrome c from a lupanine-transforming Pseudomonas putida strain is expressed in Escherichia coli during aerobic cultivation and efficiently exported and assembled in the periplasm.

Applied and environmental microbiology 69 (8)
PMID : 12902264  :   DOI  :   10.1128/aem.69.8.4727-4731.2003     PMC  :   PMC169100    
Abstract >>
We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain. Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm (approximately 4 mg of hemoprotein/liter of culture). The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine. Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program. The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence.
KeywordMeSH Terms
21. Satoh  A, Adachi  O, Tanizawa  K, Hirotsu  K,     ( 2003 )

The active site structure of quinohemoprotein amine dehydrogenase inhibited by p-nitrophenylhydrazine.

Biochimica et biophysica acta 1647 (1��2��)
PMID : 12686145  :   DOI  :   10.1016/s1570-9639(03)00069-4    
Abstract >>
Quinohemoprotein amine dehydrogenase (QH-AmDH) catalyzes the oxidative deamination of aliphatic and aromatic amines. The enzyme from Pseudomonas putida has an alpha beta gamma heterotrimeric structure with two heme c groups in the largest alpha subunit, and a novel quinone cofactor [cysteine tryptophylquinone (CTQ)] and hitherto unknown internal cross-bridges in the smallest gamma subunit. The crystal structure of the enzyme in the complex with the inhibitor [p-nitrophenylhydrazine (pNPH)] has been determined at a 2.0 A resolution.(1) The hydrazone of the cofactor with the inhibitor was nicely modeled into the omit electron density map, identifying the C6 carbonyl group as the reactive site of the cofactor. The Asp33 gamma is unambiguously determined as the catalytic base to abstract the alpha-proton from a substrate, because N beta atom of the inhibitor corresponding to the C alpha atom of the substrate amine is neighbored to Asp33 gamma. The bound inhibitor is completely enclosed in the active site pocket formed by the residues from the beta- and gamma-subunits. The cofactor-inhibitor adduct may be predominantly in the hydrazone with the azo form as a minor component. The binding of the inhibitor causes minor but important conformational changes in the residues surrounding the active site. The inhibitor may have access to the active site pocket through the water-filled crevice between the beta- and gamma-subunits.
KeywordMeSH Terms
22. Park  SM, Park  HH, Lim  WK, Shin  HJ,     ( 2003 )

A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida.

Journal of biotechnology 103 (3)
PMID : 12890609  :  
Abstract >>
A new variant type of regulatory activator and relevant promoters (designated capR, Pr and Po) involved in the metabolism of phenolic compounds were cloned from Pseudomonas putida KCTC1452 by using PCR. The deduced amino acid sequence of CapR revealed a difference in nine amino acids from the effector binding domain of DmpR. To measure effector specificity, plasmids were constructed in such a way that the expression of luc gene for firefly luciferase or lacZ for beta-galactosidase as a reporter was under the control of capR. When Escherichia coli transformed with the plasmids was exposed to phenol, dramatic increases in the activity of luciferase or beta-galactosidase were observed in a range of 0.01-1 mM. Among various phenolic compounds tested, other effective compounds included catechol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2-chlorophenol, 4-chlorophenol, 2-nitrophenol, resorcinol, and 2, 5-dimethylphenol. The results indicate that CapR has effector specificity different from other related activators, CatR and DmpR. Waste water and soil potentially containing phenolic compounds were also tested by this system and the results were compared with chemical and GC data. The present results indicate that the biosensor consisting of capR and the promoters may be utilized for the development of a phenolic compounds-specific biosensor in monitoring the environmental pollutant.
KeywordMeSH Terms
Transcription Factors
23. McLeish  MJ, Kneen  MM, Gopalakrishna  KN, Koo  CW, Babbitt  PC, Gerlt  JA, Kenyon  GL,     ( 2003 )

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.

Journal of bacteriology 185 (8)
PMID : 12670968  :   DOI  :   10.1128/jb.185.8.2451-2456.2003     PMC  :   PMC152609    
Abstract >>
The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.
KeywordMeSH Terms
24. Karlsson  A, Parales  JV, Parales  RE, Gibson  DT, Eklund  H, Ramaswamy  S,     ( 2003 )

Crystal structure of naphthalene dioxygenase: side-on binding of dioxygen to iron.

Science (New York, N.Y.) 299 (5609)
PMID : 12586937  :   DOI  :   10.1126/science.1078020    
Abstract >>
Binding of oxygen to iron is exploited in several biological and chemical processes. Although computational and spectroscopic results have suggested side-on binding, only end-on binding of oxygen to iron has been observed in crystal structures. We have determined structures of naphthalene dioxygenase that show a molecular oxygen species bound to the mononuclear iron in a side-on fashion. In a complex with substrate and dioxygen, the dioxygen molecule is lined up for an attack on the double bond of the aromatic substrate. The structures reported here provide the basis for a reaction mechanism and for the high stereospecificity of the reaction catalyzed by naphthalene dioxygenase.
KeywordMeSH Terms
25. Sentchilo  V, Zehnder  AJ, van der Meer  JR,     ( 2003 )

Characterization of two alternative promoters for integrase expression in the clc genomic island of Pseudomonas sp. strain B13.

Molecular microbiology 49 (1)
PMID : 12823813  :   DOI  :   10.1046/j.1365-2958.2003.03548.x    
Abstract >>
The clc genomic island is a 105 kb integrative and conjugative element (ICE) in Pseudomonas sp. strain B13, which encodes metabolism of 3-chlorocatechol. The clc island is integrated in a tRNAGly gene, but can excise and form a circular intermediate in which both ends are connected. The integrase gene (intB13) of the clc genomic island is located at the right end, 202 bp from the junction site facing inwards. Fragments upstream of intB13 in the circular form and in the integrated form were fused to a promoterless gfp gene for Green Fluorescent Protein and introduced in monocopy onto the chromosome of strain B13. Quantitative GFP fluorescence measurements in individual cells of the different B13-derivatives revealed that the circular form fragment contained a strong constitutive promoter (Pcirc) driving intB13 expression in all cells. By using primer extension Pcirc could be mapped near the left end of the clc element and Pcirc can therefore only control intB13 expression when left and right ends are connected as in the circular form. Expression from intB13 upstream fragments from the integrated clc element was weaker than that from Pcirc and only occurred in maximally 15% of individual cells in a culture. A promoter (Pint) could be roughly mapped in this region by using reverse-transcription PCR and by successively shortening the fragment from the 5' end. Transposon mutants in cloned left end sequences of the clc element were selected which had lost the activation potential on the Pint promoter and those which resulted in overexpression of GFP from Pint. The DNA sequence of the region of the transposon insertions pointed to a relatively well conserved area among various other genomic islands. The activator mutants mapped in an open reading frame (ORF) encoding a 175 amino acid protein without any significant similarity to functionally characterized proteins in the databases.
KeywordMeSH Terms
Genomic Islands
Promoter Regions, Genetic
26. Endoh  T, Kasuga  K, Horinouchi  M, Yoshida  T, Habe  H, Nojiri  H, Omori  T,     ( 2003 )

Characterization and identification of genes essential for dimethyl sulfide utilization in Pseudomonas putida strain DS1.

Applied microbiology and biotechnology 62 (1)
PMID : 12835925  :   DOI  :   10.1007/s00253-003-1233-7    
Abstract >>
Microbial dimethyl sulfide (DMS) conversion is thought to be involved in the global sulfur cycle. We isolated Pseudomonas putida strain DS1 from soil as a bacterium utilizing DMS as a sole sulfur source, and tried to elucidate the DMS conversion mechanism of strain DS1 at biochemical and genetic level. Strain DS1 oxidized DMS to dimethyl sulfone (DMSO(2)) via dimethyl sulfoxide, whereas the oxidation was repressed in the presence of sulfate, suggesting that a sulfate starvation response is involved in DMS utilization by strain DS1. Two of the five DMS-utilization-defective mutants isolated by transposon 5 (Tn 5) mutagenesis had a Tn 5 insertion in the ssuEADCBF operon, which has been reported to encode a two-component monooxygenase system (SsuED), an ABC-type transporter (SsuABC), and a small protein (SsuF), and also to play a key role in utilization of sulfonates and sulfate esters in another bacterium, P. putida strain S-313. Disruption of ssuD and SsuD enzymatic activity demonstrated that methanesulfonate is a metabolic intermediate of DMS and desulfonated by SsuD. Disruption of ssuC or ssuF also led to a DMS-utilization-defective phenotype. Another two mutants had a defect in a gene homologous to pa2354 from P. aeruginosa PAO1, which encodes a putative transcriptional regulator, while the remaining mutant had a defect in cysM encoding O-acetylserine (thiol)-lyase B.
KeywordMeSH Terms
Genes, Bacterial
27. Toyama  H, Fujii  T, Aoki  N, Matsushita  K, Adachi  O,     ( 2003 )

Molecular cloning of quinohemoprotein alcohol dehydrogenase, ADH IIB, from Pseudomonas putida HK5.

Bioscience, biotechnology, and biochemistry 67 (6)
PMID : 12843671  :   DOI  :   10.1271/bbb.67.1397    
Abstract >>
Molecular cloning of the gene of quinohemoprotein alcohol dehydrogenase (ADH IIB) from Pseudomonas putida HK5 was done. The gene (qbdA) was 690 amino acids in length, containing a 22-amino acid signal sequence. Another gene (qbdB) upstream of qbdA, probably in the same transcriptional unit, was found. Further upstream, a gene divergently transcribed against qbdBA had identity with NAD-dependent aldehyde dehydrogenase.
KeywordMeSH Terms
28. Parret  AH, Schoofs  G, Proost  P, De Mot  R,     ( 2003 )

Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate.

Journal of bacteriology 185 (3)
PMID : 12533465  :   DOI  :   10.1128/jb.185.3.897-908.2003     PMC  :   PMC142807    
Abstract >>
Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.
KeywordMeSH Terms
29. Senapin  S, Chaisri  U, Panyim  S, Tungpradabkul  S,     ( 1999 )

A new type of flagellin gene in Pseudomonas putida.

The Journal of general and applied microbiology 45 (3)
PMID : 12501380  :  
Abstract >>
Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.
KeywordMeSH Terms
30. Tungpradabkul  S, Senapin  S, Panyim  S,     ( 1998 )

PCR-based method for isolation of the flagellin genes from Pseudomonas species.

The Journal of general and applied microbiology 44 (3)
PMID : 12501433  :  
Abstract >>
N/A
KeywordMeSH Terms
31. Greated  A, Lambertsen  L, Williams  PA, Thomas  CM,     ( 2002 )

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida.

Environmental microbiology 4 (12)
PMID : 12534468  :  
Abstract >>
The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.
KeywordMeSH Terms
32. Lombardi  G, Luzzaro  F, Docquier  JD, Riccio  ML, Perilli  M, Colì  A, Amicosante  G, Rossolini  GM, Toniolo  A,     ( 2002 )

Nosocomial infections caused by multidrug-resistant isolates of pseudomonas putida producing VIM-1 metallo-beta-lactamase.

Journal of clinical microbiology 40 (11)
PMID : 12409373  :   DOI  :   10.1128/jcm.40.11.4051-4055.2002     PMC  :   PMC139695    
Abstract >>
Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-beta-lactamases, which confer high-level resistance to carbapenems and most other beta-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were >/=32 micro g/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-beta-lactamase activity, while molecular analysis detected in each isolate a bla(VIM-1) determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the beta-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the bla(VIM-1) determinant (named In110) showed that the bla(VIM-1)-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a bla(VIM-1)-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
33. Tanaka  N, Kusakabe  Y, Ito  K, Yoshimoto  T, Nakamura  KT,     ( 2002 )

Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases.

Journal of molecular biology 324 (3)
PMID : 12445786  :   DOI  :   10.1016/s0022-2836(02)01066-5    
Abstract >>
Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family. The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as cosubstrate) in typical alcohol dehydrogenases (ADHs), is tightly but not covalently bound to the protein and acts as a cofactor. PFDH can catalyze aldehyde dismutations without an external addition of NAD(H). The structural basis of the tightly bound cofactor of PFDH is unknown. The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 A resolution. The 170-kDa homotetrameric PFDH molecule shows 222 point group symmetry. Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed. A comparison of the present structure of PFDH with that of horse liver ADH, a typical example of an ADH, reveals that a long insertion loop of PFDH shields the adenine part of the bound NAD(+) molecule from the solvent, and a tight hydrogen bond network exists between the insertion loop and the adenine part of the cofactor, which is unique to PFDH. This insertion loop is conserved completely among the aldehyde-dismutating formaldehyde dehydrogenases, whereas it is replaced by a short turn among typical ADHs. Thus, the insertion loop specifically found among the aldehyde-dismutating formaldehyde dehydrogenases is responsible for the tight cofactor binding of these enzymes and explains why PFDH can effectively catalyze alternate oxidation and reduction of aldehydes without the release of cofactor molecule from the enzyme.
KeywordMeSH Terms
34. Fedorov  R, Ghosh  DK, Schlichting  I,     ( 2003 )

Crystal structures of cyanide complexes of P450cam and the oxygenase domain of inducible nitric oxide synthase-structural models of the short-lived oxygen complexes.

Archives of biochemistry and biophysics 409 (1)
PMID : 12464241  :   DOI  :   10.1016/s0003-9861(02)00555-6    
Abstract >>
The crystal structure of the ternary cyanide complex of P450cam and camphor was determined to 1.8A resolution and found to be identical with the structure of the active oxygen complex [I. Schlichting et al., 2000, Science 287, 1615]. Notably, cyanide binds in a bent mode and induces the active conformation that is characterized by the presence of two water molecules and a flip of the carbonyl of the conserved Asp251. The structure of the ternary complex of cyanide, L-arginine, and the oxygenase domain of inducible nitric oxide synthase was determined to 2.4A resolution. Cyanide binds essentially linearly, interacts with L-Arg, and induces the binding of a water molecule at the active site. This water is positioned by backbone interactions, located 2.8A from the nitrogen atom of cyanide, and could provide a proton required for O-O bond scission in the hydroxylation reaction of nitric oxide synthase.
KeywordMeSH Terms
35. Steidle  A, Allesen-Holm  M, Riedel  K, Berg  G, Givskov  M, Molin  S, Eberl  L,     ( 2002 )

Identification and characterization of an N-acylhomoserine lactone-dependent quorum-sensing system in Pseudomonas putida strain IsoF.

Applied and environmental microbiology 68 (12)
PMID : 12450862  :   DOI  :   10.1128/aem.68.12.6371-6382.2002     PMC  :   PMC134430    
Abstract >>
Recent reports have shown that several strains of Pseudomonas putida produce N-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promoting P. putida strain IsoF that, when present in trans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes: ppuI and ppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region between ppuI and ppuR with significant homology to rsaL from Pseudomonas aeruginosa, and a gene, designated ppuA, present upstream of ppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptional ppuA::luxAB fusion we demonstrate that expression of ppuA is AHL dependent. Furthermore, transcription of the AHL synthase ppuI is shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking the ppu gene cluster indicated that the four genes form an island in the suhB-PA3819 intergenic region of the currently sequenced P. putida strain KT2440. Moreover, we provide evidence that the ppu genes are not present in other AHL-producing P. putida strains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both a ppuI and a ppuA mutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development.
KeywordMeSH Terms
36. Weightman  AJ, Topping  AW, Hill  KE, Lee  LL, Sakai  K, Slater  JH, Thomas  AW,     ( 2002 )

Transposition of DEH, a broad-host-range transposon flanked by ISPpu12, in Pseudomonas putida is associated with genomic rearrangements and dehalogenase gene silencing.

Journal of bacteriology 184 (23)
PMID : 12426347  :   DOI  :   10.1128/jb.184.23.6581-6591.2002     PMC  :   PMC135415    
Abstract >>
Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families. The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehR(I), was isolated from strain PP3 by using the TOL plasmid pWW0. DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehR(I) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria. Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P. putida PaW340. Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P. putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing. Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.
KeywordMeSH Terms
Bacterial Proteins
37. Williams  PA, Jones  RM, Shaw  LE,     ( 2002 )

A third transposable element, ISPpu12, from the toluene-xylene catabolic plasmid pWW0 of Pseudomonas putida mt-2.

Journal of bacteriology 184 (23)
PMID : 12426346  :   DOI  :   10.1128/jb.184.23.6572-6580.2002     PMC  :   PMC135414    
Abstract >>
A 3,372-bp insertion sequence, ISPpu12, has been identified on the archetypal toluene-xylene TOL catabolic plasmid pWW0 from Pseudomonas putida mt-2. The insertion sequence element is located on the plasmid between bases 84397 and 87768 in a region which also contains the termini and transposase genes of the catabolic transposons Tn4651 and Tn4653 (A. Greated, L. Lambertson, P. A. Williams, and C. M. Thomas, Environ. Microbiol., in press). ISPpu12 has terminal inverted repeats of 24 bp with three mismatches and contains four open reading frames, a tnpA homologue and three open reading frames (lspA, orf1, and orf2) of undetermined function. After insertion in vitro of a Km(r) cassette into ISPpu12 either in the intergenic region between orf1 and orf2 or directly into the orf1 gene and ligation into a suicide vector, the modified ISPpu12-Km transposes at high frequency, often in multiple copies, into the chromosome of a P. putida recipient. Inactivation of lspA, orf1, and orf2 by introducing a 7-bp deletion into the 5' region of each gene had no major effect upon transposition, but a similar mutation of tnpA completely eliminated transposition. Analysis of the literature and of strains derived from the chlorobenzoate-degrading Pseudomonas sp. strain B13 suggests that the promiscuity of this element has played an important role in the history of plasmid pWW0. Database comparisons and the accompanying paper (A. J. Weightman, A. W. Topping, K. E. Hill, L. L. Lee, K. Sakai, J. H. Slater, and A. W. Thomas, J. Bacteriol. 184:6581-6591, 2002) show that ISPpu12 is a transposable element also found in other bacteria.
KeywordMeSH Terms
38. Kholodii  G, Gorlenko  Zh, Mindlin  S, Hobman  J, Nikiforov  V,     ( 2002 )

Tn5041-like transposons: molecular diversity, evolutionary relationships and distribution of distinct variants in environmental bacteria.

Microbiology (Reading, England) 148 (Pt 11)
PMID : 12427948  :   DOI  :   10.1099/00221287-148-11-3569    
Abstract >>
A detailed study on the geographic distribution, molecular diversity and evolutionary relationships of 24 closely related variants of the Tn5041 transposon found among 182 mercury resistant environmental Gram-negative strains from the IMG-Hg Reference Collection is reported here. RFLP analysis, followed by the determination of partial DNA sequences, identified 14 distinct types of these transposons, which differed from each other by 1-7 single-event DNA polymorphisms. No polymorphisms were detected at the right arm of the transposons except an insertion of a new mobile DNA element carrying a mer operon (named the mer2 cassette) within the Tn5041 mer operon. According to the model presented here, the insertion occurred via homologous recombination with a circular form of the mer2 cassette. A total of 8 point mutations, 1 internal deletion, 2 end-involving deletions, 3 mosaic regions and 2 insertions were detected at the left arm of the transposons. The insertions were a transposon closely related to Tn21 but lacking the integron and a new group II intron (named INT5041C). Inspection of the geographic distribution of the Tn5041 variants suggested that at least three long-distance waves of dissemination of these variants had occurred, accompanied by homologous recombination between different Tn5041 lineages. Movements of circular DNAs by homologous recombination as a source of mosaic genes and new mer genes, and formation of unusual mosaics ending or beginning at the Tn5041 att site are discussed.
KeywordMeSH Terms
Saccharomyces cerevisiae Proteins
39. Fukumori  F, Kishii  M,     ( 2002 )

Characterization of the toluene-sensitive mutants of Pseudomonas putida KT2442TOL.

The Journal of general and applied microbiology 48 (2)
PMID : 12469308  :  
Abstract >>
N/A
KeywordMeSH Terms
40. von Wallbrunn  A, Heipieper  HJ, Meinhardt  F,     ( 2002 )

Cis/trans isomerisation of unsaturated fatty acids in a cardiolipin synthase knock-out mutant of Pseudomonas putida P8.

Applied microbiology and biotechnology 60 (1��2��)
PMID : 12382061  :   DOI  :   10.1007/s00253-002-1080-y    
Abstract >>
The gene encoding cardiolipin synthase (cls) from the phenol-degrading bacterium Pseudomonas putida P8, which rapidly adapts its membrane lipids to the presence of organic solvents by cis/trans isomerisation of unsaturated fatty acids, was isolated and completely sequenced. The functionality of the predicted gene product was proven by constructing a knock-out mutant that was significantly reduced in its growth rate both at elevated temperatures and in the presence of membrane-active solvents. Though the mutant showed a clear phenotype it was still able to synthesise trace amounts of cardiolipin. As an increase in cardiolipin (diphosphatidylglycerol) content is known to function as a long term membrane adaptation mechanism in pseudomonads, we tested whether the mutant compensates for the lack of the Cls by increased cis/trans isomerisation of unsaturated fatty acids. Increase in cis/trans isomerisation of unsaturated fatty acids was observed for the mutant at zero and low concentrations of 4-chlorophenol; however, cis/trans isomerisation is not able to fully compensate for the lack of cardiolipin production. Possibly, other long-term adaptation mechanisms are instrumental in compensating for the missing cardiolipin synthesis. As the cis/trans isomerase is activated similarly in the mutant and the wildtype, cis/trans isomerisation and cardiolipin production do not display mutual dependency.
KeywordMeSH Terms
Membrane Proteins
41. Karsten  WE, Tipton  PA, Cook  PF,     ( 2002 )

Tartrate dehydrogenase catalyzes the stepwise oxidative decarboxylation of D-malate with both NAD and thio-NAD.

Biochemistry 41 (40)
PMID : 12356321  :   DOI  :   10.1021/bi026278g    
Abstract >>
Tartrate dehydrogenase catalyzes the divalent metal ion- and NAD-dependent oxidative decarboxylation of D-malate to yield CO(2), pyruvate, and NADH. The enzyme also catalyzes the metal ion-dependent oxidation of (+)-tartrate to yield oxaloglycolate and NADH. pH-rate profiles and isotope effects were measured to probe the mechanism of this unique enzyme. Data suggest a general base mechanism with likely general acid catalysis in the oxidative decarboxylation of D-malate. Of interest, the mechanism of oxidative decarboxylation of D-malate is stepwise with NAD(+) or the more oxidizing thio-NAD(+). The mechanism does not become concerted with the latter as observed for the malic enzyme, which catalyzes the oxidative decarboxylation of L-malate [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103]. It appears the change in mechanism observed with malic enzyme is specific to its transition state structure and not a generalized trait of metal ion- and NAD(P)-dependent beta-hydroxy acid oxidative decarboxylases. The V/K(malate) pH-rate profile decreases at low and high pH and exhibits pK(a) values of about 6.3 and 8.3, while that for V/K(tartrate) (measured from pH 7.5 to pH 9) exhibits a pK(a) of 8.6 on the basic side. A single pK(a) of 6.3 is observed on the acid side of the V(max) pH profile, but the pK(a) seen on the basic side of the V/K pH profiles is not observed in the V(max) pH profiles. Data suggest the requirement for a general base that accepts a proton from the 2-hydroxyl group of either substrate to facilitate hydride transfer. A second enzymatic group is also required protonated for optimum binding of substrates and may also function as a general acid to donate a proton to the enolpyruvate intermediate to form pyruvate. The (13)C isotope effect, measured on the decarboxylation of D-malate using NAD(+) as the dinucleotide substrate, decreases from a value of 1.0096 +/- 0.0006 with D-malate to 1.00787 +/- 0.00006 with D-malate-2-d, suggesting a stepwise mechanism for the oxidative decarboxylation of D-malate. Using thio-NAD(+) as the dinucleotide substrate the (13)C isotope effects are 1.0034 +/- 0.0007 and 1.0027 +/- 0.0002 with D-malate and D-malate-2-d, respectively.
KeywordMeSH Terms
42. Horne  I, Sutherland  TD, Oakeshott  JG, Russell  RJ,     ( 2002 )

Cloning and expression of the phosphotriesterase gene hocA from Pseudomonas monteilii C11.

Microbiology (Reading, England) 148 (Pt 9)
PMID : 12213915  :   DOI  :   10.1099/00221287-148-9-2687    
Abstract >>
The cloning of a gene encoding the novel phosphotriesterase from Pseudomonas monteilii C11, which enabled it to use the organophosphate (OP) coroxon as its sole phosphorus source, is described. The gene, called hocA (hydrolysis of coroxon) consists of 501 bp and encodes a protein of 19 kDa. This protein had no sequence similarity to any proteins in the SWISS-PROT/GenBank databases. When a spectinomycin-resistance cassette was placed in this gene, phosphotriesterase activity was abolished and P. monteilii C11 could no longer grow with coroxon as the sole phosphorus source. Overexpression and purification of HocA as a maltose-binding protein fusion produced a protein having a broad substrate specificity across oxon and thion OPs. Michaelis-Menten kinetics were observed with the oxon OPs, but not with the thion OPs. End-product inhibition was observed for coroxon-hydrolytic activity. Increased expression of hocA was observed from an integrative hocA-lacZ fusion when cultures were grown in the absence of phosphate, suggesting that it might be part of the Pho regulon, but the phosphate-regulated promoter was not cloned in this study. This is believed to be the first study in which a gene required for an organism to grow with OP pesticides as a phosphorus source has been isolated.
KeywordMeSH Terms
43. Adaikkalam  V, Swarup  S,     ( 2002 )

Molecular characterization of an operon, cueAR, encoding a putative P1-type ATPase and a MerR-type regulatory protein involved in copper homeostasis in Pseudomonas putida.

Microbiology (Reading, England) 148 (Pt 9)
PMID : 12213931  :   DOI  :   10.1099/00221287-148-9-2857    
Abstract >>
The authors have characterized a chromosomally localized two-gene operon, cueAR, which encodes a putative P1-type ATPase, CueA, and a MerR-type metalloregulatory protein, CueR, in Pseudomonas putida PNL-MK25. Disruption of cueAR by the insertion of mini-Tn5::gfp into the wild-type strain led to a mutant strain with a sixfold reduction in its tolerance to copper; however, the tolerance of this mutant strain to the other seven related transition metals tested was not affected. The sensitivity of the mutant strain was attributed to a higher level of accumulation of intracellular copper, suggesting the involvement of CueA in copper export. Insertion of the cloned cueAR operon into the copper-sensitive mutant strain fully restored its tolerance to copper. cueA::gfp expression studies confirmed that the cueAR operon was transcriptionally regulated by copper and CueR. Studies done on the mutant strain complemented with cueR and cueA revealed partial functional redundancy of cueA and cueR, respectively, in copper tolerance. Thus, the results of this study clearly suggest the involvement of cueAR in copper homeostasis in P. putida.
KeywordMeSH Terms
44. Petruschka  L, Adolf  K, Burchhardt  G, Dernedde  J, Jürgensen  J, Herrmann  H,     ( 2002 )

Analysis of the zwf-pgl-eda-operon in Pseudomonas putida strains H and KT2440.

FEMS microbiology letters 215 (1)
PMID : 12393206  :   DOI  :   10.1111/j.1574-6968.2002.tb11375.x    
Abstract >>
A 3.9-kb fragment of the genome of Pseudomonas putida H, containing the complete zwf-pgl-eda-operon, encoding glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase and 2-keto-3-deoxy-6-phosphogluconate-aldolase, respectively, and part of the divergently transcribed regulatory gene, hexR, was cloned and analyzed. The nucleotide sequences of these genes showed high similarities to the corresponding DNA sequences of P. putida KT2440 and also to sequences of Pseudomonas aeruginosa PAO1. Derivatives of strains H and KT2440, containing transcriptional lacZ fusions to P(zwf) were generated and used to study the expression of these operons. In both strains, this operon was induced by carbohydrates such as glucose, gluconate, fructose and glycerol. The transcription rate of the zwf-pgl-eda-operon was found to be about three times higher in the KT2440 background than in strain H. In both strains the induction of the zwf-pgl-eda-operon by carbohydrates during growth on carboxylic acids was not affected by carbon catabolite repression.
KeywordMeSH Terms
45. Goda  M, Hashimoto  Y, Takase  M, Herai  S, Iwahara  Y, Higashibata  H, Kobayashi  M,     ( 2002 )

Isonitrile hydratase from Pseudomonas putida N19-2. Cloning, sequencing, gene expression, and identification of its active acid residue.

The Journal of biological chemistry 277 (48)
PMID : 12244065  :   DOI  :   10.1074/jbc.M208571200    
Abstract >>
Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19-2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.
KeywordMeSH Terms
46. Deprez  E, Gill  E, Helms  V, Wade  RC, Hui Bon Hoa  G,     ( 2002 )

Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin.

Journal of inorganic biochemistry 91 (4)
PMID : 12237225  :  
Abstract >>
Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool binding strongly suggests that, similarly to our previous finding for cytochrome P450cam, electrostatic gates participate in the control of substrate access.
KeywordMeSH Terms
47. Fiedler  S, Steinbüchel  A, Rehm  BH,     ( 2002 )

The role of the fatty acid beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida.

Archives of microbiology 178 (2)
PMID : 12115060  :   DOI  :   10.1007/s00203-002-0444-0    
Abstract >>
In order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis, the fadBA(Po) operon of Pseudomonas oleovorans was cloned and characterized. The fadBA(Po) operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBA(Po) has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBA(Po) did not mediate PHA(MCL) biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the (R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of beta-oxidation towards PHA(MCL) biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3- Re enoyl-CoA hydratase, was identified. The co-expression of phaJ(Po/Pp) with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJ(Po/Pp) mediated biosynthesis of either PHA(MCL), contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate- co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBA(Po)does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3- Re enoyl-CoA hydratases are required to divert intermediates of fatty acid beta-oxidation towards PHA biosynthesis in P. oleovorans.
KeywordMeSH Terms
Genes, Bacterial
48. Genka  H, Nagata  Y, Tsuda  M,     ( 2002 )

Site-specific recombination system encoded by toluene catabolic transposon Tn4651.

Journal of bacteriology 184 (17)
PMID : 12169600  :   DOI  :   10.1128/jb.184.17.4757-4766.2002     PMC  :   PMC135285    
Abstract >>
The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.
KeywordMeSH Terms
DNA Transposable Elements
Recombination, Genetic
49. Park  W, Padmanabhan  P, Padmanabhan  S, Zylstra  GJ, Madsen  EL,     ( 2002 )

nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA.

Microbiology (Reading, England) 148 (Pt 8)
PMID : 12177326  :   DOI  :   10.1099/00221287-148-8-2319    
Abstract >>
In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism [Schell, M. A. & Poser, E. F. (1989). J Bacteriol 171, 837-846]. The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P. putida NCIB 9816-4 was verified. Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source. The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P. putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers. The amplified nahR homologues and the intergenic regions were cloned and sequenced. Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates. The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5'-ATTCACGCTN(2)TGAT-3'). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not. DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories. PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene. The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes. Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.
KeywordMeSH Terms
Coal Tar
Plasmids
50. Park  W, Jeon  CO, Madsen  EL,     ( 2002 )

Interaction of NahR, a LysR-type transcriptional regulator, with the alpha subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4.

FEMS microbiology letters 213 (2)
PMID : 12167532  :   DOI  :   10.1111/j.1574-6968.2002.tb11300.x    
Abstract >>
NahR, a LysR-type transcriptional regulator, is required for expression of naphthalene catabolic operons. However, detailed mechanisms of transcriptional activation by NahR are poorly understood. Many transcriptional activators make direct contact with RNA polymerase (RNAP) to initiate transcription. We investigated the hypothesis that direct contact between NahR and the alpha subunit of RNAP (alphaRNAP) may be involved in expression of the naphthalene catabolic operons in Pseudomonas putida NCIB 9816-4. Interactions between the NahR and alphaRNAP in P. putida NCIB 9816-4 were analyzed using the yeast two-hybrid system. The results obtained indicate that protein-protein interactions occur between alphaRNAP and the NahR. Gene activation by NahR is consistent with the general transcriptional mechanism of class I transcription factors, which function by contacting alphaRNAP.
KeywordMeSH Terms
51. Treviño-Quintanilla  LG, Galán-Wong  LJ, Rodríguez-Uribe  B, Soberón-Chávez  G,     ( 2002 )

Cloning and characterization of a FAD-monooxygenase gene (cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07.

Applied microbiology and biotechnology 59 (4��5��)
PMID : 12172624  :   DOI  :   10.1007/s00253-002-1040-6    
Abstract >>
A bacterium culture was isolated on the basis of its ability to degrade chloranilic acid, and was later identified as Pseudomonas putida (TQ07). Several transposon insertion mutants unable to degrade chloranilic acid were selected. The characterization of the site of insertion of one of these mutants led to the identification of the cadA gene encoding an enzyme with significant homology with FAD-monooxygenases involved in the degradation of aromatic and chloroaromatic compounds. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of "halo" formation (a zone of clearing color on agar plates around TQ07 colonies that degrade chloranilic acid) and degradation of chloranilic acid, unequivocally assigned cadA a function in the metabolism of this compound. We also found that most of the transposon insertion mutants unable to degrade chloranilic acid are clustered in a 10-kb region of the P. putidagenome that is encoded in a megaplasmid or in an unstable chromosomal region.
KeywordMeSH Terms
52. Devescovi  G, Aguilar  C, Majolini  MB, Marugg  J, Weisbeek  P, Venturi  V,     ( 2001 )

A siderophore peptide synthetase gene from plant-growth-promoting Pseudomonas putida WCS358.

Systematic and applied microbiology 24 (3)
PMID : 11822666  :   DOI  :   10.1078/0723-2020-00063    
Abstract >>
Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety. Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions. In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene. Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning. This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each. ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans. It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358. PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P. aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P. aeruginosa. It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.
KeywordMeSH Terms
Genes, Bacterial
53. Barna  T, Messiha  HL, Petosa  C, Bruce  NC, Scrutton  NS, Moody  PC,     ( 2002 )

Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme.

The Journal of biological chemistry 277 (34)
PMID : 12048188  :   DOI  :   10.1074/jbc.M202846200    
Abstract >>
The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.
KeywordMeSH Terms
Bacterial Proteins
54. Chen  ZW, Matsushita  K, Yamashita  T, Fujii  TA, Toyama  H, Adachi  O, Bellamy  HD, Mathews  FS,     ( 2002 )

Structure at 1.9 A resolution of a quinohemoprotein alcohol dehydrogenase from Pseudomonas putida HK5.

Structure (London, England : 1993) 10 (6)
PMID : 12057198  :  
Abstract >>
The type II quinohemoprotein alcohol dehydrogenase of Pseudomonas putida is a periplasmic enzyme that oxidizes substrate alcohols to the aldehyde and transfers electrons first to pyrroloquinoline quinone (PQQ) and then to an internal heme group. The 1.9 A resolution crystal structure reveals that the enzyme contains a large N-terminal eight-stranded beta propeller domain (approximately 60 kDa) similar to methanol dehydrogenase and a small C-terminal c-type cytochrome domain (approximately 10 kDa) similar to the cytochrome subunit of p-cresol methylhydoxylase. The PQQ is bound near the axis of the propeller domain about 14 A from the heme. A molecule of acetone, the product of the oxidation of isopropanol present during crystallization, appears to be bound in the active site cavity.
KeywordMeSH Terms
55. Hasona  A, York  SW, Yomano  LP, Ingram  LO, Shanmugam  KT,     ( 2002 )

Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase.

Applied and environmental microbiology 68 (6)
PMID : 12039716  :   DOI  :   10.1128/aem.68.6.2651-2659.2002     PMC  :   PMC123972    
Abstract >>
During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).
KeywordMeSH Terms
56. Mindlin  S, Kholodii  G, Gorlenko  Z, Minakhina  S, Minakhin  L, Kalyaeva  E, Kopteva  A, Petrova  M, Yurieva  O, Nikiforov  V,     ( 2001 )

Mercury resistance transposons of gram-negative environmental bacteria and their classification.

Research in microbiology 152 (9)
PMID : 11763242  :  
Abstract >>
A total of 29 mercury resistance transposons were isolated from mercury-resistant environmental strains of proteobacteria collected in different parts of Eurasia and the USA and tested for hybridization with probes specific for transposase genes of known mercury resistance transposons. 9 were related to Tn21 in this test, 12 were related to Tn5053, 4 to Tn5041 and 1 to Tn5044; three transposons were negative in this test. Restriction mapping and DNA sequencing revealed that 12 transposons were identical or nearly identical to their corresponding relatives while the rest showed varying divergence from their closest relatives. Most of these previously unknown transposons apparently arose as a result of homologous or site-specific recombination. One of these, Tn5046, was completely sequenced, and shown to be a chimera with the mer operon and the transposition module derived from the transposons related to Tn5041 and to Tn5044, respectively. Transposon Tn5070, showing no hybridization with the specific probes used in this study, was also completely sequenced. The transposition module of Tn5070 was most closely related to that of Tn3 while the mer operon was most closely related to that of plasmid pMERPH. The merR of Tn5070 is transcribed in the same direction as the mer structural genes, which is typical for mer operons of gram-positive bacteria. Our data suggest that environmental bacteria may harbor many not yet recognized mercury resistance transposons and warrant their further inventory.
KeywordMeSH Terms
Environmental Microbiology
57. Sevrioukova  IF, Poulos  TL,     ( 2002 )

Putidaredoxin reductase, a new function for an old protein.

The Journal of biological chemistry 277 (28)
PMID : 12011076  :   DOI  :   10.1074/jbc.M201110200    
Abstract >>
Properties of recombinant wild type (WT) and six-histidine tag-fused (His(6)) putidaredoxin reductase (Pdr), a FAD-containing component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, have been studied. Both WT and His(6) Pdr were found to undergo a monomer-dimer association-dissociation and were partially present as an NAD(+)-bound form. Although molecular, spectral, and electron transferring properties of recombinant His(6) Pdr to artificial and native electron acceptors were similar to those of the WT protein, the presence of eight additional C-terminal amino acid residues, Pro-Arg-His-His-His-His-His-His, had a crucial effect on the enzyme interaction with oxidized pyridine nucleotide. Under anaerobic conditions, NAD(+) induced in His(6) Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD(+). The reaction proceeded considerably faster in the presence of free histidine and thiol-reducing agents, such as dithiothreitol and reduced glutathione. In the presence of any of these three reagents, NAD(+) was capable of inducing reduction of the flavin in WT Pdr. Free thiol groups were identified as an internal source of electrons in the enzyme. The results showed that WT and His(6) Pdr were able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD(+)-dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin. Similarity of Pdr to the enzymes of the glutathione reductase family is discussed.
KeywordMeSH Terms
58. Satoh  A, Kim  JK, Miyahara  I, Devreese  B, Vandenberghe  I, Hacisalihoglu  A, Okajima  T, Kuroda  S, Adachi  O, Duine  JA, Van Beeumen  J, Tanizawa  K, Hirotsu  K,     ( 2002 )

Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges.

The Journal of biological chemistry 277 (4)
PMID : 11704672  :   DOI  :   10.1074/jbc.M109090200    
Abstract >>
The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution. The enzyme comprises three non-identical subunits: a four-domain alpha-subunit that harbors a di-heme cytochrome c, a seven-bladed beta-propeller beta-subunit that provides part of the active site, and a small gamma-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic gamma-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue gamma-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.
KeywordMeSH Terms
Indolequinones
59. Vrionis  HA, Daugulis  AJ, Kropinski  AM,     ( 2002 )

Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization.

Applied microbiology and biotechnology 58 (4)
PMID : 11954793  :   DOI  :   10.1007/s00253-001-0912-5    
Abstract >>
Two-phase partitioning bioreactors (TPPBs) comprise an aqueous phase containing all non-carbon nutrients necessary for microbial growth and a solvent phase containing high concentrations of inhibitory or toxic substrates that partition at sub-inhibitory levels to the aqueous phase in response to cellular demand. This work aimed at eliminating the growth of Pseudomonas putida ATCC 11172 on medium-chain-length (C8-C12) aliphatic alcohols, hence enabling their use as xenobiotic delivery solvents within two-phase partitioning bioreactors. Experiments resulted in the isolation of a mini-Tn5 mutant unable to utilize these alcohols. The mutation, which also eliminated growth on glycerol and ethanol, was identified to be within a homologue of the P aeruginosa agmR gene, which encodes a response regulator. Enzyme analysis of the agmR::Tn5Km mutant cell extracts revealed a 10-fold decrease in pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase activity. A knockout in a gene (exaA) encoding a PQQ-linked alcohol dehydrogenase slowed but did not eliminate growth on medium-chain-length alcohols or ethanol, suggesting metabolic redundancy within P. putida ATCC 11172. Analysis of P. putida KT2440 genome sequence data indicated the presence of two PQQ-linked alcohol dehydrogenase-encoding genes. The successful elimination of alcohol utilization in the agmR mutant indicates control by AgmR on multiple pathways and presents a useful strain for biotechnological applications requiring alcohol non-utilizing microbial catalysts.
KeywordMeSH Terms
Trans-Activators
60. Kasai  Y, Inoue  J, Harayama  S,     ( 2001 )

The TOL plasmid pWW0 xylN gene product from Pseudomonas putida is involved in m-xylene uptake.

Journal of bacteriology 183 (22)
PMID : 11673437  :   DOI  :   10.1128/JB.183.22.6662-6666.2001     PMC  :   PMC95498    
Abstract >>
The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.
KeywordMeSH Terms
61. Nam  GH, Jang  DS, Cha  SS, Lee  TH, Kim  DH, Hong  BH, Yun  YS, Oh  BH, Choi  KY,     ( 2001 )

Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B.

Biochemistry 40 (45)
PMID : 11695900  :   DOI  :   10.1021/bi015547k    
Abstract >>
Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.
KeywordMeSH Terms
62. Dunn  AR, Dmochowski  IJ, Bilwes  AM, Gray  HB, Crane  BR,     ( 2001 )

Probing the open state of cytochrome P450cam with ruthenium-linker substrates.

Proceedings of the National Academy of Sciences of the United States of America 98 (22)
PMID : 11606730  :   DOI  :   10.1073/pnas.221297998     PMC  :   PMC60069    
Abstract >>
Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-A deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.
KeywordMeSH Terms
63. Lee  K, Lim  JB, Yum  JH, Yong  D, Chong  Y, Kim  JM, Livermore  DM,     ( 2002 )

bla(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital.

Antimicrobial agents and chemotherapy 46 (4)
PMID : 11897589  :   DOI  :   10.1128/aac.46.4.1053-1058.2002     PMC  :   PMC127086    
Abstract >>
We investigated the phenotypic and genetic properties of metallo-beta-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 beta-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the bla(VIM-2) genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had bla(VIM) located downstream of a variant of aacA4. bla(VIM) also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-beta-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.
KeywordMeSH Terms
64. Santos  PM, Mignogna  G, Heipieper  HJ, Zennaro  E,     ( 2002 )

Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains.

Research in microbiology 153 (2)
PMID : 11900268  :  
Abstract >>
Pseudomonas sp. strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs). Affinity chromatography purification showed the presence of at least one GST in each studied strain. The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested. Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions. Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations. In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions. Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate. These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells. The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism. Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity.
KeywordMeSH Terms
Glutathione Transferase
65. Francis  CA, Tebo  BM,     ( 2001 )

cumA multicopper oxidase genes from diverse Mn(II)-oxidizing and non-Mn(II)-oxidizing Pseudomonas strains.

Applied and environmental microbiology 67 (9)
PMID : 11526033  :   DOI  :   10.1128/aem.67.9.4272-4278.2001     PMC  :   PMC93157    
Abstract >>
A multicopper oxidase gene, cumA, required for Mn(II) oxidation was recently identified in Pseudomonas putida strain GB-1. In the present study, degenerate primers based on the putative copper-binding regions of the cumA gene product were used to PCR amplify cumA gene sequences from a variety of Pseudomonas strains, including both Mn(II)-oxidizing and non-Mn(II)-oxidizing strains. The presence of highly conserved cumA gene sequences in several apparently non-Mn(II)-oxidizing Pseudomonas strains suggests that this gene may not be expressed, may not be sufficient alone to confer the ability to oxidize Mn(II), or may have an alternative function in these organisms. Phylogenetic analysis of both CumA and 16S rRNA sequences revealed similar topologies between the respective trees, including the presence of several distinct phylogenetic clusters. Overall, our results indicate that both the cumA gene and the capacity to oxidize Mn(II) occur in phylogenetically diverse Pseudomonas strains.
KeywordMeSH Terms
Bacterial Proteins
66. Iwaki  M, Kagamiyama  H, Nozaki  M,     ( 1979 )

The complete amino acid sequence of the beta-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Journal of biochemistry 86 (4)
PMID : 115853  :   DOI  :   10.1093/oxfordjournals.jbchem.a132612    
Abstract >>
The complete amono aicd sequence of the beta-subunit of protocatechuate 3,4-dioxygenase is presented. The beta-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated beta-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphyloccal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated beta-subunit. The calculated molecular weight was 26,588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
KeywordMeSH Terms
Oxygenases
Protocatechuate-3,4-Dioxygenase
67. Ditty  JL, Harwood  CS,     ( 2002 )

Charged amino acids conserved in the aromatic acid/H+ symporter family of permeases are required for 4-hydroxybenzoate transport by PcaK from Pseudomonas putida.

Journal of bacteriology 184 (5)
PMID : 11844776  :   DOI  :   10.1128/jb.184.5.1444-1448.2002     PMC  :   PMC134867    
Abstract >>
Charged amino acids in the predicted transmembrane portion of PcaK, a permease from Pseudomonas putida that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for P. putida to have a chemotactic response to 4-HBA. An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H+ symporter family of the major facilitator superfamily of transporters.
KeywordMeSH Terms
Membrane Transport Proteins
68. Vandenberghe  I, Kim  JK, Devreese  B, Hacisalihoglu  A, Iwabuki  H, Okajima  T, Kuroda  S, Adachi  O, Jongejan  JA, Duine  JA, Tanizawa  K, Van Beeumen  J,     ( 2001 )

The covalent structure of the small subunit from Pseudomonas putida amine dehydrogenase reveals the presence of three novel types of internal cross-linkages, all involving cysteine in a thioether bond.

The Journal of biological chemistry 276 (46)
PMID : 11555656  :   DOI  :   10.1074/jbc.M107164200    
Abstract >>
Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the beta-carbon atom of an aspartic acid, one with the gamma-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.
KeywordMeSH Terms
Indolequinones
69. Sukumar  N, Xu  Y, Gatti  DL, Mitra  B, Mathews  FS,     ( 2001 )

Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase.

Biochemistry 40 (33)
PMID : 11502180  :   DOI  :   10.1021/bi010938k    
Abstract >>
The structure of an active mutant of (S)-mandelate dehydrogenase (MDH-GOX2) from Pseudomonas putida has been determined at 2.15 A resolution. The membrane-associated flavoenzyme (S)-mandelate dehydrogenase (MDH) catalyzes the oxidation of (S)-mandelate to give a flavin hydroquinone intermediate which is subsequently reoxidized by an organic oxidant residing in the membrane. The enzyme was rendered soluble by replacing its 39-residue membrane-binding peptide segment with a corresponding 20-residue segment from its soluble homologue, glycolate oxidase (GOX). Because of their amphipathic nature and peculiar solubilization properties, membrane proteins are notoriously difficult to crystallize, yet represent a large fraction of the proteins encoded by genomes currently being deciphered. Here we present the first report of such a structure in which an internal membrane-binding segment has been replaced, leading to successful crystallization of the fully active enzyme in the absence of detergents. This approach may have general application to other membrane-bound proteins. The overall fold of the molecule is that of a TIM barrel, and it forms a tight tetramer within the crystal lattice that has circular 4-fold symmetry. The structure of MDH-GOX2 reveals how this molecule can interact with a membrane, although it is limited by the absence of a membrane-binding segment. MDH-GOX2 and GOX adopt similar conformations, yet they retain features characteristic of membrane and globular proteins, respectively. MDH-GOX2 has a distinctly electropositive surface capable of interacting with the membrane, while the opposite surface is largely electronegative. GOX shows no such pattern. MDH appears to form a new class of monotopic integral membrane protein that interacts with the membrane through coplanar electrostatic binding surfaces and hydrophobic interactions, thus combining features of both the prostaglandin synthase/squaline-hopine cyclase and the C-2 coagulation factor domain classes of membrane proteins.
KeywordMeSH Terms
70. Godoy  P, Ramos-González  MI, Ramos  JL,     ( 2001 )

Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E.

Journal of bacteriology 183 (18)
PMID : 11514511  :   DOI  :   10.1128/jb.183.18.5285-5292.2001     PMC  :   PMC95410    
Abstract >>
Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with (14)C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.
KeywordMeSH Terms
Escherichia coli Proteins
71. Ferrero  M, Llobet-Brossa  E, Lalucat  J, García-Valdés  E, Rosselló-Mora  R, Bosch  R,     ( 2002 )

Coexistence of two distinct copies of naphthalene degradation genes in Pseudomonas strains isolated from the western Mediterranean region.

Applied and environmental microbiology 68 (2)
PMID : 11823244  :   DOI  :   10.1128/aem.68.2.957-962.2002     PMC  :   PMC126682    
Abstract >>
We analyzed the occurrence of the naphthalene degradation upper-pathway (nah) genes in the western Mediterranean region. The amplification, restriction, and sequence analysis of internal fragments for several nah genes (nahAc, nahB, nahC, and nahE) from naphthalene-degrading strains isolated from this geographical area proved the coexistence of two distinct sets of nah genes.
KeywordMeSH Terms
Gene Duplication
72. Toyama  H, Aoki  N, Matsushita  K, Adachi  O,     ( 2001 )

Azurin involved in alcohol oxidation system in Pseudomonas putida HK5: expression analysis and gene cloning.

Bioscience, biotechnology, and biochemistry 65 (7)
PMID : 11515547  :   DOI  :   10.1271/bbb.65.1617    
Abstract >>
Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis. Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared. In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB. The azurin gene (azu) was cloned and sequenced. azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found. However, its relative position suggests different transcriptional regulation from that in azu of P. aeruginosa. The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.
KeywordMeSH Terms
73. Sevrioukova  IF, Hazzard  JT, Tollin  G, Poulos  TL,     ( 2001 )

Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction.

Biochemistry 40 (35)
PMID : 11524002  :   DOI  :   10.1021/bi010874d    
Abstract >>
The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam. The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system. We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx. Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)). The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin. The rate of FADH(*) decay was dependent on ionic strength and NAD(+). In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed. The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively. This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase. Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.
KeywordMeSH Terms
74. Alaminos  M, Ramos  JL,     ( 2001 )

The methionine biosynthetic pathway from homoserine in Pseudomonas putida involves the metW, metX, metZ, metH and metE gene products.

Archives of microbiology 176 (1��2��)
PMID : 11479715  :  
Abstract >>
Biosynthesis of methionine from homoserine in Pseudomonas putida takes place in three steps. The first step is the acylation of homoserine to yield an acyl-L-homoserine. This reaction is catalyzed by the products of the metXW genes and is equivalent to the first step in enterobacteria, gram-positive bacteria and fungi, except that in these microorganisms the reaction is catalyzed by a single polypeptide (the product of the metA gene in Escherichia coli and the met5 gene product in Neurospora crassa). In Pseudomonas putida, as in gram-positive bacteria and certain fungi, the second and third steps are a direct sulfhydrylation that converts the O-acyl-L-homoserine into homocysteine and further methylation to yield methionine. The latter reaction can be mediated by either of the two methionine synthetases present in the cells.
KeywordMeSH Terms
75. Kojic  M, Venturi  V,     ( 2001 )

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator.

Journal of bacteriology 183 (12)
PMID : 11371535  :   DOI  :   10.1128/JB.183.12.3712-3720.2001     PMC  :   PMC95248    
Abstract >>
The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription Factors
76. Caspi  R, Pacek  M, Consiglieri  G, Helinski  DR, Toukdarian  A, Konieczny  I,     ( 2001 )

A broad host range replicon with different requirements for replication initiation in three bacterial species.

The EMBO journal 20 (12)
PMID : 11406602  :   DOI  :   10.1093/emboj/20.12.3262     PMC  :   PMC150194    
Abstract >>
Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.
KeywordMeSH Terms
DNA Replication
Escherichia coli Proteins
Replicon
77. Futamata  H, Harayama  S, Watanabe  K,     ( 2001 )

Diversity in kinetics of trichloroethylene-degrading activities exhibited by phenol-degrading bacteria.

Applied microbiology and biotechnology 55 (2)
PMID : 11330722  :  
Abstract >>
Whole-cell kinetics of phenol- and trichloroethylene (TCE)-degrading activities expressed by 13 phenol-degrading bacteria were analyzed. The Ks (apparent affinity constant in Haldane's equation) values for TCE were unexpectedly diverse, ranging from 11 microM to over 800 microM. The Vmax/Ks values for phenol were three orders of magnitude higher than the values for TCE in all bacteria analyzed, suggesting that these bacteria preferentially degrade phenol rather than TCE. A positive correlation between Ks for phenol and Ks for TCE was found, i.e., bacteria exhibiting high Ks values for phenol showed high Ks values for TCE, and vice versa. A comparison of the Ks values allowed grouping of these bacteria into three types, i.e., low-, moderate- and high-Ks types. Pseudo-first-order degradation-rate constants for TCE at 3.8 microM were found to be adequate to rapidly discriminate among the three types of bacteria. When bacteria were grown on phenol at the initial concentration of 2 mM, Comamonas testosteroni strain R5, a representative of low-Ks bacteria, completely degraded TCE at 3.8 microM, while strain P-8, a representative of high-Ks bacteria, did not. A mixed culture of these two bacteria poorly degraded TCE under the same conditions, where P-8 outgrew R5. These results suggest that low-Ks bacteria should be selectively grown for effective bioremediation of TCE-contaminated groundwater.
KeywordMeSH Terms
78. van Beilen  JB, Panke  S, Lucchini  S, Franchini  AG, Röthlisberger  M, Witholt  B,     ( 2001 )

Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

Microbiology (Reading, England) 147 (Pt 6)
PMID : 11390693  :   DOI  :   10.1099/00221287-147-6-1621    
Abstract >>
The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS:
KeywordMeSH Terms
DNA Transposable Elements
Multigene Family
79. Bertani  I, Kojic  M, Venturi  V,     ( 2001 )

Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358.

Microbiology (Reading, England) 147 (Pt 6)
PMID : 11390692  :   DOI  :   10.1099/00221287-147-6-1611    
Abstract >>
The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WCS358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the beta-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB. In this study the identification and characterization of the pobC-pobA locus of P. putida WCS358 is presented. The p-hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated POBC: Using the pobA promoter transcriptionally fused to a promoterless lacZ gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA. This PobC-PCA response was genetically demonstrated by making use of pobC::Tn5 and pcaH::Tn5 mutants of strain WCS358 constructed in this study. In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in the pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent pobA activation was still observed. Finally, the activation of pobA by PHB varied according to the concentration and it was observed that in the pcaR::Tn5 regulatory mutant of strain WCS358 the pobA promoter activity was reduced. PcaR is a regulator involved in the regulation of several loci of the beta-ketoadipate pathway, one of which is pcaK. It was postulated that the reduction of pobA activation in pcaR::Tn5 mutants was because there was no expression of the pcaK gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell.
KeywordMeSH Terms
Membrane Transport Proteins
Transcription Factors
80. Goda  M, Hashimoto  Y, Shimizu  S, Kobayashi  M,     ( 2001 )

Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage.

The Journal of biological chemistry 276 (26)
PMID : 11306561  :   DOI  :   10.1074/jbc.M007856200    
Abstract >>
Isonitrile containing an N triple bond C triple bond was degraded by microorganism sp. N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified as Pseudomonas putida. The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K(m) value for cyclohexyl isocyanide was 16.2 mm. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.
KeywordMeSH Terms
81. Duque  E, Segura  A, Mosqueda  G, Ramos  JL,     ( 2001 )

Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida.

Molecular microbiology 39 (4)
PMID : 11251828  :   DOI  :   10.1046/j.1365-2958.2001.02310.x    
Abstract >>
Pseudomonas putida DOT-T1E grows on a water-toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
82. Olivera  ER, Carnicero  D, García  B, Miñambres  B, Moreno  MA, Cañedo  L, Dirusso  CC, Naharro  G, Luengo  JM,     ( 2001 )

Two different pathways are involved in the beta-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications.

Molecular microbiology 39 (4)
PMID : 11251808  :   DOI  :   10.1046/j.1365-2958.2001.02296.x    
Abstract >>
In Pseudomonas putida U, the degradation of n-alkanoic and n-phenylalkanoic acids is carried out by two sets of beta-oxidation enzymes (betaI and betaII). Whereas the first one (called betaI) is constitutive and catalyses the degradation of n-alkanoic and n-phenylalkanoic acids very efficiently, the other one (betaII), which is only expressed when some of the genes encoding betaI enzymes are mutated, catabolizes n-phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the betaI genes handicaps the growth of P. putida U in media containing n-alkanoic or n-phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n-alkanoates as a result of the induction of betaII, but they were still unable to catabolize n-phenylalkanoates completely, as the betaI-FadBA enzymes are essential for the beta-oxidation of certain n-phenylalkanoyl-CoA derivatives when they reach a critical size. Owing to the existence of the betaII system, mutants lacking betaIfadB/A are able to synthesize new poly 3-OH-n-alkanoates (PHAs) and poly 3-OH-n-phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.
KeywordMeSH Terms
Escherichia coli Proteins
83. O'Leary  ND, O'Connor  KE, Duetz  W, Dobson  AD,     ( 2001 )

Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3.

Microbiology (Reading, England) 147 (Pt 4)
PMID : 11283293  :   DOI  :   10.1099/00221287-147-4-973    
Abstract >>
The styrene degradative pathway in Pseudmonas putida CA-3 has previously been shown to be divided into an upper pathway involving the conversion of styrene to phenylacetic acid and a lower pathway for the subsequent degradation of phenylacetic acid. It is reported here that expression of the regulatory genes styS and styR is essential for transcription of the upper pathway, but not for degradation of the lower pathway inducer, phenylacetic acid. The presence of phenylacetic acid in the growth medium completely repressed the upper pathway enzymes even in the presence of styrene, the upper pathway inducer. This repression is mediated at the transcription level by preventing expression of the styS and styR regulatory genes. Finally, an examination was made of the various stages of the diauxic growth curve obtained when P. putida CA-3 was grown on styrene together with an additional carbon source and it is reported that catabolite repression may involve a different mechanism to transcriptional repression by an additional carbon source.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
84. Manzanera  M, Aranda-Olmedo  I, Ramos  JL, Marqués  S,     ( 2001 )

Molecular characterization of Pseudomonas putida KT2440 rpoH gene regulation.

Microbiology (Reading, England) 147 (Pt 5)
PMID : 11320135  :   DOI  :   10.1099/00221287-147-5-1323    
Abstract >>
The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor sigma(32) was cloned and sequenced, and the translated gene product was predicted to be a protein of 32.5 kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned P. putida gene complemented the growth defect, at 37 and 42 degrees C, of an Escherichia coli rpoH mutant strain. Primer extension analysis showed that in P. putida the rpoH gene is expressed from three promoters in cells growing at 30 degrees C. Two of them, P1 and P3, share homology with the sigma(70)-dependent promoters, while the third one, P2, shows a typical sigma(24)-consensus sequence. The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5' region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20 bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P. putida growing at temperatures from 18 to 42 degrees C.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Sigma Factor
85. Bartels  F, Fernández  S, Holtel  A, Timmis  KN, de Lorenzo  V,     ( 2001 )

The essential HupB and HupN proteins of Pseudomonas putida provide redundant and nonspecific DNA-bending functions.

The Journal of biological chemistry 276 (20)
PMID : 11278879  :   DOI  :   10.1074/jbc.M011295200    
Abstract >>
A protein mixture containing two major components able to catalyze a beta-recombination reaction requiring nonspecific DNA bending was obtained by fractionation of a Pseudomonas putida extract. N-terminal sequence analysis and genomic data base searches identified the major component as an analogue of HupB of Pseudomonas aeruginosa and Escherichia coli, encoding one HU protein variant. The minor component of the fraction, termed HupN, was divergent enough from HupB to predict a separate DNA-bending competence. The determinants of the two proteins were cloned and hyperexpressed, and the gene products were purified. Their activities were examined in vitro in beta-recombination assays and in vivo by complementation of the Hbsu function of Bacillus subtilis. HupB and HupN were equally efficient in all tests, suggesting that they are independent and functionally redundant DNA bending proteins. This was reflected in the maintenance of in vivo activity of the final sigma54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in DeltahupB or DeltahupN strains. However, hupB/hupN double mutants were not viable. It is suggested that the requirement for protein-facilitated DNA bending is met in P. putida by two independent proteins that ensure an adequate supply of an essential cellular activity.
KeywordMeSH Terms
86. Lee  SW, Glickmann  E, Cooksey  DA,     ( 2001 )

Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator.

Applied and environmental microbiology 67 (4)
PMID : 11282588  :   DOI  :   10.1128/AEM.67.4.1437-1444.2001     PMC  :   PMC92752    
Abstract >>
Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharacterized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, zntR, regulates zntA in E. coli, but it is not contiguous with zntA in the E. coli genome as cadA and cadR were in P. putida. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike zntA, they did not confer significant levels of lead resistance. The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress cadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.
KeywordMeSH Terms
87. Lee  DS, Park  SY, Yamane  K, Obayashi  E, Hori  H, Shiro  Y,     ( 2001 )

Structural characterization of n-butyl-isocyanide complexes of cytochromes P450nor and P450cam.

Biochemistry 40 (9)
PMID : 11258878  :   DOI  :   10.1021/bi002225s    
Abstract >>
Alkyl-isocyanides are able to bind to both ferric and ferrous iron of the heme in cytochrome P450, and the resulting complexes exhibit characteristic optical absorption spectra. While the ferric complex gives a single Soret band at 430 nm, the ferrous complex shows double Soret bands at 430 and 450 nm. The ratio of intensities of the double Soret bands in the ferrous isocyanide complex of P450 varies, as a function of pH, ionic strength, and the origin of the enzyme. To understand the structural origin of these characteristic spectral features, we examined the crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochorme P450nor, since ferrous isocyanide complex of P450cam gives a single Soret band at 453 nm, while that of P450nor gives one at 427 nm. Corresponding to the optical spectra, we observed C-N stretching of a ferrous iron-bound isocyanide at 2145 and 2116 cm(-1) for P450nor and P450cam, respectively. The crystal structures of the ferric and ferrous n-butyl isocyanide complexes of P450cam and P450nor were determined. The coordination structure of the fifth Cys thiolate was indistinguishable for the two P450s, but the coordination geometry of the isocyanide was different for the case of P450cam [d(Fe-C) = 1.86 A, angleFe-C-N = 159 degrees ] versus P450nor [d(Fe-C) = 1.85 A, angleFe-C-N = 175 degrees ]. Another difference in the structures was the chemical environment of the heme pocket. In the case of P450cam, the iron-bound isocyanide is surrounded by some hydrophobic side chains, while, for P450nor, it is surrounded by polar groups including several water molecules. On the basis of these observations, we proposed that the steric factors and/or the polarity of the environment surrounding the iron-bound isocyanide significantly effect on the resonance structure of the heme(Fe)-isocyanide moiety and that differences in these two factors are responsible for the spectral characteristics for P450s.
KeywordMeSH Terms
88. Holtwick  R, von Wallbrunn  A, Keweloh  H, Meinhardt  F,     ( 2001 )

A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector.

Microbiology (Reading, England) 147 (Pt 2)
PMID : 11158350  :   DOI  :   10.1099/00221287-147-2-337    
Abstract >>
In Pseudomonas putida P8, three cryptic circular plasmids were detected, i.e. pPP8-1 (2.5 kbp), pPP8-2 (42 kbp) and pPP8-3 (approximately 100 kbp). Cloning and complete sequencing of pPP8-1 revealed a 2534 bp element harbouring four open reading frames (ORFs A, B, C and D). No function could be attributed to the latter three ORFs, whereas the predicted ORF A gene product is homologous to replication proteins known from small multicopy plasmids of Gram-positive bacteria and single-stranded (ss) phages, genetic elements replicating via a rolling circle (RC) mechanism involving characteristic ssDNA intermediates. Consistently, a double-strand origin of replication, highly conserved in rolling-circle-replicating (RCR) elements, was identified in pPP8-1, along with a putative single-strand origin. Beyond this, ss replication intermediates were confirmed by Southern analysis and mungbean-nuclease digestion. This being the first element of this type known in pseudomonads, a kanamycin-resistance gene was ligated into pPP8-1 and the resulting vector was successfully used for the transformation of both Escherichia coli and P. putida.
KeywordMeSH Terms
DNA Replication
Genetic Vectors
89. Chablain  PA, Zgoda  AL, Sarde  CO, Truffaut  N,     ( 2001 )

Genetic and molecular organization of the alkylbenzene catabolism operon in the psychrotrophic strain Pseudomonas putida 01G3.

Applied and environmental microbiology 67 (1)
PMID : 11133479  :   DOI  :   10.1128/AEM.67.1.453-458.2001     PMC  :   PMC92599    
Abstract >>
The 11-kb sequence encompassing the alkylbenzene upper pathway in Pseudomonas putida 01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized. Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in P. putida RE204. ebd genes appeared to be preferentially induced by ethylbenzene. Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities. Close to identified genes, remnants of IS5-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster.
KeywordMeSH Terms
90. Liebert  CA, Watson  AL, Summers  AO,     ( 2000 )

The quality of merC, a module of the mer mosaic.

Journal of molecular evolution 51 (6)
PMID : 11116334  :  
Abstract >>
We examined a region of high variability in the mosaic mercury resistance (mer) operon of natural bacterial isolates from the primate intestinal microbiota. The region between the merP and merA genes of nine mer loci was sequenced and either the merC, the merF, or no gene was present. Two novel merC genes were identified. Overall nucleotide diversity, pi (per 100 sites), of the merC gene was greater (49.63) than adjacent merP (35.82) and merA (32.58) genes. However, the consequences of this variability for the predicted structure of the MerC protein are limited and putative functional elements (metal-binding ligands and transmembrane domains) are strongly conserved. Comparison of codon usage of the merTP, merC, and merA genes suggests that several merC genes are not coeval with their flanking sequences. Although evidence of homologous recombination within the very variable merC genes is not apparent, the flanking regions have higher homologies than merC, and recombination appears to be driving their overall sequence identities higher. The synonymous codon usage bias (EN(C)) values suggest greater variability in expression of the merC gene than in flanking genes in six different bacterial hosts. We propose a model for the evolution of MerC as a host-dependent, adventitious module of the mer operon.
KeywordMeSH Terms
Bacterial Proteins
Cation Transport Proteins
91. Ramos  C, Molina  L, Mølbak  L, Ramos  JL, Molin  S,     ( 2000 )

A bioluminescent derivative of Pseudomonas putida KT2440 for deliberate release into the environment.

FEMS microbiology ecology 34 (2)
PMID : 11102686  :   DOI  :   10.1111/j.1574-6941.2000.tb00758.x    
Abstract >>
Recombinant derivatives of Pseudomonas putida strain KT2440 are of potential interest as microbial inoculants to be deliberately released for agricultural applications. To facilitate tracking of this strain and its derivatives after introduction into the environment, a mini-Tn5-'luxAB transposon was introduced into the chromosome of P. putida KT2440, yielding strain P. putida S1B1. Sequencing of the DNA region located upstream of the 'luxAB genes and similarity search with the P. putida KT2440 genome sequence, localized the transposon within a 3021-bp open reading frame (ORF), whose translated sequence showed significant similarity with the hypothetical YdiJ proteins from Escherichia coli and Haemophilus influenzae. A second ORF adjacent to and divergent from the ydiJ sequence was also found and showed significant homology with various LysR-type transcriptional activator proteins from several bacteria. Disruption of the ydiJ locus in P. putida S1B1 did not affect the survival of the strain in unvegetated or vegetated soils. Bioluminescent detection of P. putida S1B1 cells enriched in selective media directly from soil allowed detection of culturable cells in soil samples over a period of at least 8 months. The addition of the luxAB biomarker facilitates tracking in the root system of several plant species grown under sterile and non-sterile conditions. The correlation of the bioluminescent phenotype with the growth activity of P. putida S1B1 cells colonizing the root system of barley and corn plants was estimated by monitoring ribosomal contents using quantitative hybridization with fluorescence-labeled ribosomal RNA probes. A correlation between inoculum density, light output, and ribosomal contents was found for P. putida cells colonizing the root system of barley seedlings grown under sterile conditions. Although ribosomal contents, and therefore growth activity, of P. putida S1B1 cells extracted from the rhizosphere of corn plants grown in non-sterile soil were similar to those found in starved cells, the luminescent system permitted non-destructive in situ detection of the strain in the upper root system.
KeywordMeSH Terms
92. Hughes  MA, Williams  PA,     ( 2001 )

Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3.

Journal of bacteriology 183 (4)
PMID : 11157934  :   DOI  :   10.1128/JB.183.4.1225-1232.2001     PMC  :   PMC94995    
Abstract >>
Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with pnbB but appears to have no function in the pathway. The central pnbR gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P. putida strain TW3.
KeywordMeSH Terms
Genes, Bacterial
93. Wery  J, Hidayat  B, Kieboom  J, de Bont  JA,     ( 2001 )

An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress.

The Journal of biological chemistry 276 (8)
PMID : 11094055  :   DOI  :   10.1074/jbc.M007687200    
Abstract >>
The novel insertion sequence ISS12 plays a key role in the tolerance of Pseudomonas putida S12 to sudden toluene stress. Under normal culturing conditions the P. putida S12 genome contained seven copies of ISS12. However, a P. putida S12 population growing to high cell density after sudden addition of a separate phase of toluene carried eight copies. The survival frequency of cells in this variant P. putida S12 population was 1000 times higher than in "normal" P. putida S12 populations. Analysis of the nucleotide sequence flanking the extra ISS12 insertion revealed integration into the srpS gene. srpS forms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC. SrpABC makes a major contribution to solvent tolerance in P. putida S12 and is induced by toluene. The basal level of srp promoter activity in the P. putida S12 variant was seven times higher than in wild-type P. putida S12. Introduction of the intact srpRS gene cluster in the variant resulted in a dramatic decrease of survival frequency after a toluene shock. These findings strongly suggest that interruption of srpS by ISS12 up-regulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic solvents. We propose that P. putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe adverse conditions.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Variation
Mutagenesis, Insertional
94. Cowles  CE, Nichols  NN, Harwood  CS,     ( 2000 )

BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida.

Journal of bacteriology 182 (22)
PMID : 11053377  :   DOI  :   10.1128/jb.182.22.6339-6346.2000     PMC  :   PMC94779    
Abstract >>
Pseudomonas putida converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded meta pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of P. putida to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of pcaK, the gene encoding the 4-HBA permease. Here we identified a gene, benR, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. A benR mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to benR were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. The benABC genes likely encode benzoate dioxygenase, and benD likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase. benK and benF were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene, benE, is not known. benR activated expression of a benA-lacZ reporter fusion in response to benzoate. It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E. coli chromosome. Third, benR was required for benzoate-mediated repression of pcaK-lacZ fusion expression. The benA promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the meta cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.
KeywordMeSH Terms
Genes, Bacterial
Genes, Regulator
95. Kajander  T, Kahn  PC, Passila  SH, Cohen  DC, Lehtiö  L, Adolfsen  W, Warwicker  J, Schell  U, Goldman  A,     ( 2000 )

Buried charged surface in proteins.

Structure (London, England : 1993) 8 (11)
PMID : 11080642  :  
Abstract >>
The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.
KeywordMeSH Terms
Protein Conformation
Static Electricity
96. Hishiki  T, Shimada  H, Nagano  S, Egawa  T, Kanamori  Y, Makino  R, Park  SY, Adachi  S, Shiro  Y, Ishimura  Y,     ( 2000 )

X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center.

Journal of biochemistry 128 (6)
PMID : 11098139  :   DOI  :   10.1093/oxfordjournals.jbchem.a022848    
Abstract >>
The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.
KeywordMeSH Terms
97. Bond  CJ, Huang  J, Hajduk  R, Flick  KE, Heath  PJ, Stoddard  BL,     ( 2000 )

Cloning, sequence and crystallographic structure of recombinant iron superoxide dismutase from Pseudomonas ovalis.

Acta crystallographica. Section D, Biological crystallography 56 (Pt 11)
PMID : 11053832  :   DOI  :   10.1107/s0907444900009537    
Abstract >>
The gene encoding the iron-dependent superoxide dismutase from Pseudomonas ovalis was cloned from a genomic library and sequenced. The ORF differs from the previously published protein sequence, which was used for the original structure determination, at 16 positions. The differences include three additional inserted residues, one deleted residue and 12 point substitutions. The gene was subcloned and the recombinant protein overexpressed, purified and crystallized in a trigonal space group. The structure was determined by molecular replacement and was refined to 2.1 A resolution.
KeywordMeSH Terms
98. Perebituk  AN, Zehnder  AJ, Sentchilo  VS,     ( 2000 )

Molecular diversity of plasmids bearing genes that encode toluene and xylene metabolism in Pseudomonas strains isolated from different contaminated sites in Belarus.

Applied and environmental microbiology 66 (7)
PMID : 10877777  :   DOI  :   10.1128/aem.66.7.2842-2852.2000     PMC  :   PMC92082    
Abstract >>
Twenty different Pseudomonas strains utilizing m-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the meta cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with xyl gene probes of pWW0. The majority of the plasmids carried xyl-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024-5029, 1997), with two distinguishable meta pathway operons, one upper pathway operon, and three xylS-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one meta pathway operon, one upper pathway operon, and one copy each of xylS and xylR. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the xyl genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the xylTE and xylA genes of the pSVS plasmids confirmed the strong homologies to the xyl genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the xyl operons are discussed in light of the diversity analysis carried out on the pSVS plasmids.
KeywordMeSH Terms
Soil Microbiology
99. Lee  SW,     ( 2000 )

Genes expressed in Pseudomonas putida during colonization of a plant-pathogenic fungus.

Applied and environmental microbiology 66 (7)
PMID : 10877766  :   DOI  :   10.1128/aem.66.7.2764-2772.2000     PMC  :   PMC92071    
Abstract >>
In vivo expression technology (IVET) was employed to study colonization of Phytophthora parasitica by a biological control bacterium, Pseudomonas putida 06909, based on a new selection marker. The pyrB gene, which encodes aspartate transcarbamoylase, an enzyme used for pyrimidine biosynthesis, was cloned from P. putida 06909. A pyrB-disrupted mutant did not grow in pyrimidine-deficient media unless it was complemented with pyrBC' behind an active promoter. Thirty clones obtained from P. putida 06909 that were expressed on fungal hyphae but not on culture media were isolated by IVET based on the promoterless transcriptional fusion between pyrBC' and lacZ. Nineteen of these clones were induced during late-stage bacterial growth in vitro, while 11 of the clones were expressed only when they were inoculated onto fungal hyphae. Restriction analysis of these 11 clones revealed that there were five unique clones. Sequence analyses of three of the five unique clones showed that the 3' ends of the clones fused to pyrB were similar to genes encoding diacylglycerol kinase (DAGK), bacterial ABC transporters, and outer membrane porins. The sequences of the two other clones were not similar to the sequences of any of the genes in the database used. A LuxR family response regulator was found upstream of DAGK, and a LysR family response regulator was found upstream of the ABC transporter. The location of the inducible promoter of two clones suggested that DAGK and the ABC transporter are induced and may play a role in colonization of the fungus P. parasitica by P. putida 06909.
KeywordMeSH Terms
Gene Expression
100. Givskov  M, Sternberg  C, Rothballer  MH, Ammendola  A, Eberl  L,     ( 2000 )

Inactivation of gltB abolishes expression of the assimilatory nitrate reductase gene (nasB) in Pseudomonas putida KT2442.

Journal of bacteriology 182 (12)
PMID : 10852866  :   DOI  :   10.1128/jb.182.12.3368-3376.2000     PMC  :   PMC101894    
Abstract >>
By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase, luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasB suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of nasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of the gltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
101. Ha  NC, Kim  MS, Lee  W, Choi  KY, Oh  BH,     ( 2000 )

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes.

The Journal of biological chemistry 275 (52)
PMID : 11007792  :   DOI  :   10.1074/jbc.M007561200    
Abstract >>
Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.
KeywordMeSH Terms
102. Baetens  M, Park  CH, Pandza  S,     ( 2000 )

The G-protein FlhF has a role in polar flagellar placement and general stress response induction in Pseudomonas putida.

Molecular microbiology 36 (2)
PMID : 10792727  :   DOI  :   10.1046/j.1365-2958.2000.01859.x    
Abstract >>
The flhF gene of Pseudomonas putida, which encodes a GTP-binding protein, is part of the flagellar-motility-chemotaxis operon. Its disruption leads to a random flagellar arrangement in the mutant (MK107) and loss of directional motility in contrast to the wild type, which has polar flagella. The return of a normal flhF allele restores polar flagella and normal motility to MK107; its overexpression triples the flagellar number but does not restore directional motility. As FlhF is homologous to the receptor protein of the signal recognition particle (SRP) pathway of membrane protein translocation, this pathway may have a role in polar flagellar placement in P. putida. MK107 is also compromised in the development of the starvation-induced general stress resistance (SGSR) and effective synthesis of several starvation and exponential phase proteins. While somewhat increased protein secretion in MK107 may contribute to its SGSR impairment, the altered protein synthesis pattern also appears to have a role.
KeywordMeSH Terms
103. Kurusu  Y, Narita  T, Suzuki  M, Watanabe  T,     ( 2000 )

Genetic analysis of an incomplete mutS gene from Pseudomonas putida.

Journal of bacteriology 182 (18)
PMID : 10960118  :   DOI  :   10.1128/jb.182.18.5278-5279.2000     PMC  :   PMC94682    
Abstract >>
We genetically characterized the Pseudomonas putida mutS gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in the mutS mutants of Escherichia coli and Bacillus subtilis. A P. putida mutS mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.
KeywordMeSH Terms
Adenosine Triphosphatases
DNA-Binding Proteins
Escherichia coli Proteins
Mutation
104. Vermeij  P, Wietek  C, James  P, Kahnert  A,     ( 2000 )

The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313.

Journal of bacteriology 182 (10)
PMID : 10781557  :   DOI  :   10.1128/jb.182.10.2869-2878.2000     PMC  :   PMC101997    
Abstract >>
Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.
KeywordMeSH Terms
105. Motoshima  H, Inagaki  K, Kumasaka  T, Furuichi  M, Inoue  H, Tamura  T, Esaki  N, Soda  K, Tanaka  N, Yamamoto  M, Tanaka  H,     ( 2000 )

Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida.

Journal of biochemistry 128 (3)
PMID : 10965031  :   DOI  :   10.1093/oxfordjournals.jbchem.a022760    
Abstract >>
L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.
KeywordMeSH Terms
106. Gorlenko  ZhM, Bass  IA, Mindlin  SZ, Kholodiĭ  GIa,     ( 2000 )

[Molecular genetic analysis of the Tn5041 transposition system].

Genetika 36 (4)
PMID : 10822806  :  
Abstract >>
A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).
KeywordMeSH Terms
DNA Transposable Elements
107. Keyhan  M, Wielinga  B, Park  CH,     ( 2000 )

Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase.

Applied and environmental microbiology 66 (5)
PMID : 10788340  :   DOI  :   10.1128/aem.66.5.1788-1795.2000     PMC  :   PMC101413    
Abstract >>
Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.
KeywordMeSH Terms
108. Salido  A, Espinosa-Urgel  M,     ( 2000 )

Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds.

Journal of bacteriology 182 (9)
PMID : 10762233  :   DOI  :   10.1128/jb.182.9.2363-2369.2000     PMC  :   PMC111295    
Abstract >>
Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.
KeywordMeSH Terms
109. Truu  J, Heinaru  E,     ( 2000 )

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds.

FEMS microbiology ecology 31 (3)
PMID : 10719200  :   DOI  :   10.1111/j.1574-6941.2000.tb00684.x    
Abstract >>
A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.
KeywordMeSH Terms
110. Goh  CB, Fong  KP,     ( 2000 )

The genes for benzene catabolism in Pseudomonas putida ML2 are flanked by two copies of the insertion element IS1489, forming a class-I-type catabolic transposon, Tn5542.

Plasmid 43 (2)
PMID : 10686128  :   DOI  :   10.1006/plas.1999.1442    
Abstract >>
Two directly repeated sequences of the IS elements IS1489v1 and IS1489v2 flank the benzene dioxygenase (bedC1C2BA) and the cis-benzene dihydrodiol dehydrogenase (bedD) genes on the catabolic plasmid pHMT112 in Pseudomonas putida ML2, forming a Class-I-type composite transposon, Tn5542. Both IS1489v1 and IS1489v2 contain an identical 1371-bp open reading frame, tnpA, that is preceded by a possible ribosome binding site. The tnpA gene of IS1489v1 is bound by a pair of 40-bp imperfect inverted repeats while that of IS1489v2 is flanked only by the left inverted repeat. The tnpA gene codes for a putative 53-kDa polypeptide of 456 amino acids bearing similarity to transposases encoded on IS elements of P. alcaligenes, P. aeruginosa, P. stutzeri, and Serratia marcescens. The basic nature of the putative TnpA protein with a deduced pI of 8.93 is typical of IS-encoded transposases. Similar to other IS elements, an outward facing promoter was detected at the right end of IS1489v1. Experiments involving the suicide vector, pKNG101, failed to show transposition of Tn5542.
KeywordMeSH Terms
Multigene Family
111. Parales  RE, Parales  JV, Choudhury  D, Kauppi  B, Karlsson  A, Carredano  E,     ( 2000 )

Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding.

Journal of molecular biology 296 (2)
PMID : 10669618  :   DOI  :   10.1006/jmbi.1999.3462    
Abstract >>
The three-dimensional structure of the aromatic hydroxylating enzyme naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was recently determined. The refinement of the structure together with cyclic averaging showed that in the active site of the enzyme there is electron density for a flat aromatic compound. This compound appears to be an indole adduct, which in Escherichia coli is derived from tryptophan present in the rich culture medium. An indole-dioxygen adduct has been built which fits the electron density convincingly. Support for this interpretation was obtained from crystals of the enzyme purified from cells grown in the absence of tryptophan which had an empty substrate pocket. These types of crystals were soaked in indole solutions and the position of indole in this complex was similar to the corresponding part in the modelled indole-oxygen adduct. This suggests that a peroxide bound to iron end-on attacks the substrate and forms this intermediate. The substrate position has implications for the substrate specificity of the enzyme. Docking studies with indole, naphthalene and biphenyl inside the substrate pocket of NDO suggest the presence of subpockets where the one close to the active site iron is reserved for the binding of the aromatic ring which is hydroxylated upon catalysis. The plausible location for the binding of dioxygen is between this pocket and the catalytic iron. This is in accordance with the enantiospecificity of the products.
KeywordMeSH Terms
112. Degrassi  G, Kojic  M,     ( 1999 )

Cloning and characterisation of the rpoS gene from plant growth-promoting Pseudomonas putida WCS358: RpoS is not involved in siderophore and homoserine lactone production.

Biochimica et biophysica acta 1489 (2��3��)
PMID : 10673044  :   DOI  :   10.1016/s0167-4781(99)00210-9    
Abstract >>
The rpoS gene which encodes a stationary phase sigma factor has been identified and characterised from the rhizosphere-colonising plant growth-promoting Pseudomonas putida strain WCS358. The predicted protein sequence has extensive homologies with the RpoS proteins form other bacteria, in particular with the RpoS sigma factors of the fluorescent pseudomonads. A genomic transposon insertion in the rpoS gene was constructed, these mutants were analysed for their ability to produce siderophore (iron-transport agent) and the autoinducer quorum-sensing molecules called homoserine lactones (AHL). It was determined that RpoS was not involved in the regulation of siderophore and AHL production, synthesis of these molecules is important for gene expression at stationary phase. P. putida WCS358 produces at least three different AHL molecules.
KeywordMeSH Terms
113. Nichols  NN,     ( 2000 )

An aerotaxis transducer gene from Pseudomonas putida.

FEMS microbiology letters 182 (1)
PMID : 10612751  :   DOI  :   10.1111/j.1574-6968.2000.tb08893.x    
Abstract >>
An aerotaxis gene, aer, was cloned from Pseudomonas putida PRS2000. A P. putida aer mutant displayed an altered aerotactic response in a capillary assay. Wild-type P. putida clustered at the air/liquid interface. In contrast, the aer mutant did not cluster at the interface, but instead formed a diffuse band at a distance from the meniscus. Wild-type aer, provided in trans, complemented the aer mutant to an aerotactic response that was stronger than wild-type. The P. putida Aer sequence is similar over its entire length to the aerotaxis (energy taxis) signal transducer protein, Aer, of Escherichia coli. The amino-terminus is similar to redox-sensing regulatory proteins, and the carboxy-terminus contains the highly conserved domain present in chemotactic transducers.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
114. Park  HS,     ( 2000 )

Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12.

Journal of bacteriology 182 (3)
PMID : 10633088  :   DOI  :   10.1128/jb.182.3.573-580.2000     PMC  :   PMC94317    
Abstract >>
Pseudomonas putida HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on two plasmids, pNB1 and pNB2. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1 kb). Meanwhile, the nbzB gene encoding hydroxylaminobenzene mutase, a second-step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8 kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in Escherichia coli strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes nbzA and nbzB are located on the 1.1-kb SmaI-SnaBI fragment of pNB1 and the 1.0-kb SspI-SphI fragment of pNB2, respectively, and their expressions were not tightly regulated. On the other hand, the genes nbzC, nbzD, and nbzE, involved in the ring cleavage pathway of 2-aminophenol, are localized on the 6.6-kb SnaBI-SmaI fragment of pNB1 and clustered in the order nbzC-nbzD-nbzE as an operon. The nbzCDE genes, which are transcribed in the opposite direction of the nbzA gene, are coordinately regulated by both nitrobenzene and a positive transcriptional regulator that seems to be encoded on pNB2.
KeywordMeSH Terms
115. Molina  L, Vílchez  S,     ( 2000 )

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates.

Journal of bacteriology 182 (1)
PMID : 10613867  :   DOI  :   10.1128/jb.182.1.91-99.2000     PMC  :   PMC94244    
Abstract >>
Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.
KeywordMeSH Terms
Amino Acid Transport Systems, Neutral
116. Lee  K, Resnick  SM, Parales  RE,     ( 2000 )

Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme.

Journal of bacteriology 182 (6)
PMID : 10692370  :   DOI  :   10.1128/jb.182.6.1641-1649.2000     PMC  :   PMC94462    
Abstract >>
The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.
KeywordMeSH Terms
Amino Acid Substitution
Mutagenesis, Site-Directed
117. Mosqueda  G,     ( 2000 )

A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism.

Journal of bacteriology 182 (4)
PMID : 10648517  :   DOI  :   10.1128/jb.182.4.937-943.2000     PMC  :   PMC94367    
Abstract >>
Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the tod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD and ttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single ttgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and ttgD mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.
KeywordMeSH Terms
Operon
118. Ha  NC, Kim  SW, Kim  DH, Choi  G,     ( 2000 )

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

Biochemistry 39 (5)
PMID : 10653633  :   DOI  :   10.1021/bi991579k    
Abstract >>
Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.
KeywordMeSH Terms
119. Chu  K, Stock  AM, Maves  SA, Benson  DE, Berendzen  J, Schlichting  I,     ( 2000 )

The catalytic pathway of cytochrome p450cam at atomic resolution.

Science (New York, N.Y.) 287 (5458)
PMID : 10698731  :   DOI  :   10.1126/science.287.5458.1615    
Abstract >>
Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.
KeywordMeSH Terms
120. Chen  ZW, Shamala  N, Mathews  FS, Cunane  LM,     ( 2000 )

Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism.

Journal of molecular biology 295 (2)
PMID : 10623531  :   DOI  :   10.1006/jmbi.1999.3290    
Abstract >>
The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enzyme first catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilizing one atom of oxygen derived from water, and yielding one molecule of reduced FAD. The reducing electron equivalents are then passed one at a time from the flavin cofactor to the heme cofactor by intramolecular electron transfer, and subsequently to cytochrome oxidase within the periplasmic membrane via one or more soluble electron carrier proteins. The product, p-hydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzaldehyde. The fully refined X-ray crystal structure of PCMH in the native state has been obtained at 2. 5 A resolution on the basis of the gene sequence. The structure of the enzyme-substrate complex has also been refined, at 2.75 A resolution, and reveals significant conformational changes in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route for substrate access to the site has been identified and is shown to be governed by a swinging-gate mechanism. Two possible proton transfer pathways, that may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydrogen bonding interactions between the flavoprotein and cytochrome subunits that stabilize the intramolecular complex and may contribute to the electron transfer process have been identified.
KeywordMeSH Terms
121. Miller  CD, Kim  YC,     ( 1999 )

Transcriptional regulation by iron of genes encoding iron- and manganese-superoxide dismutases from Pseudomonas putida.

Gene 239 (1)
PMID : 10571042  :   DOI  :   10.1016/s0378-1119(99)00369-8    
Abstract >>
Genes from Pseudomonas putida (Pp), sodA, encoding manganese-superoxide dismutase (MnSOD) and, sodB, iron-superoxide dismutase (FeSOD) were cloned by hybridization with digoxigenin (dig)-labeled PCR products generated from Pp genomic DNA. The sodB gene had a 594 bp open reading frame (ORF), corresponding to 198 amino acids (aa), and a transcript of 880 bases. The sodA gene contained a 609 bp ORF encoding 203 aa and was transcribed as part of a polycistronic operon, consisting of orfY-fumC-orfX-sodA. Pp sodA or sodB genes both restored aerobic growth, growth on paraquat, and growth on minimal medium to an Escherichia coli (Ec) mutant deficient in SOD activity. Paraquat treatment did not enhance mRNA transcription of the sod genes or increase SOD activity in Pp. The Pp sodB gene was highly expressed throughout logarithmic-(log) growth phase and stationary-phase cells grown in medium supplemented with FeCl3, but was down-regulated in iron-deficient conditions, such as in stationary-phase or generated by 2,2'-dipyridyl (DP) treatment. This is the first evidence that iron regulates expression of the sodB gene at the transcriptional level. In contrast, iron-deficient conditions, or addition of MnCl2 to the growth medium, induced transcripts (2.4 kb and 1.2 kb) from the sodA operon. Our results reveal an intricate role of iron in the transcriptional regulation of both Pp sodA and sodB genes.
KeywordMeSH Terms
122. Crane  BR, Wilker  JJ, Dmochowski  IJ,     ( 1999 )

Optical detection of cytochrome P450 by sensitizer-linked substrates.

Proceedings of the National Academy of Sciences of the United States of America 96 (23)
PMID : 10557259  :   DOI  :   10.1073/pnas.96.23.12987     PMC  :   PMC23886    
Abstract >>
The ability to detect, characterize, and manipulate specific biomolecules in complex media is critical for understanding metabolic processes. Particularly important targets are oxygenases (cytochromes P450) involved in drug metabolism and many disease states, including liver and kidney dysfunction, neurological disorders, and cancer. We have found that Ru photosensitizers linked to P450 substrates specifically recognize submicromolar cytochrome P450(cam) in the presence of other heme proteins. In the P450:Ru-substrate conjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence decay. The crystal structure of a P450(cam):Ru-adamantyl complex reveals access to the active center via a channel whose depth (Ru-Fe distance is 21 A) is virtually the same as that extracted from an analysis of the energy-transfer kinetics. Suitably constructed libraries of sensitizer-linked substrates could be employed to probe the steric and electronic properties of buried active sites.
KeywordMeSH Terms
123. Kim  J, Fuller  JH, Cronin  CN,     ( 1999 )

Organization and sequences of p-hydroxybenzaldehyde dehydrogenase and other plasmid-encoded genes for early enzymes of the p-cresol degradative pathway in Pseudomonas putida NCIMB 9866 and 9869.

DNA sequence : the journal of DNA sequencing and mapping 10 (1)
PMID : 10565539  :  
Abstract >>
The gene (designated pchA) encoding the aldehyde dehydrogenase that is required to metabolise the p-hydroxybenzaldehyde produced by the degradation of p-cresol in Pseudomonas putida NCIMB 9866 and 9869 has been identified on plasmids pRA4000 and pRA500, respectively. The gene lies immediately upstream of the pchC and pchF genes encoding the subunits of p-cresol methylhydroxylase (PCMH), the preceeding enzyme in the p-cresol degradative pathway. In pRA500 the latter genes are followed by the genes encoding the alpha (pcaG) and beta (pcaH) subunits of protocatechuate-3,4-dioxygenase, whereas in pRA4000 the genes encoding PCMH are followed by an open reading frame encoding a protein that is similar to the maturase-related protein of P. alcaligenes. A gene, designated pchX, that encodes a protein of unknown function was identified between the pchC and pchF genes in both plasmids.
KeywordMeSH Terms
Intramolecular Transferases
124. Fox  BG, Blehert  DS,     ( 1999 )

Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases.

Journal of bacteriology 181 (20)
PMID : 10515912  :   PMC  :   PMC103757    
Abstract >>
The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5alpha. The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems.
KeywordMeSH Terms
Bacterial Proteins
125. Künne  S, Fischer  F,     ( 1999 )

Bacterial 2,4-dioxygenases: new members of the alpha/beta hydrolase-fold superfamily of enzymes functionally related to serine hydrolases.

Journal of bacteriology 181 (18)
PMID : 10482514  :   PMC  :   PMC94093    
Abstract >>
1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from Pseudomonas putida 33/1 and 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from Arthrobacter ilicis R?61a catalyze an N-heterocyclic-ring cleavage reaction, generating N-formylanthranilate and N-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the alpha/beta hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 strongly suggested that Qdo and Hod are structurally related to the alpha/beta hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the alpha/beta hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed.
KeywordMeSH Terms
Dioxygenases
126. Matsushita  K, Mathews  FS, Baruch  P, Chen  Z,     ( 1999 )

Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (ADHs): a soluble monomeric ADH from Pseudomonas putida HK5 (ADH-IIB) and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS).

Acta crystallographica. Section D, Biological crystallography 55 (Pt 11)
PMID : 10531500  :   DOI  :   10.1107/s0907444999010744    
Abstract >>
Crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (ADH-IIB) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (ADH-GS) have been obtained. The ADH-IIB crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of PEG 8000, sodium citrate, HEPES buffer and 2-propanol. X-ray data were collected at 110 K to 1. 9 A resolution (R(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from Methylophilus methylotrophus W3A1) was obtained by molecular replacement. Preliminary refinement of this model (10.0-3.0 A resolution, R = 0.37, R(free) = 0.40) led to tentative identification of the two highest peaks in a native anomalous difference Fourier map as the Fe atom of the heme and a calcium ion interacting with the PQQ prosthetic group. The ADH-GS crystals are tetragonal, displaying six similar lattices, both primitive and centered, and were grown by the sitting-drop method after replacement of Triton X-100 by dodecylmaltoside or octaethylene glycol monododecyl ether in the presence of ammonium sulfate and sodium acetate buffer, with and without PEG 3500 and calcium ion. The best diffraction is obtained at 110 K where the resolution extends to about 4 A in the a and b directions and about 3 A in the c direction.
KeywordMeSH Terms
127. Sagara  Y, Aramaki  H,     ( 1999 )

Cloning and sequencing of rpoH and identification of ftsE-ftsX in Pseudomonas putida PpG1.

DNA research : an international journal for rapid publication of reports on genes and genomes 6 (4)
PMID : 10492171  :   DOI  :   10.1093/dnares/6.4.241    
Abstract >>
The rpoH gene encoding the heat-shock sigma factor of Pseudomonas putida was cloned by using its ability to complement the temperature-sensitive growth of the Escherichia coli rpoH mutant. The cloned DNA contained an open reading frame for a 284 amino acid sequence exhibiting high homology to the sigmaH proteins of P. aeruginosa and E. coli. Moreover, homologs to the cell division genes ftsX and ftsE were found immediately upstream of the rpoH gene.
KeywordMeSH Terms
ATP-Binding Cassette Transporters
Escherichia coli Proteins
128. Junker  F,     ( 1999 )

Involvement of the cis/trans isomerase Cti in solvent resistance of Pseudomonas putida DOT-T1E.

Journal of bacteriology 181 (18)
PMID : 10482510  :   PMC  :   PMC94089    
Abstract >>
Pseudomonas putida DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the cti gene of this strain, which encodes the cis/trans isomerase, termed Cti, that catalyzes the cis-trans isomerization of esterified fatty acids in phospholipids, mainly cis-oleic acid (C(16:1,9)) and cis-vaccenic acid (C(18:1,11)), in response to solvents. To determine the importance of this cis/trans isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5 degrees C, the Cti-null mutant strain grew significantly slower at temperatures above 37 degrees C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the cti gene is present in all the tested P. putida strains, including non-solvent-resistant ones, and in some other Pseudomonas species.
KeywordMeSH Terms
Bacterial Proteins
129. Jang  DS, Choi  G, Joo  S, Nam  GH, Kim  DH,     ( 1999 )

Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B.

Biochemistry 38 (42)
PMID : 10529226  :   DOI  :   10.1021/bi991040m    
Abstract >>
The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B have been characterized in their roles in steroid binding and catalysis. Kinetic and equilibrium binding analyses were carried out for the mutant enzymes with the substitutions Phe-54 --> Ala or Leu, Phe-82 --> Ala or Leu, and Trp-116 --> Ala, Phe, or Tyr. The removal of their bulky, aromatic side chains at any of these three positions results in reduced k(cat), particularly when Phe-82 or Trp-116 is replaced by Ala. The results are consistent with the binding interactions of the aromatic residues with the bound steroid contributing to catalysis. All the mutations except the F82A mutation increase K(m); the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility that the phenyl ring at position 82 might be unfavorable for substrate binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a phenyl ring at position 82, and a nonpolar aromatic or small side chain at position 116 might be favorable for binding the reaction intermediate. In contrast to the increased K(D) for equilenin, the enzymes with any substitutions at positions 54 and 116 display a decreased K(D) for 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116 might be unfavorable for product binding. The crystal structure of F82A determined to 2.1-A resolution reveals that Phe-82 is important for maintaining the active site geometry. Taken together, our results demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to the stabilization of steroid species including substrate, intermediate, and product.
KeywordMeSH Terms
130. Fernández-Valverde  M, Cañedo  LM, Prieto  MA, Miñambres  B, Olivera  ER, García  B,     ( 1999 )

Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.

The Journal of biological chemistry 274 (41)
PMID : 10506180  :   DOI  :   10.1074/jbc.274.41.29228    
Abstract >>
Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed.
KeywordMeSH Terms
131. Horikoshi  K, Takami  H, Hirayama  H, Kobata  K, Usami  R,     ( 1999 )

Outer membrane changes in a toluene-sensitive mutant of toluene-tolerant Pseudomonas putida IH-2000.

Journal of bacteriology 181 (15)
PMID : 10419944  :   PMC  :   PMC103577    
Abstract >>
We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerant Pseudomonas putida IH-2000 by transposon mutagenesis with Tn5. The gene disrupted by insertion of Tn5 was identified as cyoC, which is one of the subunits of cytochrome o. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of cyoC caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.
KeywordMeSH Terms
Cytochrome b Group
Escherichia coli Proteins
132. Harwood  CS,     ( 1999 )

Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Journal of bacteriology 181 (16)
PMID : 10438780  :   PMC  :   PMC93997    
Abstract >>
Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) by Pseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H(+) symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.
KeywordMeSH Terms
Membrane Transport Proteins
133. Hol  WG, Seger  K, Turley  S, Sokatch  JR,     ( 1999 )

Crystal structure of 2-oxoisovalerate and dehydrogenase and the architecture of 2-oxo acid dehydrogenase multienzyme complexes.

Nature structural biology 6 (8)
PMID : 10426958  :   DOI  :   10.1038/11563    
Abstract >>
The family of giant multienzyme complexes metabolizing pyruvate, 2-oxoglutarate, branched-chain 2-oxo acids or acetoin contains several of the largest and most sophisticated protein assemblies known, with molecular masses between 4 and 10 million Da. The principal enzyme components, E1, E2 and E3, are present in numerous copies and utilize multiple cofactors to catalyze a directed sequence of reactions via substrate channeling. The crystal structure of a heterotetrameric (alpha2beta2) E1, 2-oxoisovalerate dehydrogenase from Pseudomonas putida, reveals a tightly packed arrangement of the four subunits with the beta2-dimer held between the jaws of a 'vise' formed by the alpha2-dimer. A long hydrophobic channel, suitable to accommodate the E2 lipoyl-lysine arm, leads to the active site, which contains the cofactor thiamin diphosphate (ThDP) and an inhibitor-derived covalent modification of a histidine side chain. The E1 structure, together with previous structural information on E2 and E3, completes the picture of the shared architectural features of these enormous macromolecular assemblies.
KeywordMeSH Terms
134. Keweloh  H, Meinhardt  F,     ( 1999 )

cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type.

Applied and environmental microbiology 65 (6)
PMID : 10347055  :   PMC  :   PMC91390    
Abstract >>
From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.
KeywordMeSH Terms
135. Vedadi  M, Powlowski  J, Sylvestre  M,     ( 1999 )

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1, 2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates.

Biochemical and biophysical research communications 260 (1)
PMID : 10381363  :   DOI  :   10.1006/bbrc.1999.0706    
Abstract >>
Pseudomonas putida strain G7 cis-1,2-dihydro-1, 2-dihydroxynaphthalene dehydrogenase (NahB) and Comamonas testosteroni strain B-356 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) were found to be catalytically active towards cis-2,3-dihydro-2,3-dihydroxybiphenyl (specificity factors of 501 and 5850 s-1 mM-1 respectively), cis-1,2-dihydro-1, 2-dihydroxynaphthalene (specificity factors of 204 and 193 s-1 mM-1 respectively) and 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl (specificity factors of 1.6 and 4.9 s-1 mM-1 respectively). A key finding in this work is the capacity of strain B-356 BphB as well as Burkholderia cepacia strain LB400 BphB to catalyze dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl which is the metabolite resulting from the catalytic meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl by LB400 biphenyl dioxygenase.
KeywordMeSH Terms
Oxidoreductases Acting on CH-CH Group Donors
136. Hai  W, Keijers  V, de Mot  R, Willems  A, Schoofs  G,     ( 1999 )

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri.

Systematic and applied microbiology 22 (2)
PMID : 10390872  :   DOI  :   10.1016/S0723-2020(99)80068-X    
Abstract >>
The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.
KeywordMeSH Terms
137. Ishida  T, Horiike  K, Nozaki  M, Kita  S, Fujisawa  I, Inaka  K,     ( 1999 )

An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Ppseudomonas putida mt-2.

Structure (London, England : 1993) 7 (1)
PMID : 10368270  :  
Abstract >>
Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.
KeywordMeSH Terms
Dioxygenases
138. Kurusu  Y, Nishihara  H, Takamura  Y,     ( 1999 )

Cloning and characterization of mdc genes encoding malonate decarboxylase from Pseudomonas putida.

FEMS microbiology letters 174 (2)
PMID : 10339824  :   DOI  :   10.1111/j.1574-6968.1999.tb13584.x    
Abstract >>
The DNA fragment encoding malonate decarboxylase, involved in malonate assimilation, was cloned from Pseudomonas putida. The 11-kb DNA fragment contained nine open reading frames, which were designated mdcABCDEGHLM in the given order. N-terminal protein sequencing established that the mdcA, mdcC, mdcD, mdcE and mdcH genes encoded subunits alpha, delta, beta, gamma and epsilon of the malonate decarboxylase, respectively. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmid harboring the entire gene cluster or the mdc genes lacking the mdcL and mdcM genes. The mdcL and mdcM genes encode membrane proteins and disruption of the genes of P. putida by the insertion of a kanamycin resistance cassette reduced the malonate uptake activity of the organism. Thus, we conclude that MdcLM is a malonate transporter.
KeywordMeSH Terms
139. Kertesz  MA, Wietek  C, Kahnert  A, Wüest  T,     ( 1999 )

Genetic organization of sulphur-controlled aryl desulphonation in Pseudomonas putida S-313.

Molecular microbiology 32 (5)
PMID : 10361295  :   DOI  :   10.1046/j.1365-2958.1999.01398.x    
Abstract >>
Pseudomonas putida S-313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR-type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate-specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
140. Wubbolts  MG, Witholt  B, Meyer  A, Huber  CM,     ( 1999 )

An alkane-responsive expression system for the production of fine chemicals

Applied and environmental microbiology 65 (6)
PMID : 10347009  :   PMC  :   PMC91344    
Abstract >>
Membrane-located monooxygenase systems, such as the Pseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of alkS, whose activated gene product positively regulates the transcription of the structural genes alkBFGHJKL, on a 3.7-kb SalI-HpaI OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.
KeywordMeSH Terms
141. Ramos-González  MI,     ( 1999 )

Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization.

Gene 232 (1)
PMID : 10333523  :   DOI  :   10.1016/s0378-1119(99)00113-4    
Abstract >>
Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow with toluene as the sole C-source. Tn5 mutagenesis was carried out and a mutant unable to use toluene as the sole C-source was isolated. DNA was sequenced upstream and downstream of the site where the Tn5 was inserted. Analysis of the DNA revealed 13 open reading frames (ORFs) homologous to the tod genes for the toluene dioxygenase pathway of P. putida F1, which are organized in two operons: todXFC1C2BADEGIH and todST. The Tn5 was inserted at the todH gene. The role of the todXFC1C2BADEGIH operon in toluene metabolism was further confirmed in a todC1 mutant (generated by insertional inactivation), which was unable to use toluene as the sole C-source. Primer extension analysis identified a single promoter upstream from the todX gene. The -10 and -35 regions of this promoter showed no significant homology to known promoters. Expression from the todX promoter occurred in response to toluene, ethylbenzene, styrene, xylenes and other aromatic hydrocarbons. Expression from the todS gene was constitutive. Sensitivity to toluene of the todH and todC1 mutants was similar to that of the wild-type strain. This suggests that toluene metabolism is not involved in toluene tolerance.
KeywordMeSH Terms
142. Hauer  B, Lingens  F, Betz  A, Facey  S,     ( 1999 )

Cloning, sequence analysis, and expression of the Pseudomonas putida 33/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase gene, encoding a carbon monoxide forming dioxygenase.

Biochimica et biophysica acta 1431 (2)
PMID : 10350631  :   DOI  :   10.1016/s0167-4838(99)00083-7    
Abstract >>
1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from the 1H-4-oxoquinoline utilizing Pseudomonas putida strain 33/1, which catalyzes the cleavage of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilate, is devoid of any transition metal ion or other cofactor and thus represents a novel type of ring-cleavage dioxygenase. Gene qdo was cloned and sequenced. Its overexpression in Escherichia coli yielded recombinant His-tagged Qdo which was catalytically active. Qdo exhibited 36% and 16% amino acid identity to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) and atropinesterase (a serine hydrolase), respectively. Qdo as well as Hod possesses a SXSHG motif, resembling the motif GXSXG of the serine hydrolases which comprises the active-site nucleophile (X=arbitrary residue).
KeywordMeSH Terms
Dioxygenases
143. Harwood  CS,     ( 1999 )

NahY, a catabolic plasmid-encoded receptor required for chemotaxis of Pseudomonas putida to the aromatic hydrocarbon naphthalene.

Journal of bacteriology 181 (10)
PMID : 10322041  :   PMC  :   PMC93795    
Abstract >>
Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.
KeywordMeSH Terms
Chemotaxis
Naphthalenes
144. Pochapsky  TC, Heymont  J, Lyons  TA,     ( 1999 )

A refined model for the solution structure of oxidized putidaredoxin.

Biochemistry 38 (15)
PMID : 10200155  :   DOI  :   10.1021/bi983030b    
Abstract >>
A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2 ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxo solution structure. First, essentially complete assignments for diamagnetic 15N and 13C resonances of Pdxo have been made using multidimensional NMR methods, and 15N- and 13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional 13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated 15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (M?ller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 A for backbone heavy atoms and 0.83 A for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 A for backbone atoms and 0.71 A for all heavy atoms.
KeywordMeSH Terms
Models, Molecular
145. Rétey  J, Schulz  GE,     ( 1999 )

Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile.

Biochemistry 38 (17)
PMID : 10220322  :   DOI  :   10.1021/bi982929q    
Abstract >>
Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the alpha-amino group of histidine and is closely related to the important plant enzyme phenylalanine ammonia-lyase. The crystal structure of histidase from Pseudomonas putida was determined at 2.1 A resolution revealing a homotetramer with D2 symmetry, the molecular center of which is formed by 20 nearly parallel alpha-helices. The chain fold, but not the sequence, resembles those of fumarase C and related proteins. The structure shows that the reactive electrophile is a 4-methylidene-imidazole-5-one, which is formed autocatalytically by cyclization and dehydration of residues 142-144 with the sequence Ala-Ser-Gly. With respect to the first dehydration step, this modification resembles the chromophore of the green fluorescent protein. The active center is clearly established by the modification and by mutations. The observed geometry allowed us to model the bound substrate at a high confidence level. A reaction mechanism is proposed.
KeywordMeSH Terms
146. Hong  MC, Chang  MC, Wu  ML,     ( 1998 )

Expression and export of Pseudomonas putida NTU-8 creatinase by Escherichia coli using the chitinase signal sequence of Aeromonas hydrophila.

Biochemical genetics 36 (11��12��)
PMID : 10230521  :  
Abstract >>
The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.
KeywordMeSH Terms
147. Adachi  O, Yamashita  T, Aoki  N, Toyama  H,     ( 1999 )

Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5.

Biochemistry 38 (19)
PMID : 10320337  :   DOI  :   10.1021/bi990121f    
Abstract >>
A blue copper protein was purified together with a type II quinohemoprotein alcohol dehydrogenase (ADH IIB) from the soluble fraction of Pseudomonas putida HK5 grown on n-butanol. The purified blue copper protein was shown to be azurin, on the basis of several properties such as its absorption maximum (623 nm), its low molecular mass (17 500 Da), its acidic nature (pI of 4.1), its relatively high redox potential (306 mV), the presence of an intramolecular disulfide bond, and N-terminal amino acid sequence homology with respect to azurins from other sources, especially from P. putida NCIB 9869 and Pseudomonas fluorescens. Direct electron transfer from ADH IIB to azurin was shown to occur at a rate of 48-70 s-1. The apparent Km value of ADH IIB for azurin, determined by steady-state kinetics, was decreased several-fold by increasing the ionic strength. Furthermore, the extent of fluorescence quenching of ADH IIB due to the interaction with azurin was increased by increasing the ionic strength, but the binding constant for binding between ADH IIB and azurin was unchanged. The redox potential of azurin was increased 12 mV by incubation with ADH but not vice versa. Furthermore, the redox potential gap between ADH and azurin was increased from 102 to 126 mV by increasing the ionic strength. It is conceivable that a hydrophobic interaction is involved in the electron transfer between both proteins, and it is also suggested that the electron transfer may occur by a freely reversible on and off binding process but may not be related to the global binding process of both proteins. Thus, the results presented here strongly suggest that azurin works as an electron-transfer mediator in a PQQ-dependent alcohol oxidase respiratory chain in P. putida HK5.
KeywordMeSH Terms
148. Hill  KE, Marchesi  JR,     ( 1999 )

Investigation of two evolutionarily unrelated halocarboxylic acid dehalogenase gene families.

Journal of bacteriology 181 (8)
PMID : 10198020  :   PMC  :   PMC93682    
Abstract >>
Dehalogenases are key enzymes in the metabolism of halo-organic compounds. This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial alpha-halocarboxylic acid (alphaHA) dehalogenase genes, called group I and group II deh genes. The two families are evolutionarily unrelated and together represent almost all of the alphaHA deh genes described to date. We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and II deh genes. Amino acid sequences derived from 10 of 11 group I deh partial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity. The exception, DehD from a Rhizobium sp., had only two of these five residues. Group II deh gene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized. Group II dehalogenases were stereoselective, dechlorinating L- but not D-2-chloropropionic acid, and derived amino acid sequences for all of the genes except dehII degrees P11 showed conservation of previously identified essential residues. Molecular analysis of the two deh families highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group I deh genes included two putative cryptic or silent genes, dehI degrees PP3 and dehI degrees 17a, produced by different organisms. Group II deh genes included two cryptic genes and an active gene, dehIIPP3, that can be switched off and on. All alphaHA-degrading bacteria so far described were Proteobacteria, a result that may be explained by limitations either in the host range for deh genes or in isolation methods.
KeywordMeSH Terms
Evolution, Molecular
Genes, Bacterial
Multigene Family
149. de Vrind-de Jong  EW, Baysse  C, Cornelis  P, Brouwers  GJ,     ( 1999 )

cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1.

Applied and environmental microbiology 65 (4)
PMID : 10103278  :   PMC  :   PMC91248    
Abstract >>
Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.
KeywordMeSH Terms
150. Kok  RG, Young  DM,     ( 1999 )

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation.

Applied and environmental microbiology 65 (4)
PMID : 10103267  :   PMC  :   PMC91237    
Abstract >>
Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.
KeywordMeSH Terms
Dioxygenases
Transformation, Bacterial
151. Thompson  LC, Ladner  JE, Codreanu  SG, Harp  J, Gilliland  GL, Armstrong  RN,     ( 2007 )

2-Hydroxychromene-2-carboxylic acid isomerase: a kappa class glutathione transferase from Pseudomonas putida.

Biochemistry 46 (23)
PMID : 17508726  :   DOI  :   10.1021/bi700356u    
Abstract >>
The enzyme 2-hydroxychromene-2-carboxylic acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion (Keq = 1.5) of HCCA and trans-o-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The dimeric protein binds one molecule of GSH very tightly (Kd approximately 5 nM) and a second molecule of GSH with much lower affinity (Kd approximately 2 to 11 microM). The enzyme is unstable in the absence of GSH. The turnover number in the forward direction (47 s(-1) at 25 degrees C) greatly exceeds off rates for GSH (koff approximately 10(-3) to 10(-2) s(-1) at 10 degrees C), suggesting that GSH acts as a tightly bound cofactor in the reaction. The crystal structure of the enzyme at 1.7 A resolution reveals that the isomerase is closely related to class kappa GSH transferases. Diffraction quality crystals could only be obtained in the presence of GSH and HCCA/tHBPA. Clear electron density is seen for GSH. Electron density for the organic substrates is located near the GSH and is best modeled to include both HCCA and tHBPA at occupancies of 0.5 for each. Although there is no electron density connecting the sulfur of GSH to the organic substrates, the sulfur is located very close (2.78 A) to C7 of HCCA. Taken together, the results suggest that the isomerization reaction involves a short-lived covalent adduct between the sulfur of GSH and C7 of the substrate.
KeywordMeSH Terms
152. Shi  YW, Cui  LF, Yuan  JM,     ( 2007 )

Gene cloning, expression, and substrate specificity of an imidase from the strain Pseudomonas putida YZ-26.

Current microbiology 55 (1)
PMID : 17534563  :   DOI  :   10.1007/s00284-005-0455-6    
Abstract >>
A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni(2+)-NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.
KeywordMeSH Terms
153. Yamada  M, Okada  Y, Yoshida  T, Nagasawa  T,     ( 2007 )

Purification, characterization and gene cloning of isoeugenol-degrading enzyme from Pseudomonas putida IE27.

Archives of microbiology 187 (6)
PMID : 17516050  :   DOI  :   10.1007/s00203-007-0218-9    
Abstract >>
An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k (cat) for isoeugenol were 175 muM and 5.18 s(-1), respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-alpha,beta-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.
KeywordMeSH Terms
Mixed Function Oxygenases
154. El Azhari  N, Chabaud  S, Percept  A, Bru  D, Martin-Laurent  F,     ( 2007 )

pcaH, a molecular marker for estimating the diversity of the protocatechuate-degrading bacterial community in the soil environment.

Pest management science 63 (5)
PMID : 17427160  :   DOI  :   10.1002/ps.1357    
Abstract >>
Microorganisms degrading phenolic compounds play an important role in soil carbon cycling as well as in pesticide degradation. The pcaH gene encoding a key ring-cleaving enzyme of the beta-ketoadipate pathway was selected as a functional marker. Using a degenerate primer pair, pcaH fragments were cloned from two agricultural soils. Restriction fragment length polymorphism (RFLP) screening of 150 pcaH clones yielded 68 RFLP families. Comparison of 86 deduced amino acid sequences displayed 70% identity to known PcaH sequences. Phylogenetic analysis results in two major groups mainly related to PcaH sequences from Actinobacteria and Proteobacteria phyla. This confirms that the developed primer pair targets a wide diversity of pcaH sequences, thereby constituting a suitable molecular marker to estimate the response of the pca community to agricultural practices.
KeywordMeSH Terms
Soil Microbiology
155. Chen  SX, Shi  BZ,     ( 2007 )

[Screening of strain producing an novel esterase with high enantioselectivity and molecular cloning of the enzyme gene].

Wei sheng wu xue bao = Acta microbiologica Sinica 47 (3)
PMID : 17672304  :  
Abstract >>
A novel strain producing an enantioselective lipolytic enzyme was isolated from soil samples, and identified as Pseudomonas putida NH33. A genomic library of P. putida NH33 was constructed and screened for esterase activity in E. coli. One positive clone was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.7kb DNA fragment carrying esterase gene. The nucleotide sequence of the DNA was found to contain an open reading frame of 1142 nucleotides encoding esterase of 381 amino acid residues and designated PPEst. The primary structure of the esterase exhibited 35%-40% homology to those of related enzymes from various sources and 80%-90% homology to esterases from the genus Pseudomonas. Amino acid sequence deduced from the nucleotide sequence contains of the consensus active site sequence, GXSXG, of serine esterase. The PPEst fragments were cloned into the expression vector pET-22b(+) and transformed into E. coli BL21 (DE3), and the recombinant protein fused with 6 x His at its C-terminus was purified to homogeneity by a single immobilized metal ion affinity chromatographic step. The molecular mass of the esterase was determined to be approximately 42kDa by SDS-PAGE. The purified enzyme could convert ethyl esters of 2-arylpropanoic acid to S-isomer of 2-arylpropanoic acids with an optical purity of > 99%. The result suggests that this esterase is excellent biocatalyst for synthesis of chiral pharmaceuticals. The enzyme is an novel esterase, and its nucleotide sequence has been submitted to GenBank under accession number AY896293.
KeywordMeSH Terms
Cloning, Molecular
156. Yano  H, Garruto  CE, Sota  M, Ohtsubo  Y, Nagata  Y, Zylstra  GJ, Williams  PA, Tsuda  M,     ( 2007 )

Complete sequence determination combined with analysis of transposition/site-specific recombination events to explain genetic organization of IncP-7 TOL plasmid pWW53 and related mobile genetic elements.

Journal of molecular biology 369 (1)
PMID : 17408691  :   DOI  :   10.1016/j.jmb.2007.02.098    
Abstract >>
Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.
KeywordMeSH Terms
Sequence Analysis, DNA
157. Mendes  RE, Castanheira  M, Toleman  MA, Sader  HS, Jones  RN, Walsh  TR,     ( 2007 )

Characterization of an integron carrying blaIMP-1 and a new aminoglycoside resistance gene, aac(6')-31, and its dissemination among genetically unrelated clinical isolates in a Brazilian hospital.

Antimicrobial agents and chemotherapy 51 (7)
PMID : 17470660  :   DOI  :   10.1128/AAC.00838-06     PMC  :   PMC1913225    
Abstract >>
Seven bla(IMP-1)-harboring Acinetobacter sp. isolates and one Pseudomonas putida clinical isolate were recovered from hospitalized patients. All isolates possessed a class 1 integron, named In86, carrying the same cassette array [bla(IMP1), aac(6')-31, and aadA1], which was plasmid located in five of the isolates. This report describes the ability of nonfermentative nosocomial pathogens to acquire and disseminate antimicrobial resistance determinants.
KeywordMeSH Terms
Genes, Bacterial
158. Drevland  RM, Waheed  A, Graham  DE,     ( 2007 )

Enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in Methanocaldococcus jannaschii.

Journal of bacteriology 189 (12)
PMID : 17449626  :   DOI  :   10.1128/JB.00166-07     PMC  :   PMC1913355    
Abstract >>
The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of beta-isopropylmalate, beta-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.
KeywordMeSH Terms
159. Bernal  P, Muñoz-Rojas  J, Hurtado  A, Ramos  JL, Segura  A,     ( 2007 )

A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality.

Environmental microbiology 9 (5)
PMID : 17472630  :   DOI  :   10.1111/j.1462-2920.2006.01236.x    
Abstract >>
Biological membranes have evolved different mechanisms to modify their composition in response to chemical stimuli in a process called 'homeoviscous adaptation'. Among these mechanisms, modifications in the ratio of saturated/unsaturated fatty acids and in cis/trans fatty acid isomers, cyclopropanation and changes in the phospholipids head group composition have been observed. To further understand the role of phospholipid head groups in solvent stress adaptation, we knocked out the cls (cardiolipin synthase) gene in Pseudomonas putida DOT-T1E. As expected, cls mutant membranes contained less cardiolipin than those of the wild-type strain. Although no significant growth rate defect was observed in the cls mutant compared with the wild-type strain, mutant cells were significantly smaller than the wild-type cells. The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant. Membranes of the cls mutant strain seemed to be more rigid than those of the parental strain, as observed by measurements of fluorescence polarization using the DPH probe, which intercalates into the membranes. Ethidium bromide is pumped out in Pseudomonas putida by at least one RND efflux pump involved in antibiotic and solvent resistance, and the higher rate of accumulation of ethidium bromide inside mutant cells indicated that functioning of the efflux pumps was compromised as a consequence of the alteration in phospholipid head group composition.
KeywordMeSH Terms
160. Vo  MT, Lee  KW, Kim  TK, Lee  YH,     ( 2007 )

Utilization of fadA knockout mutant Pseudomonas putida for overproduction of medium chain-length-polyhydroxyalkanoate.

Biotechnology letters 29 (12)
PMID : 17653511  :   DOI  :   10.1007/s10529-007-9476-5    
Abstract >>
The fadBA operon in the fatty acid beta-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l(-1) stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l(-1) with a cell dry weight of 10.3 g l(-1).
KeywordMeSH Terms
161. Kouzuma  A, Endoh  T, Omori  T, Nojiri  H, Yamane  H, Habe  H,     ( 2007 )

The ptsP gene encoding the PTS family protein EI(Ntr) is essential for dimethyl sulfone utilization by Pseudomonas putida.

FEMS microbiology letters 275 (1)
PMID : 17711452  :   DOI  :   10.1111/j.1574-6968.2007.00882.x    
Abstract >>
Many bacteria living in soil have developed the ability to use a wide variety of organosulfur compounds. Pseudomonas putida strain DS1 is able to utilize dimethyl sulfide as a sulfur source via a series of oxidation reactions that sequentially produce dimethyl sulfoxide, dimethyl sulfone (DMSO2), methanesulfonate, and sulfite. To isolate novel genes involved in DMSO2 utilization, a transposon-based mutagenesis of DS1 was performed. Of c. 10,000 strains containing mini-Tn5 inserts, 11 mutants lacked the ability to utilize DMSO2, and their insertion sites were determined. In addition to the cysNC, cysH, and cysM genes involved in sulfate assimilation, the ptsP gene encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family protein EI(Ntr) was identified, which is necessary for DMSO2 utilization. Using quantitative reverse transcriptase-polymerase chain reaction analysis, it was demonstrated that the expression of the sfn genes, necessary for DMSO2 utilization, was impaired in the ptsP disruptant. To the authors' knowledge, this is the first report of a PTS protein that is involved in bacterial assimilation of organosulfur compounds.
KeywordMeSH Terms
162. Neidle  E, Hartnett  C, Ornston  LN, Bairoch  A, Rekik  M, Harayama  S,     ( 1992 )

cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily.

European journal of biochemistry 204 (1)
PMID : 1740120  :   DOI  :   10.1111/j.1432-1033.1992.tb16612.x    
Abstract >>
In the aerobic degradation of benzoate by bacteria, benzoate is first dihydroxylated by a ring-hydroxylating dioxygenase to form a cis-diol (1,2-dihydroxycyclohexa-3,4-diene carboxylate) which is subsequently transformed to a catechol by an NAD(+)-dependent cis-diol dehydrogenase. The structural gene for this dehydrogenase, encoded on TOL plasmid pWW0 of Pseudomonas putida (xylL) and that encoded on the chromosome of Acinetobacter calcoaceticus (benD), were sequenced. They encode polypeptides of about 28 kDa in size. These proteins are similar to each other, exhibiting 58% sequence identity. They are also similar to other proteins of at least 20 different functions, which are members of the short-chain alcohol dehydrogenase family. The alignment of these proteins suggest two amino acids, lysine and tyrosine, as catalytically important residues.
KeywordMeSH Terms
Genes, Bacterial
Oxidoreductases Acting on CH-CH Group Donors
Plasmids
163. Rivera-Cancel  G, Bocioaga  D, Hay  AG,     ( 2007 )

Bacterial degradation of N,N-diethyl-m-toluamide (DEET): cloning and heterologous expression of DEET hydrolase.

Applied and environmental microbiology 73 (9)
PMID : 17337538  :   DOI  :   10.1128/AEM.02765-06     PMC  :   PMC1892861    
Abstract >>
Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.
KeywordMeSH Terms
164. Shingler  V, Powlowski  J, Marklund  U,     ( 1992 )

Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.

Journal of bacteriology 174 (3)
PMID : 1732207  :   DOI  :   10.1128/jb.174.3.711-724.1992     PMC  :   PMC206147    
Abstract >>
The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.
KeywordMeSH Terms
165. Bertani  I, Rampioni  G, Leoni  L, Venturi  V,     ( 2007 )

The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation.

BMC microbiology 7 (N/A)
PMID : 17655747  :   DOI  :   10.1186/1471-2180-7-71     PMC  :   PMC1949823    
Abstract >>
In Pseudomonas putida and Pseduomonas aeruginosa, the similar PpuR/RsaL/PpuI and LasR/RsaL/LasI acyl homoserine lactones (AHLs) quorum sensing (QS) systems have been shown to be under considerable regulation by other global regulators. A major regulator is the RsaL protein which strongly directly represses the transcription of the P. putida ppuI and P. aeruginosa lasI AHL synthases. In this study we screened a transposon mutant bank of P. putida in order to identify if any other regulators were involved in negative regulation of AHL QS. In our screen we identified three Tn5 mutants which displayed overproduction of AHLs in P. putida strain WCS358. Two of the mutants had a Tn5 located in the rsaL gene, whereas in one mutant the transposon was located in the lon protease gene. Lon proteases play important roles in protein quality control via degradation of misfolded proteins. It was determined that in the P. putida lon mutant, AHL levels, PpuR levels and ppuI promoter activity all increased significantly; we therefore postulated that PpuR is a target for Lon. The Lon protease had no effect on AHL production in P. aeruginosa. The Lon protease is a negative regulator of AHL production in P. putida WCS358. The Lon protease has also been shown by others to influence AHL QS in Vibrio fischeri and Agrobacterium tumefaciens and can thus become an important regulator of AHL QS timing and regulation in bacteria.
KeywordMeSH Terms
Quorum Sensing
166. Palmer  JA, Madhusudhan  KT, Hatter  K, Sokatch  JR,     ( 1991 )

Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida.

European journal of biochemistry 202 (2)
PMID : 1722146  :   DOI  :   10.1111/j.1432-1033.1991.tb16367.x    
Abstract >>
The third lipoamide dehydrogenase structural gene of Pseudomonas putida, lpd3, was isolated from a library of P. putida PpG2 DNA cloned in Escherichia coli TB1. The nucleotide sequence of lpd3 and its flanking regions indicate that lpd3 is not part of an operon, which is unique for a prokaryotic lipoamide dehydrogenase. An open reading frame was found 207 bases upstream from the start of transcription, but is encoded on the strand opposite lpd3. There is no evidence of an open reading frame immediately downstream from lpd3. The coding region of lpd3 consists of 1401 bp, providing for 466 amino acids plus a stop codon with a G/C content of 62.4%. The transcriptional start site was located 33-bp upstream from the start of translation. The third lipoamide dehydrogenase (LPD-3) shares amino acid identity with the other two lipoamide dehydrogenases of P. putida, 45% with that of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes, and 45.9% with the lipoamide dehydrogenase of the branched-chain oxoacid complex. LPD-3 is more closely related to eukaryotic lipoamide dehydrogenases since it has 53.6% amino acid sequence identity with pig and human lipoamide dehydrogenases and 51.1% identity with yeast lipoamide dehydrogenase. LPD-3 was not produced in wild-type P. putida PpG2 under a variety of growth conditions. However, LPD-3 was produced in P. putida PpG2 carrying pSP14, a pKT240-based clone with the entire lpd3 gene plus 104 bases of the leader. The only demonstrated role of LPD-3 in P. putida is as a substitute for lipoamide dehydrogenase of the 2-oxoglutarate dehydrogenase and pyruvate multienzyme complexes when the latter is inactive or missing.
KeywordMeSH Terms
Genes, Bacterial
Transcription, Genetic
167. Ercolini  D, Russo  F, Blaiotta  G, Pepe  O, Mauriello  G, Villani  F,     ( 2007 )

Simultaneous detection of Pseudomonas fragi, P. lundensis, and P. putida from meat by use of a multiplex PCR assay targeting the carA gene.

Applied and environmental microbiology 73 (7)
PMID : 17293505  :   DOI  :   10.1128/AEM.02603-06     PMC  :   PMC1855653    
Abstract >>
Species-specific primers and a multiplex PCR assay were developed for the simultaneous identification and differentiation of Pseudomonas fragi, P. lundensis, and P. putida based on the coamplification of different portions of the small subunit of the carbamoyl phosphate synthase gene (carA). The carA multiplex PCR was used to detect the presence of the three Pseudomonas species from beef, chicken, and pork samples and proved to be effective in showing their evolution during the storage of meat.
KeywordMeSH Terms
168. Achour  AR, Bauda  P, Billard  P,     ( 2007 )

Diversity of arsenite transporter genes from arsenic-resistant soil bacteria.

Research in microbiology 158 (1��2��)
PMID : 17258434  :   DOI  :   10.1016/j.resmic.2006.11.006    
Abstract >>
A PCR approach was developed to assess the occurrence and diversity of arsenite transporters in arsenic-resistant bacteria. For this purpose, three sets of degenerate primers were designed for the specific amplification of approximately 750bp fragments from arsB and two subsets of ACR3 (designated ACR3(1) and ACR3(2)) arsenite carrier gene families. These primers were used to screen a collection of 41 arsenic-resistant strains isolated from two soil samples with contrasting amounts of arsenic. PCR results showed that 70.7% of the isolates contained a gene related to arsB or ACR3, with three of them carrying both arsB and ACR3-like genes. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsB in Firmicutes and Gammaproteobacteria, while ACR3(1) and ACR3(2) were mostly present in Actinobacteria and Alphaproteobacteria, respectively. In addition to validating the use of degenerate primers for the identification of arsenite transporter genes in a taxonomically wide range of bacteria, the study describes a novel collection of strains displaying interesting features of resistance to arsenate, arsenite and antimonite, and the ability to oxidize arsenite.
KeywordMeSH Terms
Soil Microbiology
169. Kudou  D, Misaki  S, Yamashita  M, Tamura  T, Takakura  T, Yoshioka  T, Yagi  S, Hoffman  RM, Takimoto  A, Esaki  N, Inagaki  K,     ( 2007 )

Structure of the antitumour enzyme L-methionine gamma-lyase from Pseudomonas putida at 1.8 A resolution.

Journal of biochemistry 141 (4)
PMID : 17289792  :   DOI  :   10.1093/jb/mvm055    
Abstract >>
l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.
KeywordMeSH Terms
170. Takeo  M, Prabu  SK, Kitamura  C, Hirai  M, Takahashi  H, Kato  D, Negoro  S,     ( 2006 )

Characterization of alkylphenol degradation gene cluster in Pseudomonas putida MT4 and evidence of oxidation of alkylphenols and alkylcatechols with medium-length alkyl chain.

Journal of bioscience and bioengineering 102 (4)
PMID : 17116584  :   DOI  :   10.1263/jbb.102.352    
Abstract >>
Alkylphenols (APs) are ubiquitous contaminants in aquatic environments and have endocrine disrupting and toxic effects on aquatic organisms. To investigate biodegradation mechanisms of APs, an AP degradation gene cluster was cloned from a butylphenol (BP)-degrading bacterium, Pseudomonas putida MT4. The gene cluster consisted of 13 genes named bupBA1A2A3A4A5A6CEHIFG. From the nucleotide sequences, bupA1A2A3A4A5A6 were predicted to encode a multicomponent phenol hydroxylase (PH), whereas bupBCEHIFG were expected to encode meta-cleavage pathway enzymes. A partial sequence of a putative NtrC-type regulatory gene, bupR, was also found upstream of the gene bupB. This result indicates that APs can be initially oxidized into alkylcatechols (ACs), followed by the meta-cleavage of the aromatic rings. To confirm this pathway, AP degradation tests were carried out using the recombinant P. putida KT2440 harboring the PH genes (bupA1A2A3A4A5A6). The recombinant strain oxidized 4-n-APs with an alkyl chain of up to C7 (< or = C7) efficiently and also several BPs including those with an alkyl chain with some degree of branching. Therefore, it was found that PH had a broad substrate specificity for APs with a medium-length alkyl chain (C3-C7). Moreover, the cell extract of a recombinant Escherichia coli harboring bupB (a catechol 2,3-dioxygenase gene) converted 4-n-ACs with an alkyl chain of < or = C9 into yellow meta-cleavage products with a maximum absorbance at 379 nm, indicating that the second step enzyme in this pathway is also responsible for the degradation of ACs with a medium-length alkyl chain. These results suggest that MT4 is a very useful strain in the biodegradation of a wide range of APs with a medium-length alkyl chain, which known nonylphenol-degrading Sphingomonas strains have never degraded.
KeywordMeSH Terms
171. Kosti?  T, Weilharter  A, Rubino  S, Delogu  G, Uzzau  S, Rudi  K, Sessitsch  A, Bodrossy  L,     ( 2007 )

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens.

Analytical biochemistry 360 (2)
PMID : 17123456  :   DOI  :   10.1016/j.ab.2006.09.026    
Abstract >>
A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.
KeywordMeSH Terms
172. Hu  N, Zhao  B,     ( 2007 )

Key genes involved in heavy-metal resistance in Pseudomonas putida CD2.

FEMS microbiology letters 267 (1)
PMID : 17166231  :   DOI  :   10.1111/j.1574-6968.2006.00505.x    
Abstract >>
A cadmium-resistant bacterium Pseudomonas putida CD2 was isolated from sewage sludge samples. Strain CD2 exhibited high maximal tolerant concentrations (MTC) for a large spectrum of divalent metals. Screening a library obtained using Tn5-B21 insertion mutagenesis resulted in identification of 12 mutants with a substantial decrease in resistance to 3 mM cadmium. The DNA sequences of the contiguous region from the Tn5 insertion sites were determined by inverse PCR. Six genes involved in cadmium resistance were identified. These genes were from three gene clusters: czcCBA1, cadA2R and colRS. The homologs of the first two gene clusters were predicted to be metal efflux systems, whereas the products of colRS, ColR and ColS, were thought to be a two-component signal transduction (TCST) system. In this study, we have demonstrated that ColRS also function in regulating multi-metal resistance using genetic complementation.
KeywordMeSH Terms
173. Duque  E, Rodríguez-Herva  JJ, de la Torre  J, Domínguez-Cuevas  P, Muñoz-Rojas  J, Ramos  JL,     ( 2007 )

The RpoT regulon of Pseudomonas putida DOT-T1E and its role in stress endurance against solvents.

Journal of bacteriology 189 (1)
PMID : 17071759  :   DOI  :   10.1128/JB.00950-06     PMC  :   PMC1797225    
Abstract >>
Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents. Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon with the preceding gene and with the gene located downstream. The translated gene product of the open reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar DeltarpoT mutant was generated by homologous recombination, and survival of the mutant was tested under various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H(2)O(2), and benzoate. In the DeltarpoT mutant background, expression of around 50 transcriptional units was affected: 31 cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to the fact that in the DeltarpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.
KeywordMeSH Terms
Toluene
174. Henning  H, Leggewie  C, Pohl  M, Müller  M, Eggert  T, Jaeger  KE,     ( 2006 )

Identification of novel benzoylformate decarboxylases by growth selection.

Applied and environmental microbiology 72 (12)
PMID : 17012586  :   DOI  :   10.1128/AEM.01541-06     PMC  :   PMC1694272    
Abstract >>
A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the beta-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.
KeywordMeSH Terms
Culture Media
175. Jussila  MM, Zhao  J, Suominen  L, Lindström  K,     ( 2007 )

TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo.

Environmental pollution (Barking, Essex : 1987) 146 (1��2��)
PMID : 17000041  :   DOI  :   10.1016/j.envpol.2006.07.012    
Abstract >>
Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids.
KeywordMeSH Terms
Industrial Oils
176. Fessenmaier  M, Frank  R, Retey  J, Schubert  C,     ( 1991 )

Cloning and sequencing the urocanase gene (hutU) from Pseudomonas putida.

FEBS letters 286 (1��2��)
PMID : 1677899  :   DOI  :   10.1016/0014-5793(91)80938-y    
Abstract >>
A clone harbouring the entire urocanase gene (hutU) was obtained from a genomic library of Pseudomonas putida using oligonucleotide probes synthesised on the basis of known flanking sequences. One subunit of urocanase consists of 556 amino acids and has a molecular mass of 60,771 Da.
KeywordMeSH Terms
177. Coll  M, Knof  SH, Ohga  Y, Messerschmidt  A, Huber  R, Moellering  H, Rüssmann  L, Schumacher  G,     ( 1990 )

Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures.

Journal of molecular biology 214 (2)
PMID : 1696320  :   DOI  :   10.1016/0022-2836(90)90201-v    
Abstract >>
Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. An enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the general base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the N(3) atom of the guanidinium group. OH- 377 adds to the C(1) atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.
KeywordMeSH Terms
178. Lee  K, Ryu  EK, Choi  KS, Cho  MC, Jeong  JJ, Choi  EN, Lee  SO, Yoon  DY, Hwang  I, Kim  CK,     ( 2006 )

Identification and expression of the cym, cmt, and tod catabolic genes from Pseudomonas putida KL47: expression of the regulatory todST genes as a factor for catabolic adaptation.

Journal of microbiology (Seoul, Korea) 44 (2)
PMID : 16728956  :  
Abstract >>
Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene (C(1)-C(4)), biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99%) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
179. Benjamin  RC, Voss  JA, Kunz  DA,     ( 1991 )

Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7.

Journal of bacteriology 173 (8)
PMID : 1672868  :   DOI  :   10.1128/jb.173.8.2724-2728.1991     PMC  :   PMC207846    
Abstract >>
Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids. The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus. A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus. The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.
KeywordMeSH Terms
Dioxygenases
180. Rothmel  RK, Aldrich  TL, Houghton  JE, Coco  WM, Ornston  LN, Chakrabarty  AM,     ( 1990 )

Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family.

Journal of bacteriology 172 (2)
PMID : 1688844  :   DOI  :   10.1128/jb.172.2.922-931.1990     PMC  :   PMC208520    
Abstract >>
Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS.
KeywordMeSH Terms
Bacterial Proteins
Carbon-Carbon Double Bond Isomerases
Genes, Bacterial
Genes, Regulator
Intramolecular Lyases
Operon
181. Sota  M, Yano  H, Ono  A, Miyazaki  R, Ishii  H, Genka  H, Top  EM, Tsuda  M,     ( 2006 )

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase.

Journal of bacteriology 188 (11)
PMID : 16707697  :   DOI  :   10.1128/JB.00185-06     PMC  :   PMC1482893    
Abstract >>
The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.
KeywordMeSH Terms
DNA Transposable Elements
Plasmids
182. Xiao  Y, Wu  JF, Liu  H, Wang  SJ, Liu  SJ, Zhou  NY,     ( 2006 )

Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73.

Applied microbiology and biotechnology 73 (1)
PMID : 16642329  :   DOI  :   10.1007/s00253-006-0441-3    
Abstract >>
The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520-6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.
KeywordMeSH Terms
183. Merimaa  M, Heinaru  E, Liivak  M, Vedler  E, Heinaru  A,     ( 2006 )

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes.

Archives of microbiology 186 (4)
PMID : 16906406  :   DOI  :   10.1007/s00203-006-0143-3    
Abstract >>
Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.
KeywordMeSH Terms
184. Danko  AS, Saski  CA, Tomkins  JP, Freedman  DL,     ( 2006 )

Involvement of coenzyme M during aerobic biodegradation of vinyl chloride and ethene by Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD.

Applied and environmental microbiology 72 (5)
PMID : 16672529  :   DOI  :   10.1128/AEM.72.5.3756-3758.2006     PMC  :   PMC1472362    
Abstract >>
The involvement of coenzyme M in aerobic biodegradation of vinyl chloride and ethene in Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD was demonstrated using PCR, hybridization, and enzyme assays. The results of this study extend the range of eubacteria known to use epoxyalkane:coenzyme M transferase.
KeywordMeSH Terms
185. Tasaki  Y, Yoshikawa  H, Tamura  H,     ( 2006 )

Isolation and characterization of an alcohol dehydrogenase gene from the octylphenol polyethoxylate degrader Pseudomonas putida S-5.

Bioscience, biotechnology, and biochemistry 70 (8)
PMID : 16926497  :   DOI  :   10.1271/bbb.60009    
Abstract >>
Octylphenol polyethoxylate (OPEO(n)) biodegradation by Pseudomonas putida S-5 under aerobic conditions is initiated by the oxidation of its terminal alcohol group by alcohol dehydrogenase. A DNA fragment, containing an alcohol dehydrogenase gene (adh1), was isolated using a combination of degenerate PCR and inverse PCR. The predicted translation product of adh1 showed significant sequence similarity to bacterial alcohol dehydrogenases. Furthermore, a flavin-binding motif and signature patterns conserved in type III FAD-dependent alcohol oxidases were detected. Two open reading frames (ORFs) were found upstream of adh1, encoding a putative acyl-CoA synthetase and a putative esterase. Downstream of adh1 and located on the opposite strand was an ORF encoding a putative aldehyde dehydrogenase. Transcription analysis using RT-PCR showed that adh1 is cotranscribed with the putative acyl-CoA synthetase and esterase genes during growth on OPEO(n). ADH1 overproduced in Escherichia coli exhibited activity not only toward various alcohols, including OPEO(n)s, but also toward primary aliphatic and aromatic aldehydes.
KeywordMeSH Terms
186. Panas  P, Ternan  NG, Dooley  JS, McMullan  G,     ( 2006 )

Detection of phosphonoacetate degradation and phnA genes in soil bacteria from distinct geographical origins suggest its possible biogenic origin.

Environmental microbiology 8 (5)
PMID : 16623750  :   DOI  :   10.1111/j.1462-2920.2005.00974.x    
Abstract >>
Phosphonoacetate is regarded as an antiviral xenobiotic whose mineralization can be catalysed by an enzyme, phosphonoacetate hydrolase, encoded by the phnA gene. To date the enzyme's activity has been detected in only a limited number of bacteria. Its expression has been shown to occur in a manner independent of the phosphate status of the cell, in direct contrast to the general rule of organophosphonate metabolism being under the control of the pho regulon. In this study the environmental occurrence of the phnA gene was evaluated by polymerase chain reaction amplification of DNA extracts obtained directly from various soil environments. Sensitivity of this method was improved such that a positive result was routinely obtained with soil spiked with as few as 6 colony-forming units (cfu) per gram of soil of Pseudomonas fluorescens 23F (phnA(+)). When total DNA from a variety of Northern Irish, Greek and Bolivian soils was tested, all were positive for phnA. Bacteria capable of utilizing phosphonoacetate as sole carbon, energy and phosphorus source, with the release of essentially equimolar concentrations of phosphate to the culture supernatant, were isolated from all soil samples tested. Analysis of three such isolates revealed all to be species of Pseudomonas sensu stricto, possessing phosphonoacetate hydrolase activity in cell-free extracts. Sequence determination of the phnA gene revealed a similarity of the putative protein sequences at levels of 98.3-99.3% between the Pseudomonas strains. This is the first study to use molecular methods to investigate the distribution of a gene encoding organophosphonate metabolism, and indicates that the phnA gene is ubiquitous within soils from geographically distinct regions. Such an observation supports the proposition that phosphonoacetate is a compound that may also have a biogenic origin.
KeywordMeSH Terms
Genes, Bacterial
Soil Microbiology
187. Dubern  JF, Lugtenberg  BJ, Bloemberg  GV,     ( 2006 )

The ppuI-rsaL-ppuR quorum-sensing system regulates biofilm formation of Pseudomonas putida PCL1445 by controlling biosynthesis of the cyclic lipopeptides putisolvins I and II.

Journal of bacteriology 188 (8)
PMID : 16585751  :   DOI  :   10.1128/JB.188.8.2898-2906.2006     PMC  :   PMC1447005    
Abstract >>
Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C(10)-N-acyl homoserine lactone (3-oxo-C(10)-AHL) or 3-oxo-C(12)-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.
KeywordMeSH Terms
Adaptation, Physiological
188. Poirel  L, Cabanne  L, Collet  L, Nordmann  P,     ( 2006 )

Class II transposon-borne structure harboring metallo-beta-lactamase gene blaVIM-2 in Pseudomonas putida.

Antimicrobial agents and chemotherapy 50 (8)
PMID : 16870796  :   DOI  :   10.1128/AAC.00398-06     PMC  :   PMC1538670    
Abstract >>
A plasmid-encoded class II transposon element was identified in a carbapenem-resistant Pseudomonas putida isolate. Tn1332, closely related to Tn1331, harbored the metallo-beta-lactamase gene bla(VIM-2) in addition to four other antibiotic resistance genes, aacA4, aadA1, bla(OXA-9), and bla(TEM-1), and two novel insertion sequences, ISPpu17 and ISPpu18.
KeywordMeSH Terms
Genes, Bacterial
189. Zhen  D, Liu  H, Wang  SJ, Zhang  JJ, Zhao  F, Zhou  NY,     ( 2006 )

Plasmid-mediated degradation of 4-chloronitrobenzene by newly isolated Pseudomonas putida strain ZWL73.

Applied microbiology and biotechnology 72 (4)
PMID : 16583229  :   DOI  :   10.1007/s00253-006-0345-2    
Abstract >>
A strain of Pseudomonas putida ZWL73 was isolated from soil contaminated with chloronitrobenzenes and identified by 16S rDNA sequencing. This bacterium released chloride and ammonia into the medium when grown on 4-chloronitrobenzene (4CNB) as the sole source of carbon, nitrogen and energy. A plasmid designated pZWL73 of approximately 100 kb in this strain was found to be responsible for 4CNB degradation. This was based on the fact that the plasmid-cured strains showed 4CNB- phenotype and the 4CNB+ phenotype could be conjugally transferred. The cell-free extracts of strain ZWL73 exhibited chloronitrobenzene nitroreductase and 2-amino-5-chlorophenol 1, 6-dioxygenase (2A5CPDO) activities, but neither activity was found from that of the plasmid-cured strain. We have also cloned a 4.9-kb EcoRI fragment exhibiting 2A5CPDO activity. Sequencing results revealed beta-subunit (cnbCa) and alpha subunit (cnbCb) of a meta-cleavage dioxygenase, which were subsequently expressed in E. coli with 2A5CPDO activity. The phylogenetic analysis suggested that 2A5CPDO may form a new subgroup in class III meta-cleavage dioxygenase with its close homologs.
KeywordMeSH Terms
190. Mooney  A, O'Leary  ND, Dobson  AD,     ( 2006 )

Cloning and functional characterization of the styE gene, involved in styrene transport in Pseudomonas putida CA-3.

Applied and environmental microbiology 72 (2)
PMID : 16461680  :   DOI  :   10.1128/AEM.72.2.1302-1309.2006     PMC  :   PMC1392900    
Abstract >>
A 1.5-kb region immediately downstream of the styABCD operon involved in styrene degradation in Pseudomonas putida CA-3 has been cloned. Sequence analysis revealed a 1,296-bp open reading frame, designated styE, and BLAST P database comparisons of the deduced StyE amino acid sequence revealed 33 to 98% identity with several membrane-associated ATPase-dependent kinase proteins involved in the active transport of aromatic hydrocarbons across bacterial membranes and also with FadL, an outer membrane protein necessary for the uptake of long-chain fatty acids in Escherichia coli. Transcription of styE is styrene dependent, and the gene is cotranscribed with the styABCD structural genes. StyE appears to be membrane associated, with a corresponding 45.9-kDa band being identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane preparations from styrene-grown cells. P. putida CA-3 cells in which the styE gene had been interrupted were no longer capable of growth on styrene. In contrast, overexpression of styE in P. putida CA-3 resulted in a 4.2-fold increase in styrene monooxygenase activity compared with wild-type cells grown on styrene, with a concomitant 8-fold increase in styA mRNA transcript levels. Experiments with the classic, ATPase inhibitor vanadate revealed that growth of wild-type cells on styrene was inhibited at a concentration of 1 mM, while 1.75 mM was required to achieve a similar effect in the StyE overexpression strain. Growth of either strain on citrate was not inhibited in the presence of up to 7 mM vanadate. These findings suggest a role for StyE in the active transport of styrene in Pseudomonas putida CA-3 and identify styrene transport as a potentially limiting factor with respect to mRNA transcript levels and associated enzymatic activity of the styrene degradative pathway.
KeywordMeSH Terms
Genes, Bacterial
191. Bitter  W, Marugg  JD, de Weger  LA, Tommassen  J, Weisbeek  PJ,     ( 1991 )

The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein.

Molecular microbiology 5 (3)
PMID : 1646376  :   DOI  :   10.1111/j.1365-2958.1991.tb00736.x    
Abstract >>
The initial step in the uptake of iron via ferric pseudobactin by the plant-growth-promoting Pseudomonas putida strain WCS358 is binding to a specific outer-membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N-terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB-dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55Fe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
Receptors, Peptide
Receptors, Virus
192. Gaillard  M, Vallaeys  T, Vorhölter  FJ, Minoia  M, Werlen  C, Sentchilo  V, Pühler  A, van der Meer  JR,     ( 2006 )

The clc element of Pseudomonas sp. strain B13, a genomic island with various catabolic properties.

Journal of bacteriology 188 (5)
PMID : 16484212  :   DOI  :   10.1128/JB.188.5.1999-2013.2006     PMC  :   PMC1426575    
Abstract >>
Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.
KeywordMeSH Terms
193. Karlsson  A, Parales  JV, Parales  RE, Gibson  DT, Eklund  H, Ramaswamy  S,     ( 2005 )

NO binding to naphthalene dioxygenase.

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 10 (5)
PMID : 15942729  :   DOI  :   10.1007/s00775-005-0657-1    
Abstract >>
Nitric oxide (NO) is commonly used as an analogue for dioxygen in structural and spectroscopic studies of oxygen binding and oxygen activation. In this study, crystallographic structures of naphthalene dioxygenase (NDO) in complex with nitric oxide are reported. In the presence of the aromatic substrate indole, NO is bound end-on to the active-site mononuclear iron of NDO. The structural observations correlate well with spectroscopic measurements of NO binding to NDO in solution. However, the end-on binding of NO is in contrast to the recently reported structure of oxygen to the active-site iron of NDO that binds side-on. While NO is a good oxygen analogue with many similarities to O(2), the different binding mode of NO to the active-site iron atom leads to different mechanistic implications. Hence, caution needs to be used in extrapolating NO as an analogue to O(2) binding.
KeywordMeSH Terms
194. Hendrickx  B, Junca  H, Vosahlova  J, Lindner  A, Rüegg  I, Bucheli-Witschel  M, Faber  F, Egli  T, Mau  M, Schlömann  M, Brennerova  M, Brenner  V, Pieper  DH, Top  EM, Dejonghe  W, Bastiaens  L, Springael  D,     ( 2006 )

Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site.

Journal of microbiological methods 64 (2)
PMID : 15949858  :   DOI  :   10.1016/j.mimet.2005.04.018    
Abstract >>
Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.
KeywordMeSH Terms
Soil Microbiology
195. Ait Tayeb  L, Ageron  E, Grimont  F, Grimont  PA,     ( N/A )

Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates.

Research in microbiology 156 (5��6��)
PMID : 15950132  :   DOI  :   10.1016/j.resmic.2005.02.009    
Abstract >>
Phylogenetic relationships within the genus Pseudomonas were examined by comparing partial (about 1000 nucleotides) rpoB gene sequences. A total of 186 strains belonging to 75 species of Pseudomonas sensu stricto and related species were studied. The phylogenetic resolution of the rpoB tree was approximately three times higher than that of the rrs tree. Ribogroups published earlier correlated well with rpoB sequence clusters. The rpoB sequence database generated by this study was used for identification. A total of 89 isolates (79.5%) were identified to a named species, while 16 isolates (14.3%) corresponded to unnamed species, and 7 isolates (6.2%) had uncertain affiliation. rpoB sequencing is now being used for routine identification of Pseudomonas isolates in our laboratory.
KeywordMeSH Terms
Phylogeny
196. Martins  BM, Svetlitchnaia  T, Dobbek  H,     ( 2005 )

2-Oxoquinoline 8-monooxygenase oxygenase component: active site modulation by Rieske-[2Fe-2S] center oxidation/reduction.

Structure (London, England : 1993) 13 (5)
PMID : 15893671  :   DOI  :   10.1016/j.str.2005.03.008    
Abstract >>
2-Oxoquinoline 8-monooxygenase is a Rieske non-heme iron oxygenase that catalyzes the NADH-dependent oxidation of the N-heterocyclic aromatic compound 2-oxoquinoline to 8-hydroxy-2-oxoquinoline in the soil bacterium Pseudomonas putida 86. The crystal structure of the oxygenase component of 2-oxoquinoline 8-monooxygenase shows a ring-shaped, C3-symmetric arrangement in which the mononuclear Fe(II) ion active site of one monomer is at a distance of 13 A from the Rieske-[2Fe-2S] center of a second monomer. Structural analyses of oxidized, reduced, and substrate bound states reveal the molecular bases for a new function of Fe-S clusters. Reduction of the Rieske center modulates the mononuclear Fe through a chain of conformational changes across the subunit interface, resulting in the displacement of Fe and its histidine ligand away from the substrate binding site. This creates an additional coordination site at the mononuclear Fe(II) ion and can open a pathway for dioxygen to bind in the substrate-containing active site.
KeywordMeSH Terms
197. Faizal  I, Dozen  K, Hong  CS, Kuroda  A, Takiguchi  N, Ohtake  H, Takeda  K, Tsunekawa  H, Kato  J,     ( 2005 )

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system.

Journal of industrial microbiology & biotechnology 32 (11��12��)
PMID : 15947959  :   DOI  :   10.1007/s10295-005-0253-y    
Abstract >>
Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.
KeywordMeSH Terms
Pseudomonas putida
198. Ingmer  H, Atlung  T,     ( 1992 )

Expression and regulation of a dnaA homologue isolated from Pseudomonas putida.

Molecular & general genetics : MGG 232 (3)
PMID : 1588913  :   DOI  :   10.1007/bf00266248    
Abstract >>
A gene homologous to the Escherichia coli dnaA gene was isolated from Pseudomonas putida and its transcription was investigated in E. coli as well as in P. putida. In both species the P. putida dnaA gene is transcribed from two promoters, one of which shows strong homology to promoters recognized by the sigma 54 factor found in both bacteria. In E. coli transcription of the P. putida dnaA gene can be repressed by overproduction of E. coli DnaA protein, presumably due to the presence of several DnaA-box-like sequences found in the promoter region. Likewise the P. putida DnaA protein is able to regulate expression of the E. coli dnaA gene but we failed to demonstrate autoregulation of the P. putida dnaA gene. A point mutation was introduced into the P. putida dnaA gene, equivalent to the ATP binding site mutation present in E. coli dnaA5 and dnaA46 mutants, and this alteration abolished the ability of the protein to repress the expression of the E. coli dnaA gene. These results indicate that DnaA proteins from other species than E. coli have maintained the ability to recognize the DnaA box sequence and that the conservation between the DnaA proteins reflects functionally similar domains.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
Promoter Regions, Genetic
199. Elmi  F, Lee  HT, Huang  JY, Hsieh  YC, Wang  YL, Chen  YJ, Shaw  SY, Chen  CJ,     ( 2005 )

Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis.

Journal of bacteriology 187 (24)
PMID : 16321951  :   DOI  :   10.1128/JB.187.24.8470-8476.2005     PMC  :   PMC1317000    
Abstract >>
Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket approximately 12 A deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.
KeywordMeSH Terms
200. Adaikkalam  V, Swarup  S,     ( 2005 )

Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain.

Canadian journal of microbiology 51 (3)
PMID : 15920618  :   DOI  :   10.1139/w04-135    
Abstract >>
We describe an operon, copABCD, that encodes copper-binding and sequestering proteins for copper homeostasis in the copper-sensitive strain Pseudomonas putida PNL-MK25. This is the second operon characterized as being involved in copper homeostasis, in addition to a P1-type ATPase encoded by cueAR, which was previously shown to be active in the same strain. In this study, 3 copper-responsive mutants were obtained through mini-Tn5::gfp mutagenesis and were found to exhibit reduced tolerance to copper. Sequencing analysis of the transposon-tagged region in the 3 mutants revealed insertions in 2 genes of an operon homologous to the copABCD of P. syringae and pcoABCD of Escherichia coli. Gene expression studies demonstrated that the P. putida copABCD is inducible starting from 3 micromol/L copper levels. Copper-sensitivity studies revealed that the tolerance of the mutant strains was reduced only marginally (only 0.16-fold) in comparison to a 6-fold reduced tolerance of the cueAR mutant. Thus, the cop operon in this strain has a minimal role when compared with its role both in other copper-resistant strains, such as P. syringae pv. syringae, and in the cueAR operon of the same strain. We propose that the reduced function of the copABCD operon is likely to be due to the presence of fewer metal-binding domains in the encoded proteins.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
201. Arias-Barrau  E, Sandoval  A, Naharro  G, Olivera  ER, Luengo  JM,     ( 2005 )

A two-component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate in Pseudomonas putida.

The Journal of biological chemistry 280 (28)
PMID : 15866873  :   DOI  :   10.1074/jbc.M501988200    
Abstract >>
The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, l-Phe, and l-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).
KeywordMeSH Terms
202. Leveau  JH, Lindow  SE,     ( 2005 )

Utilization of the plant hormone indole-3-acetic acid for growth by Pseudomonas putida strain 1290.

Applied and environmental microbiology 71 (5)
PMID : 15870323  :   DOI  :   10.1128/AEM.71.5.2365-2371.2005     PMC  :   PMC1087548    
Abstract >>
We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.
KeywordMeSH Terms
203. Jones  DH, Barth  PT, Byrom  D, Thomas  CM,     ( 1992 )

Nucleotide sequence of the structural gene encoding a 2-haloalkanoic acid dehalogenase of Pseudomonas putida strain AJ1 and purification of the encoded protein.

Journal of general microbiology 138 (4)
PMID : 1588303  :   DOI  :   10.1099/00221287-138-4-675    
Abstract >>
The nucleotide sequence of a gene encoding an L-2-haloalkanoic acid halidohydrolase from Pseudomonas putida strain AJ1 was determined. The ORF (hadL) codes for a polypeptide of 227 amino acids (Mr 25,687) which has significant homology to two other L-2-haloalkanoic acid halidohydrolases of Pseudomonas sp., DehcI and DehcII; these show 38% and 51% amino acid identity respectively to HadL. All three enzymes produce products of an opposite optical configuration to that of the substrates. Comparison of the three sequences shows several highly conserved motifs which indicate the possible position of the enzyme active site. The enzyme was purified to homogeneity and appears to exist as a tetramer.
KeywordMeSH Terms
Genes, Bacterial
204. Parales  RE, Harwood  CS,     ( 1992 )

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

Journal of bacteriology 174 (14)
PMID : 1624453  :   DOI  :   10.1128/jb.174.14.4657-4666.1992     PMC  :   PMC206261    
Abstract >>
beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.
KeywordMeSH Terms
205. Hontzeas  N, Richardson  AO, Belimov  A, Safronova  V, Abu-Omar  MM, Glick  BR,     ( 2005 )

Evidence for horizontal transfer of 1-aminocyclopropane-1-carboxylate deaminase genes.

Applied and environmental microbiology 71 (11)
PMID : 16269802  :   DOI  :   10.1128/AEM.71.11.7556-7558.2005     PMC  :   PMC1287689    
Abstract >>
PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.
KeywordMeSH Terms
Gene Transfer, Horizontal
206. Yun  YS, Nam  GH, Kim  YG, Oh  BH, Choi  KY,     ( 2005 )

Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B.

The FEBS journal 272 (8)
PMID : 15819891  :   DOI  :   10.1111/j.1742-4658.2005.04627.x    
Abstract >>
A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent-exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B (KSI-PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side-chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI-PI, mutants of those amino acids associated with the SEHC were prepared. The W92A, L125A/V127A, and W92A/L125A/V127A mutations largely decreased the conformational stability, while the L125F/V127F mutation slightly increased the stability, indicating that hydrophobic packing by the SEHC is important in maintaining stability. The crystal structure of W92A revealed that the decreased stability caused by the removal of the bulky side-chain of Trp92 could be attributed to the destabilization of the surface hydrophobic layer consisting of a solvent-exposed beta-sheet. Consistent with the structural data, the binding affinities for three different steroids showed that the surface hydrophobic layer stabilized by SEHC is required for KSI-PI to efficiently recognize hydrophobic steroids. Unfolding kinetics based on analysis of the Phi(U) value also indicated that the SEHC in the native state was resistant to the unfolding process, despite its solvent-exposed site. Taken together, our results demonstrate that the SEHC plays a key role in the structural integrity that is needed for KSI-PI to stabilize the hydrophobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic steroids in water solution.
KeywordMeSH Terms
207. Buell  CR, Anderson  AJ,     ( N/A )

Genetic analysis of the aggA locus involved in agglutination and adherence of Pseudomonas putida, a beneficial fluorescent pseudomonad.

Molecular plant-microbe interactions : MPMI 5 (2)
PMID : 1617198  :  
Abstract >>
An isolate of Pseudomonas putida, which rapidly adheres to plant roots, is agglutinated by a glycoprotein from root surfaces. Agglutination is prevented and adherence to the root surface is diminished by Tn5 insertion in mutant 5123. Two cosmid clones from wild type P. putida and a 2.7-kbp EcoRI-HindIII subclone present in both cosmid clones restored agglutinable to wild type levels in transconjugants of the nonagglutinable (Agg-) Tn5 mutant 5123. These three clones increased agglutinability in transconjugants of the parental Agg+ isolate. The 2.7-kbp EcoRI-HindIII subclone restored adherence to bean root surfaces of 5123 to wild type levels in a short-term binding assay. Deletion analysis of the 2.7-kbp fragment indicated only 1.45 kbp was necessary for complementation of agglutinability in 5123. This sequence, termed the aggA locus, contains an open reading frame of 1,356 nucleotides encoding a predicted 50,509-Da protein. The distribution of the aggA locus in plant-associated bacteria, as detected through Southern hybridization, is limited to bacteria that express the agglutination phenotype.
KeywordMeSH Terms
208. Wu  H, Kosaka  H, Kato  J, Kuroda  A, Ikeda  T, Takiguchi  N, Ohtake  H,     ( 1999 )

Cloning and characterization of Pseudomonas putida genes encoding the phosphate-specific transport system.

Journal of bioscience and bioengineering 87 (3)
PMID : 16232467  :  
Abstract >>
The pstSCAB genes of Pseudomonas putida PRS2000, encoding the phosphate (Pi)-specific transport (Pst) system, were cloned. The pstS gene of Pseudomonas aeruginosa PAO1, of which the pstCAB genes had been cloned previously, was also cloned (Nikata, T. et al., Mol. Gen. Genet., 250, 692-698, 1996). The predicted translation products of the P. putida pstSCAB genes showed 83, 75, 78 and 88% amino acid identity with their P. aeruginosa counterparts. Two well-conserved Pho box sequences were found in the region upstream of the pstS gene (15/18 base identity with the consensus Pho box sequence) and in the intercistronic region between the pstS and pstC genes (11/18 base identity) of P. putida PRS2000. To investigate the functions of PstSCAB, the pstSC genes were inactivated by inserting a kanamycin resistance gene cassette into the chromosome of P. putida PRS2000. The resultant mutant, designated PNT1, failed to take up 32Pi even under conditions of Pi limitation. Strain PNT1 was also constitutive for alkaline phosphatase synthesis, as well as chemotaxis toward Pi, indicating that the Pst system is involved in the negative regulation of the pho regulon in P. putida. Although overexpression of the pstSCAB genes in P. putida PRS2000 resulted in decreased cell growth, this recombinant strain could remove Pi at a rate similar to that seen with the control strain.
KeywordMeSH Terms
209. Yonaha  K, Nishie  M, Aibara  S,     ( 1992 )

The primary structure of omega-amino acid:pyruvate aminotransferase.

The Journal of biological chemistry 267 (18)
PMID : 1618757  :  
Abstract >>
The complete amino acid sequence of bacterial omega-amino acid:pyruvate aminotransferase (omega-APT) was determined from its primary structure. The enzyme protein was fragmented by CNBr cleavage, trypsin, and Staphylococcus aureus V8 digestions. The peptides were purified and sequenced by Edman degradation. omega-ATP is composed of four identical subunits of 449 amino acids each. The calculated molecular weight of the enzyme subunit is 48,738 and that of the enzyme tetramer is 194,952. No disulfide bonds or bound sugar molecules were found in the enzyme structure, although 6 cysteine residues were determined per enzyme subunit. Sequence homologies were found between an omega-aminotransferase, i.e. mammalian and yeast ornithine delta-aminotransferases, fungal gamma-aminobutyrate aminotransferase and 7,8-diaminoperalgonate aminotransferase, and 2,2-dialkylglycine decarboxylase. The enzyme structure is not homologous to those of aspartate aminotransferases (AspATs) including the enzymes of Escherichia coli and Sufolobus salfactaricus, though significant homology in the three-dimensional structures around the cofactor binding site has been found between omega-APT and AspATs (Watanabe, N., Sakabe, K., Sakabe, N., Higashi, T., Sasaki, K., Aibara, S., Morita, Y., Yonaha, K., Toyama, S., and Fukutani, H. (1989) J. Biochem. 105, 1-3).
KeywordMeSH Terms
210. Earhart  CA, Vetting  MW, Gosu  R, Michaud-Soret  I, Que  L, Ohlendorf  DH,     ( 2005 )

Structure of catechol 1,2-dioxygenase from Pseudomonas arvilla.

Biochemical and biophysical research communications 338 (1)
PMID : 16171781  :   DOI  :   10.1016/j.bbrc.2005.08.221    
Abstract >>
Catechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the betabeta isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429-440.] that the catechol 1,2-dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the alpha and beta polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (alphaalpha, alphabeta, and betabeta) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203-212) is proposed to be central in communicating between the lipid binding site and the active site.
KeywordMeSH Terms
211. Block  DW, Lingens  F,     ( 1992 )

Microbial metabolism of quinoline and related compounds. XIII. Purification and properties of 1H-4-oxoquinoline monooxygenase from Pseudomonas putida strain 33/1.

Biological chemistry Hoppe-Seyler 373 (5)
PMID : 1627263  :  
Abstract >>
1H-4-Oxoquinoline monooxygenase was purified to homogeneity from Pseudomonas putida strain 33/1 which can use 1H-4-oxoquinoline as sole source of carbon and energy. The apparent M(r) of the native enzyme was determined to be 126,000 by gel chromatography. SDS polyacrylamide gel electrophoresis of the enzyme revealed one protein band corresponding to M(r) 42,000. The enzyme consists of three probably identical subunits with a relative molecular mass of about 42,000. The enzyme requires oxygen and NADH for the reaction and is significantly inhibited by metal ions like Cu2+, Zn2+, Hg2+. The enzyme is specific only for 1H-4-oxoquinoline and the Km values of the enzyme for NADH and 1H-4-oxoquinoline were determined to be 87 microM and 25 microM, respectively.
KeywordMeSH Terms
212. Mihara  H, Muramatsu  H, Kakutani  R, Yasuda  M, Ueda  M, Kurihara  T, Esaki  N,     ( 2005 )

N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily.

The FEBS journal 272 (5)
PMID : 15720386  :   DOI  :   10.1111/j.1742-4658.2004.04541.x    
Abstract >>
We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.
KeywordMeSH Terms
213. Sevrioukova  IF,     ( 2005 )

Redox-dependent structural reorganization in putidaredoxin, a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida.

Journal of molecular biology 347 (3)
PMID : 15755454  :   DOI  :   10.1016/j.jmb.2005.01.047    
Abstract >>
Putidaredoxin (Pdx), a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida, transfers electrons from NADH-putidaredoxin reductase to cytochrome P450cam. Pdx exhibits redox-dependent binding affinities for P450cam and is thought to play an effector role in the monooxygenase reaction catalyzed by this hemoprotein. To understand how the reduced form of Pdx is stabilized and how reduction of the [2Fe-2S] cluster affects molecular properties of the iron-sulfur protein, crystal structures of reduced C73S and C73S/C85S Pdx were solved to 1.45 angstroms and 1.84 angstroms resolution, respectively, and compared to the corresponding 2.0 angstroms and 2.03 angstroms X-ray models of the oxidized mutants. To prevent photoreduction, the latter models were determined using in-house radiation source and the X-ray dose received by Pdx crystals was significantly decreased. Structural analysis showed that in reduced Pdx the Cys45-Ala46 peptide bond flip initiates readjustment of hydrogen bonding interactions between the [2Fe-2S] cluster, the Sgamma atoms of the cysteinyl ligands, and the backbone amide nitrogen atoms that results in tightening of the Cys39-Cys48 metal cluster binding loop around the prosthetic group and shifting of the metal center toward the Cys45-Thr47 peptide. From the metal center binding loop, the redox changes are transmitted to the linked Ile32-Asp38 peptide triggering structural rearrangement between the Tyr33-Asp34, Ser7-Asp9 and Pro102-Asp103 fragments of Pdx. The newly established hydrogen bonding interactions between Ser7, Asp9, Tyr33, Asp34, and Pro102, in turn, not only stabilize the tightened conformation of the [2Fe-2S] cluster binding loop but also assist in formation of a specific structural patch on the surface of Pdx that can be recognized by P450cam. This redox-linked change in surface properties is likely to be responsible for different binding affinity of oxidized and reduced Pdx to the hemoprotein.
KeywordMeSH Terms
Protein Structure, Tertiary
214. Kuznetsov  VY, Blair  E, Farmer  PJ, Poulos  TL, Pifferitti  A, Sevrioukova  IF,     ( 2005 )

The putidaredoxin reductase-putidaredoxin electron transfer complex: theoretical and experimental studies.

The Journal of biological chemistry 280 (16)
PMID : 15716266  :   DOI  :   10.1074/jbc.M500771200    
Abstract >>
Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 A. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp(38) and Trp(106), and at the edge of the protein-protein interface, Tyr(33) and Arg(66), were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Delta106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Delta106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp(106) and Tyr(33)/Arg(66) substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr(33), Arg(66), and Trp(106) prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp(106) but are likely to include Asp(38) and Cys(39).
KeywordMeSH Terms
215. Dubern  JF, Lagendijk  EL, Lugtenberg  BJ, Bloemberg  GV,     ( 2005 )

The heat shock genes dnaK, dnaJ, and grpE are involved in regulation of putisolvin biosynthesis in Pseudomonas putida PCL1445.

Journal of bacteriology 187 (17)
PMID : 16109938  :   DOI  :   10.1128/JB.187.17.5967-5976.2005     PMC  :   PMC1196155    
Abstract >>
Pseudomonas putida PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn5luxAB library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5luxAB flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a dnaK homologue which is located downstream of grpE and upstream of dnaJ. Analysis of putisolvin production and expression studies indicate that dnaK, together with the dnaJ and grpE heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires dnaK and dnaJ but not grpE. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that dnaK is positively regulated by GacA/GacS.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
216. Nölting  B, Jung  C, Snatzke  G,     ( 1992 )

Multichannel circular dichroism investigations of the structural stability of bacterial cytochrome P-450.

Biochimica et biophysica acta 1100 (2)
PMID : 1610873  :   DOI  :   10.1016/0005-2728(92)90078-g    
Abstract >>
The thermal unfolding of cytochrome P-450 LIN and P-450 CAM measured in presence and absence of their specific substrates was analyzed by circular dichroism (CD) and the alpha-helix content was estimated. Both proteins show, independent of the presence or absence of the substrates, nearly the same amount of loss of the CD in the peptide region. The comparison of the half transition temperatures determined from different chromophores and different methods indicates a non-two-state transition of the thermal unfolding. For such analysis we developed a new spectrometer, which is capable of measuring the CD simultaneously at all wavelengths in a limited wavelength region.
KeywordMeSH Terms
217. Segura  A, Godoy  P, van Dillewijn  P, Hurtado  A, Arroyo  N, Santacruz  S, Ramos  JL,     ( 2005 )

Proteomic analysis reveals the participation of energy- and stress-related proteins in the response of Pseudomonas putida DOT-T1E to toluene.

Journal of bacteriology 187 (17)
PMID : 16109935  :   DOI  :   10.1128/JB.187.17.5937-5945.2005     PMC  :   PMC1196166    
Abstract >>
Pseudomonas putida DOT-T1E is tolerant to toluene and other toxic hydrocarbons through extrusion of the toxic compounds from the cell by means of three efflux pumps, TtgABC, TtgDEF, and TtgGHI. To identify other cellular factors that allow the growth of P. putida DOT-T1E in the presence of high concentrations of toluene, we performed two-dimensional gel analyses of proteins extracted from cultures grown on glucose in the presence and in the absence of the organic solvent. From a total of 531 spots, 134 proteins were observed to be toluene specific. In the absence of toluene, 525 spots were clearly separated and 117 proteins were only present in this condition. Moreover, 35 proteins were induced by at least twofold in the presence of toluene whereas 26 were repressed by at least twofold under these conditions. We reasoned that proteins that were highly induced could play a role in toluene tolerance. These proteins, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, were classified into four categories: 1, proteins involved in the catabolism of toluene; 2, proteins involved in the channeling of metabolic intermediates to the Krebs cycle and activation of purine biosynthesis; 3, proteins involved in sugar transport; 4, stress-related proteins. The set of proteins in groups 2 and 3 suggests that the high energy demand required for solvent tolerance is achieved via activation of cell metabolism. The role of chaperones that facilitate the proper folding of newly synthesized proteins under toluene stress conditions was analyzed in further detail. Knockout mutants revealed that CspA, XenA, and Tuf-1 play a role in solvent tolerance in Pseudomonas, although this role is probably not specific to toluene, as indicated by the fact that all mutants grew more slowly than the wild type without toluene.
KeywordMeSH Terms
Proteome
218. Sun  YC, Chen  YC, Tian  ZX, Li  FM, Wang  XY, Zhang  J, Xiao  ZL, Lin  M, Gilmartin  N, Dowling  DN, Wang  YP,     ( 2005 )

Novel AroA with high tolerance to glyphosate, encoded by a gene of Pseudomonas putida 4G-1 isolated from an extremely polluted environment in China.

Applied and environmental microbiology 71 (8)
PMID : 16085874  :   DOI  :   10.1128/AEM.71.8.4771-4776.2005     PMC  :   PMC1183292    
Abstract >>
Glyphosate has been used globally as a safe herbicide for weed control. It inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA), which is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. A Pseudomonas putida strain, 4G-1, was isolated from a soil heavily contaminated by glyphosate in China. Its AroA-encoding gene (aroA) has been cloned, sequenced, and expressed in Escherichia coli. Phylogenetic analysis revealed that this AroA belongs neither to class I nor to class II AroA enzymes. When compared with E. coli AroA, 4G-1 AroA shows similar values for K(m)[PEP], K(m)[S3P], and specific enzyme activity. Moreover, 4G-1 AroA exhibits high tolerance to glyphosate, which indicates a protein with a high potential for structural and functional studies of AroA in general and its potential usage for the generation of transgenic crops resistant to the herbicide.
KeywordMeSH Terms
Drug Resistance, Bacterial
Soil Microbiology
219. Misono  H, Maeda  H, Tuda  K, Ueshima  S, Miyazaki  N, Nagata  S,     ( 2005 )

Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24-1.

Applied and environmental microbiology 71 (8)
PMID : 16085854  :   DOI  :   10.1128/AEM.71.8.4602-4609.2005     PMC  :   PMC1183316    
Abstract >>
An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.
KeywordMeSH Terms
Aldehyde-Lyases
220. Toyama  H, Chen  ZW, Fukumoto  M, Adachi  O, Matsushita  K, Mathews  FS,     ( 2005 )

Molecular cloning and structural analysis of quinohemoprotein alcohol dehydrogenase ADH-IIG from Pseudomonas putida HK5.

Journal of molecular biology 352 (1)
PMID : 16061256  :   DOI  :   10.1016/j.jmb.2005.06.078    
Abstract >>
Depending on the alcohols used as growth substrates, Pseudomonas putida HK5 produces two distinct quinohemoprotein alcohol dehydrogenases, ADH-IIB and ADH-IIG, both of which contain pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups but show different substrate specificities, especially for diol substrates. Molecular cloning of the gene of ADH-IIB and its crystal structure are already reported. Here, molecular cloning of the gene, qgdA, and solution of the three-dimensional structure of ADH-IIG are reported. The enzyme consists of 718 amino acid residues including a signal sequence of 29 amino acid residues. The PQQ domain is highly homologous to other quinoproteins, especially to quinohemoproteins. The crystal structure of ADH-IIG, determined at 2.2A resolution, shows that the overall structure and the amino acid residues involved in PQQ binding are quite similar to ADH-IIB and to another quinohemoprotein ADH, qhEDH from Comamonas testosteroni. However, the lengths of the linker regions connecting the PQQ and the cytochrome domains are different from each other, leading to a significant difference in orientation of the cytochrome domain with respect to the PQQ domain. Apart from ADH-IIB and qhEDH, ADH-IIG has an extra 12-residue helix within loop 3 in the PQQ domain and an extra 3(10) helix in the C terminus of the cytochrome domain, and both helices appear parallel and linked by a hydrogen bond. The amino acid residues contacting substrate/product in the crystal structures are also different among them. In the crystal structure of ADH-IIG with 1,2-propanediol, one of the hydroxyl groups of the substrate forms a hydrogen bond with O5 of PQQ and OD1 of Asp300, and the other interacts with a water molecule and with NE2 of Trp386, the corresponding residue of which is not found in ADH-IIB and qhEDH, and might be the residue responsible for making ADH-IIG prefer diol substrates.
KeywordMeSH Terms
Protein Structure, Tertiary
221. Kim  JY, Kim  JK, Lee  SO, Kim  CK, Lee  K,     ( 2005 )

Multicomponent phenol hydroxylase-catalysed formation of hydroxyindoles and dyestuffs from indole and its derivatives.

Letters in applied microbiology 41 (2)
PMID : 16033515  :   DOI  :   10.1111/j.1472-765X.2005.01734.x    
Abstract >>
To establish multicomponent phenol hydroxylases (mPHs) as novel biocatalysts for producing dyestuffs and hydroxyindoles such as 7-hydroxyindole (7-HI) from indole and its derivatives. We have isolated Pseudomonas sp. KL33, which possesses a phenol degradation pathway similar to that found in Pseudomonas sp. CF600. Pseudomonas sp. KL28 is a strain that can grow on n-alkylphenols as a carbon and energy source. Escherichia coli strains expressing mPH from strain KL28 (mPH(KL28)) and strain KL33 (mPH(KL33)) catalysed the formation of indigo and 7-HI, respectively, from indole. In addition, both mPHs catalysed the production of dyestuffs and hydroxyindoles from indole derivatives. The mPH(KL28) has proved to be one of the most versatile biocatalysts that can accommodate a wide range of indole derivatives for catalysing the formation of dyestuffs. The present work provides a new approach in producing various dyestuffs and hydroxyindoles from indole and its derivatives by mPHs. These results indicate that mPHs may serve as potential agents for organic syntheses as well as bioremediation.
KeywordMeSH Terms
222. Endoh  T, Habe  H, Nojiri  H, Yamane  H, Omori  T,     ( 2005 )

The sigma54-dependent transcriptional activator SfnR regulates the expression of the Pseudomonas putida sfnFG operon responsible for dimethyl sulphone utilization.

Molecular microbiology 55 (3)
PMID : 15661012  :   DOI  :   10.1111/j.1365-2958.2004.04431.x    
Abstract >>
Pseudomonas putida DS1 is able to utilize dimethyl sulphide through dimethyl sulphoxide, dimethyl sulphone (DMSO2), methanesulphonate (MSA) and sulphite as a sulphur source. We previously demonstrated that sfnR encoding a sigma54-dependent transcriptional regulator is essential for DMSO2 utilization by P. putida DS1. To identify the target genes of SfnR, we carried out transposon mutagenesis on an sfnR disruptant (DMSO2-utilization-defective phenotype) using mini-Tn5, which contains two outward-facing constitutively active promoters; as a result, we obtained a mutant that restored the ability to utilize DMSO2. The DMSO2-positive mutant carried a mini-Tn5 insertion in the intergenic region between two opposite-facing operons, sfnAB and sfnFG. Both sfnA and sfnB products were similar to acyl-CoA dehydrogenase family proteins, whereas sfnF and sfnG encoded a putative NADH-dependent FMN reductase (SfnF) and an FMNH2-dependent monooxygenase (SfnG). Disruption and complementation of the sfn genes indicated that the sfnG product is essential for DMSO2 utilization by P. putida DS1. Furthermore, an enzyme assay demonstrated that SfnG is an FMNH2-dependent DMSO2 monooxygenase that converts DMSO2 to MSA. It was revealed that the expression of the sfnFG operon is directly activated by the binding of SfnR at its upstream region. Site-directed mutagenesis of the SfnR binding sequences allowed us to define a potential recognition sequence for SfnR. These results provided insight into regulation of sulphate starvation-induced genes in bacteria.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
223. O'Leary  ND, O'Connor  KE, Ward  P, Goff  M, Dobson  AD,     ( 2005 )

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3.

Applied and environmental microbiology 71 (8)
PMID : 16085828  :   DOI  :   10.1128/AEM.71.8.4380-4387.2005     PMC  :   PMC1183336    
Abstract >>
Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
224. Choy  WK, Zhou  L, Syn  CK, Zhang  LH, Swarup  S,     ( 2004 )

MorA defines a new class of regulators affecting flagellar development and biofilm formation in diverse Pseudomonas species.

Journal of bacteriology 186 (21)
PMID : 15489433  :   DOI  :   10.1128/JB.186.21.7221-7228.2004     PMC  :   PMC523210    
Abstract >>
Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in Pseudomonas putida. MorA is conserved among diverse Pseudomonas species, and homologues are present in all Pseudomonas genomes sequenced thus far. In P. putida, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In Pseudomonas aeruginosa, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of P. putida, the motility of the P. aeruginosa mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of Pseudomonas species.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
225. Caballero  A, Esteve-Núñez  A, Zylstra  GJ, Ramos  JL,     ( 2005 )

Assimilation of nitrogen from nitrite and trinitrotoluene in Pseudomonas putida JLR11.

Journal of bacteriology 187 (1)
PMID : 15601726  :   DOI  :   10.1128/JB.187.1.396-399.2005     PMC  :   PMC538816    
Abstract >>
Pseudomonas putida JLR11 releases nitrogen from the 2,4,6-trinitrotoluene (TNT) ring as nitrite or ammonium. These processes can occur simultaneously, as shown by the observation that a nasB mutant impaired in the reduction of nitrite to ammonium grew at a slower rate than the parental strain. Nitrogen from TNT is assimilated via the glutamine syntethase-glutamate synthase (GS-GOGAT) pathway, as evidenced by the inability of GOGAT mutants to use TNT. This pathway is also used to assimilate ammonium from reduced nitrate and nitrite. Three mutants that had insertions in ntrC, nasT, and cnmA, which encode regulatory proteins, failed to grow on nitrite but grew on TNT, although slower than the wild type.
KeywordMeSH Terms
226. Ogasawara  N, Yoshikawa  H,     ( 1992 )

Genes and their organization in the replication origin region of the bacterial chromosome.

Molecular microbiology 6 (5)
PMID : 1552862  :   DOI  :   10.1111/j.1365-2958.1992.tb01510.x    
Abstract >>
Genes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram-positive and Gram-negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram-positive bacteria were diverged from the Gram-negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non-translatable region containing multiple repeats of DnaA-box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.
KeywordMeSH Terms
Chromosomes, Bacterial
227. Müller  I, Stückl  C, Wakeley  J, Kertesz  M, Usón  I,     ( 2005 )

Succinate complex crystal structures of the alpha-ketoglutarate-dependent dioxygenase AtsK: steric aspects of enzyme self-hydroxylation.

The Journal of biological chemistry 280 (7)
PMID : 15542595  :   DOI  :   10.1074/jbc.M410840200    
Abstract >>
The alkylsulfatase AtsK from Pseudomonas putida S-313 is a member of the non-heme iron(II)-alpha-ketoglutarate-dependent dioxygenase superfamily. In the initial step of their catalytic cycle, enzymes belonging to this widespread and versatile family coordinate molecular oxygen to the iron center in the active site. The subsequent decarboxylation of the cosubstrate alpha-ketoglutarate yields carbon dioxide, succinate, and a highly reactive ferryl (IV) species, which is required for substrate oxidation via a complex mechanism involving the transfer of radical species. Non-productive activation of oxygen may lead to harmful side reactions; therefore, such enzymes need an effective built-in protection mechanism. One of the ways of controlling undesired side reactions is the self-hydroxylation of an aromatic side chain, which leads to an irreversibly inactivated species. Here we describe the crystal structure of the alkylsulfatase AtsK in complexes with succinate and with Fe(II)/succinate. In the crystal structure of the AtsK-Fe(II)-succinate complex, the side chain of Tyr(168) is co-ordinated to the iron, suggesting that Tyr(168) is the target of enzyme self-hydroxylation. This is the first structural study of an Fe(II)-alpha-ketoglutarate-dependent dioxygenase that presents an aromatic side chain coordinated to the metal center, thus allowing structural insight into this protective mechanism of enzyme self-inactivation.
KeywordMeSH Terms
228. Barth  PT, Bolton  L, Thomson  JC,     ( 1992 )

Cloning and partial sequencing of an operon encoding two Pseudomonas putida haloalkanoate dehalogenases of opposite stereospecificity.

Journal of bacteriology 174 (8)
PMID : 1556080  :   DOI  :   10.1128/jb.174.8.2612-2619.1992     PMC  :   PMC205901    
Abstract >>
We have cloned fragments of DNA (up to 13 kb), from Pseudomonas putida AJ1, that code for two stereospecific haloalkanoate dehalogenases. These enzymes are highly specific for D and L substrates. The two genes, designated hadD and hadL, have been isolated and independently expressed in Escherichia coli and P. putida hosts by using broad-host-range vectors. They are closely adjacent and inducible in what appears to be an operon with an upstream open reading frame of unknown function. Nucleotide sequence determination of hadD predicts a mature, cytoplasmic protein of 300 amino acid residues (molecular weight of 33,601). This has no significant homology with the L-specific haloalkanoate dehalogenases from Pseudomonas sp. strain CBS3 (B. Schneider, R. Muller, R. Frank, and F. Lingens, J. Bacteriol. 173:1530-1535, 1991) nor with any other known DNA or protein sequences.
KeywordMeSH Terms
229. Muramatsu  H, Mihara  H, Kakutani  R, Yasuda  M, Ueda  M, Kurihara  T, Esaki  N,     ( 2005 )

The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline.

The Journal of biological chemistry 280 (7)
PMID : 15561717  :   DOI  :   10.1074/jbc.M411918200    
Abstract >>
A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as Delta(1)-piperideine-2-carboxylate and Delta(1)-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme Delta(1)-piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.
KeywordMeSH Terms
230. Hõrak  R, Ilves  H, Pruunsild  P, Kuljus  M, Kivisaar  M,     ( 2004 )

The ColR-ColS two-component signal transduction system is involved in regulation of Tn4652 transposition in Pseudomonas putida under starvation conditions.

Molecular microbiology 54 (3)
PMID : 15491368  :   DOI  :   10.1111/j.1365-2958.2004.04311.x    
Abstract >>
Bacteria use two-component signal transduction pathways to sense both extracellular and intracellular environment and to coordinate cellular events according to changing conditions. Adaptation can be either physiological or genetical. Here, we present evidence that a genome reorganization process such as transposition can be controlled by certain environmental cues sensed by a two-component signal transduction system. We demonstrate that transposition-dependent accumulation of phenol-utilizing mutants is severely decreased in Pseudomonas putida defective in a two-component system colRS. Translocation of Tn4652 is decreased both in colR- and colS-defective strains, indicating that signal transduction from a histidine kinase ColS to a response regulator ColR is necessary for the activation of Tn4652 in bacteria starving on phenol. However, overexpression of ColR in a colS-defective strain restores Tn4652 transposition, suggesting that absence of the signal from ColS can be compensated by an elevated amount of ColR. In vitro analysis of purified ColR and ColS proteins evidenced that they constitute a functional phosphorelay. Site-directed mutagenesis revealed that a conserved H221 can be the phosphoryl-accepting residue in ColS and that aspartate residues D8 and D51 of ColR are necessary for the phosphotransfer from ColS to ColR. To our knowledge, Tn4652 is the first bacterial transposon regulated by a two-component system. This finding indicates that transpositional activity can respond to signals sensed and processed by the host.
KeywordMeSH Terms
DNA Transposable Elements
231. Bertani  I, Venturi  V,     ( 2004 )

Regulation of the N-acyl homoserine lactone-dependent quorum-sensing system in rhizosphere Pseudomonas putida WCS358 and cross-talk with the stationary-phase RpoS sigma factor and the global regulator GacA.

Applied and environmental microbiology 70 (9)
PMID : 15345437  :   DOI  :   10.1128/AEM.70.9.5493-5502.2004     PMC  :   PMC520884    
Abstract >>
Quorum sensing is a cell population-density dependent regulatory system which in gram-negative bacteria often involves the production and detection of N-acyl homoserine lactones (AHLs). Some Pseudomonas putida strains have been reported to produce AHLs, and one quorum-sensing locus has been identified. However, it appears that the majority of strains do not produce AHLs. In this study we report the identification and regulation of the AHL-dependent system of rhizosphere P. putida WCS358. This system is identical to the recently identified system of P. putida strain IsoF and very similar to the las system of Pseudomonas aeruginosa. It is composed of three genes, the luxI family member ppuI, the putative repressor rsaL, and the luxR family member ppuR. A genomic ppuR::Tn5 mutant of strain WCS358 was identified by its inability to produce AHLs when it was cross-streaked in close proximity to an AHL biosensor, whereas an rsaL::Tn5 genomic mutant was identified by its ability to overproduce AHL molecules. Using transcriptional promoter fusions, we studied expression profiles of the rsaL, ppuI, and ppuR promoters in various genetic backgrounds. At the onset of the stationary phase, the autoinducer synthase ppuI gene expression is under positive regulation by PpuR-AHL and under negative regulation by RsaL, indicating that the molecules could be in competition for binding at the ppuI promoter. In genomic rsaL::Tn5 mutants ppuI expression and production of AHL levels increased dramatically; however, both processes were still under growth phase regulation, indicating that RsaL is not involved in repressing AHL production at low cell densities. The roles of the global response regulator GacA and the stationary-phase sigma factor RpoS in the regulation of the AHL system at the onset of the stationary phase were also investigated. The P. putida WCS358 gacA gene was cloned and inactivated in the genome. It was determined that the three global regulatory systems are closely linked, with quorum sensing and RpoS regulating each other and GacA positively regulating ppuI expression. Studies of the regulation of AHL quorum-sensing systems have lagged behind other studies and are important for understanding how these systems are integrated into the overall growth phase and metabolic status of the cells.
KeywordMeSH Terms
232. Sandler  SJ, Chackerian  B, Li  JT, Clark  AJ,     ( 1992 )

Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.

Nucleic acids research 20 (4)
PMID : 1542576  :   DOI  :   10.1093/nar/20.4.839     PMC  :   PMC312026    
Abstract >>
We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92% and 38% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.
KeywordMeSH Terms
Escherichia coli Proteins
233. Duque  E, García  V, de la Torre  J, Godoy  P, Bernal  P, Ramos  JL,     ( 2004 )

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida.

Environmental microbiology 6 (10)
PMID : 15344927  :   DOI  :   10.1111/j.1462-2920.2004.00621.x    
Abstract >>
The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.
KeywordMeSH Terms
234. Hontzeas  N, Zoidakis  J, Glick  BR, Abu-Omar  MM,     ( 2004 )

Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion.

Biochimica et biophysica acta 1703 (1)
PMID : 15588698  :   DOI  :   10.1016/j.bbapap.2004.09.015    
Abstract >>
The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to alpha-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity and high activity towards the substrate ACC: K(M)=3.4+/-0.2 mM and k(cat)=146+/-5 min(-1) at pH 8.0 and 22 degrees C. The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10. The major component of the enzyme's secondary structure was determined to be alpha-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 degrees C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site. This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared.
KeywordMeSH Terms
235. Yang  TH, Pan  JG, Seo  YS, Rhee  JS,     ( 2004 )

Use of Pseudomonas putida EstA as an anchoring motif for display of a periplasmic enzyme on the surface of Escherichia coli.

Applied and environmental microbiology 70 (12)
PMID : 15574889  :   DOI  :   10.1128/AEM.70.12.6968-6976.2004     PMC  :   PMC535197    
Abstract >>
The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme beta-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.
KeywordMeSH Terms
236. Rezzonico  F, Défago  G, Moënne-Loccoz  Y,     ( 2004 )

Comparison of ATPase-encoding type III secretion system hrcN genes in biocontrol fluorescent Pseudomonads and in phytopathogenic proteobacteria.

Applied and environmental microbiology 70 (9)
PMID : 15345390  :   DOI  :   10.1128/AEM.70.9.5119-5131.2004     PMC  :   PMC520869    
Abstract >>
Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.
KeywordMeSH Terms
237. Ye  XM, Pochapsky  TC, Pochapsky  SS,     ( 1992 )

1H NMR sequential assignments and identification of secondary structural elements in oxidized putidaredoxin, an electron-transfer protein from Pseudomonas.

Biochemistry 31 (7)
PMID : 1536837  :   DOI  :   10.1021/bi00122a009    
Abstract >>
Sequential 1H resonance assignments and secondary structural features of putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, are reported. No crystal structure has been obtained for Pdx or for any closely homologous protein. The sequentially assigned resonances represent ca. 83% of all the protons in Pdx and a large majority of those protons which are unaffected by the paramagnetism of the iron-sulfur cluster. A total of three alpha-helices, two beta-sheets, and two type I beta-turns have been identified from NOE (nuclear Overhauser effect) patterns. Besides the extensive beta-sheet described previously, a second sheet is identified, consisting of two short antiparallel strands (Ile 89-Thr 91 and Val 21-Leu 23), one of which ends in a tight type I turn (Thr 91-Pro 92-Glu 93-Leu 94). One short helix (Ala 26-Gly 31) and a second longer helical region (Glu 54-Cys 73) are present. This second helical region is discontinuous, breaking at Pro 61, resuming at Glu 65, and ending at Cys 73. The functionally important C-terminal tryptophan residue has been identified, and some structural constraints on this residue are described. Previously reported functional data concerning Pdx are discussed in light of present structural information. Finally, approaches to the determination of a high-resolution solution structure of the protein are discussed.
KeywordMeSH Terms
238. Bonin  I, Martins  BM, Purvanov  V, Fetzner  S, Huber  R, Dobbek  H,     ( 2004 )

Active site geometry and substrate recognition of the molybdenum hydroxylase quinoline 2-oxidoreductase.

Structure (London, England : 1993) 12 (8)
PMID : 15296736  :   DOI  :   10.1016/j.str.2004.05.014    
Abstract >>
The soil bacterium Pseudomonas putida 86 uses quinoline as a sole source of carbon and energy. Quinoline 2-oxidoreductase (Qor) catalyzes the first metabolic step converting quinoline to 2-oxo-1,2-dihydroquinoline. Qor is a member of the molybdenum hydroxylases. The molybdenum ion is coordinated by two ene-dithiolate sulfur atoms, two oxo-ligands, and a catalytically crucial sulfido-ligand, whose position in the active site was controversial. The 1.8 A resolution crystal structure of Qor indicates that the sulfido-ligand occupies the equatorial position at the molybdenum ion. The structural comparison of Qor with the allopurinol-inhibited xanthine dehydrogenase from Rhodobacter capsulatus allows direct insight into the mechanism of substrate recognition and the identification of putative catalytic residues. The active site protein variants QorE743V and QorE743D were analyzed to assess the catalytic role of E743.
KeywordMeSH Terms
239. Muryoi  N, Sato  M, Kaneko  S, Kawahara  H, Obata  H, Yaish  MW, Griffith  M, Glick  BR,     ( 2004 )

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2.

Journal of bacteriology 186 (17)
PMID : 15317770  :   DOI  :   10.1128/JB.186.17.5661-5671.2004     PMC  :   PMC516810    
Abstract >>
The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
240. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
241. Koh  TH, Wang  GC, Sng  LH,     ( 2004 )

IMP-1 and a novel metallo-beta-lactamase, VIM-6, in fluorescent pseudomonads isolated in Singapore.

Antimicrobial agents and chemotherapy 48 (6)
PMID : 15155248  :   DOI  :   10.1128/AAC.48.6.2334-2336.2004     PMC  :   PMC415586    
Abstract >>
Four carbapenem-resistant Pseudomonas spp. were isolated from patients in Singapore. One Pseudomonas putida isolate contained a bla(IMP-1) identical to that first described in Japan. The sequence of a variant bla(IMP-1) in Pseudomonas fluorescens contained four silent mutations compared with the original sequence. The remaining P. putida isolates contained bla(VIM-6), a novel VIM gene variant.
KeywordMeSH Terms
242. Park  W, Madsen  EL,     ( 2004 )

Characterization in Pseudomonas putida Cg1 of nahR and its role in bacterial survival in soil.

Applied microbiology and biotechnology 66 (2)
PMID : 15278309  :   DOI  :   10.1007/s00253-004-1630-6    
Abstract >>
Sequencing, RFLP analyses and experiments utilizing a lacZ transcriptional reporter fused to the promoter regions of nahR and nahG in Pseudomonas putida Cg1 confirmed that regulation of naphthalene degradation in both P. putida Cg1 and the type strain, P. putida NCIB 9816-4, is consistent with that of NAH7 from P. putida G7. Two nahR knockout strains (RK1 and Cg1-NAHR from P. putida NCIB 9816-4 and Cg1, respectively) showed a growth defect in the presence of naphthalene as sole carbon and energy source. We hypothesized that nahR influences ecological fitness of bacteria in naphthalene-contaminated soil and tested this hypothesis using both parent and nahR-knockout strains introduced to soil microcosms with and without added naphthalene. After 21 days, loss of cell viability was pronounced in the presence of added naphthalene crystals for nahR mutants of both test bacteria, relative to the wild types. Diminished viable counts were attributed to toxicity. Thus, our data indicated that NahR in P. putida Cg1 is virtually identical to its homologues in other pseudomonads and that nahR is required for resistance to naphthalene toxicity, hence the persistence of bacterial cells in soil with high concentrations of naphthalene.
KeywordMeSH Terms
243. Block  DW, Lingens  F,     ( 1992 )

Microbial metabolism of quinoline and related compounds. XIV. Purification and properties of 1H-3-hydroxy-4-oxoquinoline oxygenase, a new extradiol cleavage enzyme from Pseudomonas putida strain 33/1.

Biological chemistry Hoppe-Seyler 373 (6)
PMID : 1515060  :  
Abstract >>
1H-3-Hydroxy-4-oxoquinoline oxygenase was purified to apparent homogeneity from Pseudomonas putida strain 33/1 which can use 1H-4-oxoquinoline as sole source of carbon. The molecular mass of the enzyme was determined to 26,000 Da by gel chromatography and by SDS polyacrylamide gel electrophoresis. The enzyme is labile at temperatures above 30 degrees C and has a pH optimum of 8.0. It requires oxygen for the reaction and is significantly inhibited by metal ions like Cu2+, Zn2+, Hg2+ and by 4-chloromercuribenzoate. The enzyme is specific only for 1H-3-Hydroxy-4-oxoquinoline; the apparent Km value for this substrate is 24 microM.
KeywordMeSH Terms
244. Dennis  JJ, Zylstra  GJ,     ( 2004 )

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4.

Journal of molecular biology 341 (3)
PMID : 15288784  :   DOI  :   10.1016/j.jmb.2004.06.034    
Abstract >>
The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.
KeywordMeSH Terms
245. Ohnishi  K, Okuta  A, Ju  J, Hamada  T, Misono  H, Harayama  S,     ( 2004 )

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol.

Journal of biochemistry 135 (3)
PMID : 15113829  :   DOI  :   10.1093/jb/mvh037    
Abstract >>
3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.
KeywordMeSH Terms
DNA Shuffling
246. Arias-Barrau  E, Olivera  ER, Luengo  JM, Fernández  C, Galán  B, García  JL, Díaz  E, Miñambres  B,     ( 2004 )

The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida.

Journal of bacteriology 186 (15)
PMID : 15262943  :   DOI  :   10.1128/JB.186.15.5062-5077.2004     PMC  :   PMC451635    
Abstract >>
Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Multigene Family
247. Carl  B, Arnold  A, Hauer  B, Fetzner  S,     ( 2004 )

Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation.

Gene 331 (N/A)
PMID : 15094204  :   DOI  :   10.1016/j.gene.2004.02.020    
Abstract >>
Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet. A gene cluster involved in quinoline degradation was characterized. Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located. The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU). The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor. Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR. ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters. Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline. Insertional inactivation of oxoS abolished quinoline-induced transcription. However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS. The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor. Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR. Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM. The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.
KeywordMeSH Terms
248. Smith  N, Mayhew  M, Holden  MJ, Kelly  H, Robinson  H, Heroux  A, Vilker  VL, Gallagher  DT,     ( 2004 )

Structure of C73G putidaredoxin from Pseudomonas putida.

Acta crystallographica. Section D, Biological crystallography 60 (Pt 5)
PMID : 15103126  :   DOI  :   10.1107/S0907444904003348    
Abstract >>
The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein. The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s. The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d. < 0.3 A for the 106 C(alpha) atoms). The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts. The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants. In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution. The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.
KeywordMeSH Terms
249. Sevrioukova  IF, Li  H, Poulos  TL,     ( 2004 )

Crystal structure of putidaredoxin reductase from Pseudomonas putida, the final structural component of the cytochrome P450cam monooxygenase.

Journal of molecular biology 336 (4)
PMID : 15095867  :   DOI  :   10.1016/j.jmb.2003.12.067    
Abstract >>
The crystal structure of recombinant putidaredoxin reductase (Pdr), an FAD-containing NADH-dependent flavoprotein component of the cytochrome P450cam monooxygenase from Pseudomonas putida, has been determined to 1.90 A resolution. The protein has a fold similar to that of disulfide reductases and consists of the FAD-binding, NAD-binding, and C-terminal domains. Compared to homologous flavoenzymes, the reductase component of biphenyl dioxygenase (BphA4) and apoptosis-inducing factor, Pdr lacks one of the arginine residues that compensates partially for the negative charge on the pyrophosphate of FAD. This uncompensated negative charge is likely to decrease the electron-accepting ability of the flavin. The aromatic side-chain of the "gatekeeper" Tyr159 is in the "out" conformation and leaves the nicotinamide-binding site of Pdr completely open. The presence of electron density in the NAD-binding channel indicates that NAD originating from Escherichia coli is partially bound to Pdr. A structural comparison of Pdr with homologous flavoproteins indicates that an open and accessible nicotinamide-binding site, the presence of an acidic residue in the middle part of the NAD-binding channel that binds the nicotinamide ribose, and multiple positively charged arginine residues surrounding the entrance of the NAD-binding channel are the special structural elements that assist tighter and more specific binding of the oxidized pyridine nucleotide by the BphA4-like flavoproteins. The crystallographic model of Pdr explains differences in the electron transfer mechanism in the Pdr-putidaredoxin redox couple and their mammalian counterparts, adrenodoxin reductase and adrenodoxin.
KeywordMeSH Terms
Protein Structure, Tertiary
250. Hilario  E, Buckley  TR, Young  JM,     ( 2004 )

Improved resolution on the phylogenetic relationships among Pseudomonas by the combined analysis of atp D, car A, rec A and 16S rDNA.

Antonie van Leeuwenhoek 86 (1)
PMID : 15103237  :   DOI  :   10.1023/B:ANTO.0000024910.57117.16    
Abstract >>
A study of representatives of the bacterial genus Pseudomonas, analysing a combined data set of four molecular sequences with completely different properties and evolutionary constraints, is reported. The best evolutionary model was obtained with a hierarchical hypothesis testing program to describe each data set and the combined data set is presented and analysed under the likelihood criterion. The resolution among Pseudomonas taxa based on the combined data set analysis of the different lineages increased due to a synergistic effect of the individual data sets. The unresolved fluorescens lineage, as well as other weakly supported lineages in the single data set trees, should be revised in detail at the biochemical and molecular level. The taxonomic status of biovars of P. putida is discussed.
KeywordMeSH Terms
251. Gopalakrishna  KN, Stewart  BH, Kneen  MM, Andricopulo  AD, Kenyon  GL, McLeish  MJ,     ( 2004 )

Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family.

Biochemistry 43 (24)
PMID : 15196015  :   DOI  :   10.1021/bi049907q    
Abstract >>
A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.
KeywordMeSH Terms
252. Müller  I, Kahnert  A, Pape  T, Sheldrick  GM, Meyer-Klaucke  W, Dierks  T, Kertesz  M, Usón  I,     ( 2004 )

Crystal structure of the alkylsulfatase AtsK: insights into the catalytic mechanism of the Fe(II) alpha-ketoglutarate-dependent dioxygenase superfamily.

Biochemistry 43 (11)
PMID : 15023059  :   DOI  :   10.1021/bi035752v    
Abstract >>
The alkylsulfatase AtsK from Pseudomonas putida S-313 belongs to the widespread and versatile non-heme iron(II) alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes the oxygenolytic cleavage of a variety of different alkyl sulfate esters to the corresponding aldehyde and sulfate. The enzyme is only expressed under sulfur starvation conditions, providing a selective advantage for bacterial growth in soils and rhizosphere. Here we describe the crystal structure of AtsK in the apo form and in three complexes: with the cosubstrate alpha-ketoglutarate, with alpha-ketoglutarate and iron, and finally with alpha-ketoglutarate, iron, and an alkyl sulfate ester used as substrate in catalytic studies. The overall fold of the enzyme is closely related to that of the taurine/alpha-ketoglutarate dioxygenase TauD and is similar to the fold observed for other members of the enzyme superfamily. From comparison of these structures with the crystal structure of AtsK and its complexes, we propose a general mechanism for the catalytic cycle of the alpha-ketoglutarate-dependent dioxygenase superfamily.
KeywordMeSH Terms
253. Yoshimoto  T, Tanaka  N, Kanada  N, Inoue  T, Nakajima  Y, Haratake  M, Nakamura  KT, Xu  Y, Ito  K,     ( 2004 )

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism.

Journal of molecular biology 337 (2)
PMID : 15003455  :   DOI  :   10.1016/j.jmb.2004.01.022    
Abstract >>
Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.
KeywordMeSH Terms
254. Segura  A, Duque  E, Rojas  A, Godoy  P, Delgado  A, Hurtado  A, Cronan  J, Ramos  JL,     ( 2004 )

Fatty acid biosynthesis is involved in solvent tolerance in Pseudomonas putida DOT-T1E.

Environmental microbiology 6 (4)
PMID : 15008818  :  
Abstract >>
The unusual tolerance of Pseudomonas putida DOT-T1E to toluene is based on the extrusion of this solvent by constitutive and inducible efflux pumps and rigidification of its membranes via phospholipid alterations. Pseudomonas putida DOT-T1E-109 is a solvent-sensitive mutant. Mutant cells were less efficient in solvent extrusion than the wild-type cells, as shown by the limited efflux of 14C-1,2,4-trichlorobenzene from the cell membranes, despite the fact that the efflux pumps are overexpressed as a result of increased expression of the ttgDEF and ttgGHI efflux pump operons. This limitation could be the result of alterations in the outer membrane because the mutant cells released more beta-lactamase to the external medium than the wild-type cells. The mutant P. putida DOT-T1E-109 showed negligible synthesis of fatty acids in the presence of sublethal concentrations of toluene as revealed by analysis of 13CH3-13COOH incorporation into fatty acids. In contrast, the mutant strain in the absence of solvents, and the wild-type strain, both in the presence and in the absence of toluene, incorporated 13CH3-13COOH at a high rate into de novo synthesized lipids. The mutation in P. putida DOT-T1E-109 increases sensitivity to the solvent because of a limited efflux of the solvent from the cell membranes with the concomitant inhibition of fatty acid biosynthesis.
KeywordMeSH Terms
255. Lewis  TA, Leach  L, Morales  S, Austin  PR, Hartwell  HJ, Kaplan  B, Forker  C, Meyer  JM,     ( 2004 )

Physiological and molecular genetic evaluation of the dechlorination agent, pyridine-2,6-bis(monothiocarboxylic acid) (PDTC) as a secondary siderophore of Pseudomonas.

Environmental microbiology 6 (2)
PMID : 14756880  :  
Abstract >>
The bacterial metabolite and transition metal chelator pyridine-2,6-dithiocarboxylic acid (PDTC), promotes a novel and effective means of dechlorination of the toxic and carcinogenic pollutant, carbon tetrachloride. Pyridine-2,6-dithiocarboxylic acid has been presumed to act as a siderophore in the Pseudomonas strains known to produce it. To explore further the physiological function of PDTC production, we have examined its regulation, the phenotype of PDTC-negative (pdt) mutants, and envelope proteins associated with PDTC in P. putida strain DSM 3601. Aspects of the regulation of PDTC production and outer membrane protein composition were consistent with siderophore function. Pyridine-2,6-dithiocarboxylic acid production was coordinated with production of the well-characterized siderophore pyoverdine; exogenously added pyoverdine led to decreased PDTC production, and added PDTC led to decreased pyoverdine production. Positive regulation of a chromosomal pdtI-xylE transcriptional fusion, and of a 66 kDa outer membrane protein (IROMP), was seen in response to exogenous PDTC. Tests with transition metal chelators indicated that PDTC could provide a benefit under conditions of metal limitation; the loss of PDTC biosynthetic capacity caused by a pdtI transposon insertion resulted in increased sensitivity to 1,10-phenanthroline, a chelator that has high affinity for a range of divalent transition metals (e.g. Fe(2+), Cu(2+), Zn(2+)). Exogenously added PDTC could also suppress a phenotype of pyoverdine-negative (Pvd-) mutants, that of sensitivity to EDDHA, a chelator with higher affinity and specificity for Fe(3+). Measurement of 59Fe incorporation showed uptake from 59Fe:PDTC by DSM 3601 grown in low-iron medium, but not by cells grown in high iron medium, or by the pdtI mutant, which did not show expression of the 66 kDa envelope protein. These data verified a siderophore function for PDTC, and have implicated it in the uptake of transition metals in addition to iron.
KeywordMeSH Terms
Oligopeptides
256. Garriga  X, Calero  S, Barbé  J,     ( 1992 )

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Molecular & general genetics : MGG 236 (1)
PMID : 1494343  :   DOI  :   10.1007/bf00279651    
Abstract >>
The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 bp in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 bp in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84--Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.
KeywordMeSH Terms
Serine Endopeptidases
257. Syn  CK, Magnuson  JK, Kingsley  MT, Swarup  S,     ( 2004 )

Characterization of Pseudomonas putida genes responsive to nutrient limitation.

Microbiology (Reading, England) 150 (Pt 6)
PMID : 15184552  :   DOI  :   10.1099/mic.0.26657-0    
Abstract >>
The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation and biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high availability of nutrients and oxygen. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 were examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7 and NRM17, were selected for identification of the tagged genes. In strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased 4.9-26.4-fold under various low-nutrient conditions. In NRM7, expression of the novel NADPH : quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low-nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low-nutrient conditions but its absolute expression level was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in Escherichia coli.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Soil Microbiology
258. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
259. de Souza  JT, Mazzola  M, Raaijmakers  JM,     ( 2003 )

Conservation of the response regulator gene gacA in Pseudomonas species.

Environmental microbiology 5 (12)
PMID : 14641577  :  
Abstract >>
The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp. In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments. Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas. Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp. by group-specific primers. Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp. These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas. Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively. Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering. These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas.
KeywordMeSH Terms
Conserved Sequence
Genes, Regulator
260. Kuiper  I, Lagendijk  EL, Pickford  R, Derrick  JP, Lamers  GE, Thomas-Oates  JE, Lugtenberg  BJ, Bloemberg  GV,     ( 2004 )

Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms.

Molecular microbiology 51 (1)
PMID : 14651614  :   DOI  :   10.1046/j.1365-2958.2003.03751.x    
Abstract >>
Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.
KeywordMeSH Terms
261. Sukumar  N, Dewanti  AR, Mitra  B, Mathews  FS,     ( 2004 )

High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (S)-mandelate dehydrogenase.

The Journal of biological chemistry 279 (5)
PMID : 14604988  :   DOI  :   10.1074/jbc.M310049200    
Abstract >>
The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-A resolution. The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176-195 of glycolate oxidase from spinach. Both structures permit full tracing of the polypeptide backbone chain from residues 4-356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 A resolution. The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction. The only other structural changes within the protein interior are a 10 degrees rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 A. Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme. The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group. Implications for the low oxidase activity of the reduced enzyme are also discussed.
KeywordMeSH Terms
262. Tipton  PA, Peisach  J,     ( 1990 )

Characterization of the multiple catalytic activities of tartrate dehydrogenase.

Biochemistry 29 (7)
PMID : 2184888  :   DOI  :   10.1021/bi00459a013    
Abstract >>
Tartrate dehydrogenase (TDH) has been purified to apparent homogeneity from Pseudomonas putida and has been demonstrated to catalyze three different NAD(+)-dependent reactions. TDH catalyzes the oxidation of (+)-tartrate to form oxaloglycolate and the oxidative decarboxylation of D-malate to form pyruvate and CO2. D-Glycerate and CO2 are formed from meso-tartrate in a reaction that is formally a decarboxylation with no net oxidation or reduction. The steady-state kinetics of the first two reactions have been investigated and found to follow primarily ordered mechanisms. The pH dependence of V and V/K was determined and indicates that catalysis requires that a base on the enzyme with a pK of 6.7 be unprotonated. TDH activity requires a divalent and a monovalent cation. Kinetic data suggest that the cations function in substrate binding and facilitation of the decarboxylation of beta-ketoacid intermediates.
KeywordMeSH Terms
263. Kadow  M, Sa?  S, Schmidt  M, Bornscheuer  UT,     ( 2011 )

Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007.

AMB Express 1 (1)
PMID : 21906366  :   DOI  :   10.1186/2191-0855-1-13     PMC  :   PMC3222318    
Abstract >>
Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit.
KeywordMeSH Terms
264. Peterson  JA, Lorence  MC, Amarneh  B,     ( 1990 )

Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins.

The Journal of biological chemistry 265 (11)
PMID : 2180940  :  
Abstract >>
The oxidation of camphor by cytochrome P-450cam requires the participation of a flavoprotein, putidaredoxin reductase, and an iron-sulfur protein, putidaredoxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation. A 2.2-kilobase pair BamHI-StuI fragment from whole cell DNA of camphor-grown Pseudomonas putida has been cloned and sequenced. Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin. In the case of putidaredoxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K., and Gunsalus, I. C. (1974) J. Biol. Chem. 249, 3689-3701) with the exception of one amino acid. Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide. A potential transcription termination site was found 3' to the putidaredoxin coding region. The "FAD-binding" amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occurrence of this sequence was found beginning with amino acid 156. The second sequence could represent the NAD-binding site. The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and independently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively. Site-directed mutagenesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells. The construction of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450cam.
KeywordMeSH Terms
Cloning, Molecular
Cloning, Molecular
265. Meyer  JB, Frapolli  M, Keel  C, Maurhofer  M,     ( 2011 )

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

Applied and environmental microbiology 77 (20)
PMID : 21856827  :   DOI  :   10.1128/AEM.05434-11     PMC  :   PMC3194850    
Abstract >>
Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.
KeywordMeSH Terms
266. Tsuda  M, Iino  T,     ( 1990 )

Naphthalene degrading genes on plasmid NAH7 are on a defective transposon.

Molecular & general genetics : MGG 223 (1)
PMID : 2175388  :   DOI  :   10.1007/bf00315794    
Abstract >>
A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class II transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.
KeywordMeSH Terms
DNA Transposable Elements
Genes
Plasmids
267. Zhang  W, Chen  L, Liu  D,     ( 2012 )

Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction.

Applied microbiology and biotechnology 93 (3)
PMID : 21751007  :   DOI  :   10.1007/s00253-011-3454-5    
Abstract >>
The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 �gM HgCl?. SP1 was also highly resistant to other metals, including CdCl?, CoCl?, CrCl?, CuCl?, PbCl?, and ZnSO?, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant determinants were all located on the bacterial chromosome. Cold vapor atomic absorption spectrometry and a volatilization test indicated that the isolated P. putida SP1 was able to volatilize almost 100% of the total mercury it was exposed to and could potentially be used for bioremediation in marine environments. The optimal pH for the growth of P. putida SP1 in the presence of HgCl? and the removal of HgCl? by P. putida SP1 was between 8.0 and 9.0, whereas the optimal pH for the expression of merA, the mercuric reductase enzyme in mer operon that reduces reactive Hg?? to volatile and relatively inert monoatomic Hg? vapor, was around 5.0. LD?? of P. putida SP1 to flounder and turbot was 1.5 �� 10? CFU. Biofilm developed by P. putida SP1 was 1- to 3-fold lower than biofilm developed by an aquatic pathogen Pseudomonas fluorescens TSS. The results of this study indicate that P. putida SP1 is a low virulence strain that can potentially be applied in the bioremediation of HgCl? contamination over a broad range of pH.
KeywordMeSH Terms
268. Nogales  J, Canales  A, Jiménez-Barbero  J, Serra  B, Pingarrón  JM, García  JL, Díaz  E,     ( 2011 )

Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida.

Molecular microbiology 79 (2)
PMID : 21219457  :   DOI  :   10.1111/j.1365-2958.2010.07448.x    
Abstract >>
Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.
KeywordMeSH Terms
Multigene Family
269. Tuan  NN, Hsieh  HC, Lin  YW, Huang  SL,     ( 2011 )

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes.

Bioresource technology 102 (5)
PMID : 21227686  :   DOI  :   10.1016/j.biortech.2010.12.067    
Abstract >>
Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.
KeywordMeSH Terms
Environmental Microbiology
270. Parales  RE, Harwood  CS,     ( 1990 )

Nucleotide sequence of the gyrB gene of Pseudomonas putida.

Nucleic acids research 18 (19)
PMID : 2170947  :   DOI  :   10.1093/nar/18.19.5880     PMC  :   PMC332329    
Abstract >>
N/A
KeywordMeSH Terms
271. Yee  TW, Smith  DW,     ( 1990 )

Pseudomonas chromosomal replication origins: a bacterial class distinct from Escherichia coli-type origins.

Proceedings of the National Academy of Sciences of the United States of America 87 (4)
PMID : 2106132  :   DOI  :   10.1073/pnas.87.4.1278     PMC  :   PMC53457    
Abstract >>
The bacterial origins of DNA replication have been isolated from Pseudomonas aeruginosa and Pseudomonas putida. These origins comprise a second class of bacterial origins distinct from enteric-type origins: both origins function in both Pseudomonas species, and neither functions in Escherichia coli; enteric origins do not function in either pseudomonad. Both cloned sequences hybridize to chromosomal fragments that show properties expected of replication origins. These origin plasmids are highly unstable, are present at low copy number, and show mutual incompatibility properties. DNA sequence analysis shows that both origins contain several 9-base-pair (bp) E. coli DnaA protein binding sites; four of these are conserved in position and orientation, two of which resemble the R1 and R4 sites of the E. coli origin. Conserved 13-bp direct repeats adjacent to the analogous R1 site are also found. No GATC sites are in the P. aeruginosa origin and only four are in the P. putida origin; no other 4-bp sequence is present in high abundance. Both origins are found between sequences similar to the E. coli and Bacillus subtilis dnaA, dnaN, rpmH, and rnpA genes, a gene organization identical to that for B. subtilis and unlike that of E. coli. A second autonomously replicating sequence was obtained from P. aeruginosa that has some properties of bacterial origins.
KeywordMeSH Terms
DNA Replication
272. Yun  CS, Suzuki  C, Naito  K, Takeda  T, Takahashi  Y, Sai  F, Terabayashi  T, Miyakoshi  M, Shintani  M, Nishida  H, Yamane  H, Nojiri  H,     ( 2010 )

Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function.

Journal of bacteriology 192 (18)
PMID : 20639326  :   DOI  :   10.1128/JB.00591-10     PMC  :   PMC2937398    
Abstract >>
Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.
KeywordMeSH Terms
273. Hayase  N, Taira  K, Furukawa  K,     ( 1990 )

Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria.

Journal of bacteriology 172 (2)
PMID : 2105297  :   DOI  :   10.1128/jb.172.2.1160-1164.1990     PMC  :   PMC208555    
Abstract >>
We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.
KeywordMeSH Terms
Cloning, Molecular
Dioxygenases
Gene Expression
Genes, Bacterial
Operon
274. Summers  RM, Louie  TM, Yu  CL, Subramanian  M,     ( 2011 )

Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source.

Microbiology (Reading, England) 157 (Pt 2)
PMID : 20966097  :   DOI  :   10.1099/mic.0.043612-0    
Abstract >>
N-Demethylation of many xenobiotics and naturally occurring purine alkaloids such as caffeine and theobromine is primarily catalysed in higher organisms, ranging from fungi to mammals, by the well-studied membrane-associated cytochrome P450s. In contrast, there is no well-characterized enzyme for N-demethylation of purine alkaloids from bacteria, despite several reports on their utilization as sole source of carbon and nitrogen. Here, we provide what we believe to be the first detailed characterization of a purified N-demethylase from Pseudomonas putida CBB5. The soluble N-demethylase holoenzyme is composed of two components, a reductase component with cytochrome c reductase activity (Ccr) and a two-subunit N-demethylase component (Ndm). Ndm, with a native molecular mass of 240 kDa, is composed of NdmA (40 kDa) and NdmB (35 kDa). Ccr transfers reducing equivalents from NAD(P)H to Ndm, which catalyses an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Paraxanthine and 7-methylxanthine were determined to be the best substrates, with apparent K(m) and k(cat) values of 50.4��6.8 �gM and 16.2��0.6 min(-1), and 63.8��7.5 �gM and 94.8��3.0 min(-1), respectively. Ndm also displayed activity towards caffeine, theobromine, theophylline and 3-methylxanthine, all of which are growth substrates for this organism. Ndm was deduced to be a Rieske [2Fe-2S]-domain-containing non-haem iron oxygenase based on (i) its distinct absorption spectrum and (ii) significant identity of the N-terminal sequences of NdmA and NdmB with the gene product of an uncharacterized caffeine demethylase in P. putida IF-3 and a hypothetical protein in Janthinobacterium sp. Marseille, both predicted to be Rieske non-haem iron oxygenases.
KeywordMeSH Terms
275. Sajben  E, Manczinger  L, Nagy  A, Kredics  L, Vágvölgyi  C,     ( 2011 )

Characterization of pseudomonads isolated from decaying sporocarps of oyster mushroom.

Microbiological research 166 (4)
PMID : 20627228  :   DOI  :   10.1016/j.micres.2010.05.002    
Abstract >>
Pleurotus ostreatus is one of the most extensively cultivated mushrooms in the world; however, the success of cultivation often depends on the proliferation of different bacterial pathogens. Pseudomonas tolaasii is thought as the major cause of brown blotch disease of Agaricus bisporus and yellowing of Pleurotus ostreatus. In this study we examined the pathogenicity and assessed the industrial damage causing effect of 41 Pseudomonas strains isolated from deformed, yellowing oyster mushroom (P. ostreatus) sporocarps. Identification of the isolates at species level by the partial sequence analysis of the hypervariable region of the rpoB gene revealed nine Pseudomonas sps. We analyzed the presence of the tolaasin gene-cluster, the production of fluorescent pigments, the oxidase- and nitrite reductase activities, the growth at restrictive temperatures and the carbon source utilizing abilities of each strain. Complex lipopeptide production (including tolaasin) was examined with thin layer chromatography and a novel in vitro necrosis-test was developed and evaluated for the investigation of the pathogenic effect of Pseudomonas strains. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process. Strong correlations were found between the secretion of trypsin-like proteases and lipases and the necrotic effect of these bacteria. All the results clearly established that besides Ps. tolaasii, Ps. fluorescens biovar V strains were pathogenic to P. ostreatus and cause serious losses during mushroom production. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process, especially the trypsin-like proteases and lipases.
KeywordMeSH Terms
276. Shen  W, Liu  W, Zhang  J, Tao  J, Deng  H, Cao  H, Cui  Z,     ( 2010 )

Cloning and characterization of a gene cluster involved in the catabolism of p-nitrophenol from Pseudomonas putida DLL-E4.

Bioresource technology 101 (19)
PMID : 20466541  :   DOI  :   10.1016/j.biortech.2010.04.052    
Abstract >>
A 9.2-kb DNA fragment encoding the enzymes of a p-nitrophenol (PNP) catabolic pathway from Pseudomonas putida DLL-E4 was cloned and sequenced. Ten open reading frames (ORFs) were found and five ORFs were functionally verified. The pnpA and pnpC gene products were purified to homogeneity by Ni-NTA chromatography. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase which converts p-nitrophenol to para-benzoquinone in the presence of NADH and FAD. PnpC is a 1,2,4-trihydroxybenzene (BT) 1,2-dioxygenase which converts BT to maleylacetate. The hydroquinone (HQ) dioxygenase (PnpC1C2) multi-component protein complex was expressed in Escherichia coli via plasmid pET-pnpC1C2 containing pnpC1 and pnpC2. This complex converts HQ to gamma-hydroxymuconic semialdehyde. pnpR is a lysR-type regulator gene. PnpR is a positive regulator involved in HQ degradation in pnp gene cluster. These results demonstrate that a pathway encoded by the pnp gene cluster is involved in degradation of HQ and BT in P. putida DLL-E4.
KeywordMeSH Terms
277. Spiegelhauer  O, Werther  T, Mende  S, Knauer  SH, Dobbek  H,     ( 2010 )

Determinants of substrate binding and protonation in the flavoenzyme xenobiotic reductase A.

Journal of molecular biology 403 (2)
PMID : 20826164  :   DOI  :   10.1016/j.jmb.2010.08.047    
Abstract >>
Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NAD(P)H-dependent reduction of various �\,�]-unsaturated carbonyl compounds and is a member of the old yellow enzyme family. The reaction of XenA follows a ping-pong mechanism, implying that its active site has to accommodate and correctly position the various substrates to be oxidized (NADH/NADPH) and to be reduced (different �\,�]-unsaturated carbonyl compounds) to enable formal hydride transfers between the substrate and the isoalloxazine ring. The active site of XenA is lined by two tyrosine (Tyr27, Tyr183) and two tryptophan (Trp302, Trp358) residues, which were proposed to contribute to substrate binding. We analyzed the individual contributions of the four residues, using site-directed mutagenesis, steady-state and transient kinetics, redox potentiometry and crystal structure analysis. The Y183F substitution decreases the affinity of XenA for NADPH and reduces the rate of the oxidative half-reaction by two to three orders of magnitude, the latter being in agreement with its function as a proton donor in the oxidative half-reaction. Upon reduction of the flavin, Trp302 swings into the active site of XenA (in-conformation) and decreases the extent of the substrate-binding pocket. Its exchange against alanine induces substrate inhibition at elevated NADPH concentrations, indicating that the in-conformation of Trp302 helps to disfavor the nonproductive NADPH binding in the reduced state of XenA. Our analysis shows that while the principal catalytic mechanism of XenA, for example, type of proton donor, is analogous to that of other members of the old yellow enzyme family, its strategy to correctly position and accommodate different substrates is unprecedented.
KeywordMeSH Terms
278. Li  D, Yu  T, Zhang  Y, Yang  M, Li  Z, Liu  M, Qi  R,     ( 2010 )

Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river.

Applied and environmental microbiology 76 (11)
PMID : 20400569  :   DOI  :   10.1128/AEM.02964-09     PMC  :   PMC2876458    
Abstract >>
We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as Gammaproteobacteria. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (tet) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with tet(A) being the most common (67.0%), followed by tet(W), tet(C), tet(J), tet(L), tet(D), tet(Y), and tet(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the tet genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Water Microbiology
Water Purification
279. El-Bassi  L, Iwasaki  H, Oku  H, Shinzato  N, Matsui  T,     ( 2010 )

Biotransformation of benzothiazole derivatives by the Pseudomonas putida strain HKT554.

Chemosphere 81 (1)
PMID : 20692014  :   DOI  :   10.1016/j.chemosphere.2010.07.024    
Abstract >>
We examined the biotransformation of benzothiazole derivatives (BTHs) by an axenic microbial culture. A Gram-negative bacterium, tentatively named as strain HKT554 and identified as Pseudomonas putida, was able to transform not only benzothiazole and 2-mercaptobenzothiazole but also 2-methylthiobenzothiazole, which was previously reported as the dead-end product of wastewater treatment. GC/MS analysis of the solid-phase extract of the culture broth showed the formation of 2-(3H)-benzothiazolone/2-hydroxybenzothiazole from benzothiazole. By transposon mutagenesis, a mutant library containing ca. 5000 insertion mutants was constructed from the P. putida strain HKT554. Analysis of the disrupted gene from one of the mutants showing BTHs transformation deficiency revealed that the knocked-out gene was naphthalene dioxygenase. To our knowledge, this is the first report on the biotransformation of BTHs by Gram-negative bacteria.
KeywordMeSH Terms
280. Stoddard  BL, Ringe  D, Petsko  GA,     ( 1990 )

The structure of iron superoxide dismutase from Pseudomonas ovalis complexed with the inhibitor azide.

Protein engineering 4 (2)
PMID : 2075185  :  
Abstract >>
The 2.9 A resolution structure of iron superoxide dismutase (FeSOD) (EC 1.15.1.1) from Pseudomonas ovalis complexed with the inhibitor azide was solved. Comparison of this structure with free enzyme shows that the inhibitor is bound at the open coordination position of the iron, with a bond length of 2.0 A. The metal moves by 0.4 A into the trigonal plane to produce an orthogonal geometry at the iron. Binding of the inhibitor also causes a movement of the axial ligand (histidine 26) away from the metal, a lengthening of the iron-histidine bond, and a rotation of the histidine 74 ring. The inhibitor possesses contacts in the binding pocket with a pair of conserved tryptophan residues and with the side chains of tyrosine 34 and glutamine 70. This glutamine is conserved between all FeSODs, but is absent in MnSOD. Comparisons with MnSOD show that a different glutamine which possesses the same interactions in the active site as Gln70 in FeSOD is conserved at position 154 in the overall SOD sequence, implying that while manganese and FeSODs are structural homologues in a global sense, their functional and evolutionary relationship is that of second-site mutation revertants.
KeywordMeSH Terms
281. Spiegelhauer  O, Mende  S, Dickert  F, Knauer  SH, Ullmann  GM, Dobbek  H,     ( 2010 )

Cysteine as a modulator residue in the active site of xenobiotic reductase A: a structural, thermodynamic and kinetic study.

Journal of molecular biology 398 (1)
PMID : 20206186  :   DOI  :   10.1016/j.jmb.2010.02.044    
Abstract >>
Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NADH/NADPH-dependent reduction of various substrates, including 2-cyclohexenone and 8-hydroxycoumarin. XenA is a member of the old yellow enzyme (OYE) family of flavoproteins and is structurally and functionally similar to other bacterial members of this enzyme class. A characteristic feature of XenA is the presence of a cysteine residue (Cys25) in the active site, where in most members of the OYE family a threonine residue is found that modulates the reduction potential of the FMN/FMNH(-) couple. We investigated the role of Cys25 by studying two variants in which the residue has been exchanged for a serine and an alanine residue. While the exchange against alanine has a remarkably small effect on the reduction potential, the reactivity and the structure of XenA, the exchange against serine increases the reduction potential by +82 mV, increases the rate constant of the reductive half-reaction and decreases the rate constant in the oxidative half-reaction. We determined six crystal structures at high to true atomic resolution (d(min) 1.03-1.80 A) of the three XenA variants with and without the substrate coumarin bound in the active site. The atomic resolution structure of XenA in complex with coumarin reveals a compressed active site geometry in which the isoalloxazine ring is sandwiched between coumarin and the protein backbone. The structures further reveal that the conformation of the active site and substrate interactions are preserved in the two variants, indicating that the observed changes are due to local effects only. We propose that Cys25 and the residues in its place determine which of the two half-reactions is rate limiting, depending on the substrate couple. This might help to explain why the genome of Pseudomonas putida encodes multiple xenobiotic reductases containing either cysteine, threonine or alanine in the active site.
KeywordMeSH Terms
Thermodynamics
282. Baelum  J, Jacobsen  CS, Holben  WE,     ( 2010 )

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders.

Systematic and applied microbiology 33 (2)
PMID : 20206455  :   DOI  :   10.1016/j.syapm.2010.01.001    
Abstract >>
31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.
KeywordMeSH Terms
283. Pochapsky  TC, Ye  XM,     ( 1991 )

1H NMR identification of a beta-sheet structure and description of folding topology in putidaredoxin.

Biochemistry 30 (16)
PMID : 2018758  :   DOI  :   10.1021/bi00230a007    
Abstract >>
Putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, is the physiological reductant of P-450cam, which in turn catalyzes the monohydroxylation of camphor by molecular oxygen. No crystal structure has been obtained for Pdx or for any closely homologous protein. The application of two-dimensional 1H NMR methods to the problem of structure determination in Pdx is reported. A beta-sheet consisting of five short strands and one beta-turn has been identified from distinctive nuclear Overhauser effect patterns. All of the backbone resonances and a majority of the side-chain resonances corresponding to protons in the beta-sheet have been assigned sequence specifically. The sheet contains one parallel and three antiparallel strand orientations. Hydrophobic side chains in the beta-sheet face primarily toward the protein interior, except for a group of three valine side chains that are apparently solvent exposed. The potential significance of this "hydrophobic patch" in terms of biological activity is discussed. The folding topology, as determined by the constraints of the beta-sheet, is compared with that of other [2Fe-2S] proteins for which folding topologies are known.
KeywordMeSH Terms
284. Li  Q, Wang  X, Yin  G, Gai  Z, Tang  H, Ma  C, Deng  Z, Xu  P,     ( 2009 )

New metabolites in dibenzofuran cometabolic degradation by a biphenyl-cultivated Pseudomonas putida strain B6-2.

Environmental science & technology 43 (22)
PMID : 20028064  :   DOI  :   10.1021/es901991d    
Abstract >>
A biphenyl (BP)-utilizing bacterium, designated B6-2, was isolated from soil and identified as Pseudomonas putida. BP-grown B6-2 cells were capable of transforming dibenzofuran (DBF) via a lateral dioxygenation and meta-cleavage pathway. The ring cleavage product 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid (HOBB) was detected as a major metabolite. B6-2 growing cells could also cometabolically degrade DBF using BP as a primary substrate. A recombinant Escherichia coli strain DH10B (pUC118bphABC) expressing BP dioxygenase, BP-dihydrodiol dehydrogenase, and dihydroxybiphenyl dioxygenase was shown to be capable of transforming DBF to HOBB. Using purified HOBB that was produced by the recombinant as the substrate for B6-2, we newly identified a series of benzofuran derivatives as metabolites. The structures of these metabolites indicate that an unreported HOBB degradation pathway is employed by strain B6-2. In this pathway, HOBB is proposed to be transformed to 2-oxo-4-(3'-oxobenzofuran-2'-yl)butanoic acid and 2-hydroxy-4-(3'-oxobenzofuran-2'-yl)butanoic acid (D4) through two sequential double-bond hydrogenation steps. D4 is suggested to undergo reactions including decarboxylation and oxidation to produce 3-(3'-oxobenzofuran-2'-yl)propanoic acid (D6). 3-Hydroxy-3-(3'-oxobenzofuran-2'-yl)propanoic acid (D7) and 2-(3'-oxobenzofuran-2'-yl)acetic acid (D8) would represent metabolites involved in the processes of beta- and alpha-oxidation of D6, respectively. D7 and D8 are suggested to be transformed to their respective products 3-hydroxy-2,3-dihydrobenzofuran-2-carboxylic acid (D10) and 2-(3'-hydroxy-2',3'-dihydrobenzofuran-2'-yl)acetic acid. D10 is proposed to be transformed to salicylic acid (D14) via 2,3-dihydro-2,3-dihydroxybenzofuran, 2-oxo-2-(2'-hydroxyphenyl)acetic acid and 2-hydroxy-2-(2'-hydroxyphenyl)acetic acid. Further experimental results revealed that B6-2 was capable of growing with D14 as the sole carbon source. Because benzofuran derivatives may have biological, pharmacological, and toxic properties, the elucidation of this new pathway should be significant from both biotechnological and environmental views.
KeywordMeSH Terms
285. Santos  C, Caetano  T, Ferreira  S, Mendo  S,     ( 2010 )

Tn5090-like class 1 integron carrying bla(VIM-2) in a Pseudomonas putida strain from Portugal.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 16 (10)
PMID : 20121819  :   DOI  :   10.1111/j.1469-0691.2010.03165.x    
Abstract >>
Three Pseudomonas putida strains containing bla(VIM-2) were isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro. A novel class 1 integron was found in strain Pp2 (aacA4/bla(VIM-2)/aac6'-IIc disrupted by an insertion sequence IS1382), and strain Pp1 was found to carry a class 1 integron (aacA7/bla(VIM-2)/aacC1/aacA4), which is described for the first time in this species. Strain PF1 carries a class 1 integron associated with a Tn5090-like transposon, constituting the first finding of this type of arrangement in a strain from Portugal. This association highlights further dissemination of bla(VIM-2) in environmental hospital isolates.
KeywordMeSH Terms
Environmental Microbiology
Integrons
286. Yamashita  K, Nakajima  Y, Matsushita  H, Nishiya  Y, Yamazawa  R, Wu  YF, Matsubara  F, Oyama  H, Ito  K, Yoshimoto  T,     ( 2010 )

Substitution of Glu122 by glutamine revealed the function of the second water molecule as a proton donor in the binuclear metal enzyme creatininase.

Journal of molecular biology 396 (4)
PMID : 20043918  :   DOI  :   10.1016/j.jmb.2009.12.045    
Abstract >>
Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity. We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl(2) or MnCl(2). In the E122Q mutant, k(cat) is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.
KeywordMeSH Terms
287. Ukaegbu  UE, Kantz  A, Beaton  M, Gassner  GT, Rosenzweig  AC,     ( 2010 )

Structure and ligand binding properties of the epoxidase component of styrene monooxygenase .

Biochemistry 49 (8)
PMID : 20055497  :   DOI  :   10.1021/bi901693u     PMC  :   PMC2827657    
Abstract >>
Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 A resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA approximately 8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker approximately 2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.
KeywordMeSH Terms
288. Sevrioukova  IF, Poulos  TL, Churbanova  IY,     ( 2010 )

Crystal structure of the putidaredoxin reductase x putidaredoxin electron transfer complex.

The Journal of biological chemistry 285 (18)
PMID : 20179327  :   DOI  :   10.1074/jbc.M110.104968     PMC  :   PMC2859523     DOI  :   10.1074/jbc.M110.104968     PMC  :   PMC2859523    
Abstract >>
In the camphor monooxygenase system from Pseudomonas putida, the [2Fe-2S]-containing putidaredoxin (Pdx) shuttles electrons between the NADH-dependent putidaredoxin reductase (Pdr) and cytochrome P450(cam). The mechanism of the Pdr.Pdx redox couple has been investigated by a variety of techniques. One of the exceptions is x-ray crystallography as the native partners associate weakly and resist co-crystallization. Here, we present the 2.6-A x-ray structure of a catalytically active complex between Pdr and Pdx C73S/C85S chemically cross-linked via the Lys(409Pdr)-Glu(72Pdx) pair. The 365 A(2) Pdr-Pdx interface is predominantly hydrophobic with one central Arg(310Pdr)-Asp(38Pdx) salt bridge, likely assisting docking and orienting the partners optimally for electron transfer, and a few peripheral hydrogen bonds. A predicted 12-A-long electron transfer route between FAD and [2Fe-2S] includes flavin flanking Trp(330Pdr) and the iron ligand Cys(39Pdx). The x-ray model agrees well with the experimental and theoretical results and suggests that the linked Pdx must undergo complex movements during turnover to accommodate P450(cam), which could limit the Pdx-to-P450(cam) electron transfer reaction.
KeywordMeSH Terms
Models, Molecular
Models, Molecular
289. Juan  C, Zamorano  L, Mena  A, Albertí  S, Pérez  JL, Oliver  A,     ( 2010 )

Metallo-beta-lactamase-producing Pseudomonas putida as a reservoir of multidrug resistance elements that can be transferred to successful Pseudomonas aeruginosa clones.

The Journal of antimicrobial chemotherapy 65 (3)
PMID : 20071364  :   DOI  :   10.1093/jac/dkp491    
Abstract >>
To study the prevalence, nature, involved genetic elements and epidemiology of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa and Pseudomonas putida isolated in a Spanish hospital between 2005 and 2008. Etests were used for susceptibility testing and screening for MBLs, confirmed through bla(VIM) PCRs and sequencing. Clonal relatedness was evaluated by PFGE and multilocus sequence typing (MLST). MBL-carrying plasmids were characterized by restriction fragment length polymorphism, Southern blot and electroporation. MBL genetic elements were studied by cloning and sequencing. MBL-producing P. putida was detected in eight patients (one clone each; two harbouring bla(VIM-1) and six harbouring bla(VIM-2)), representing 14% of all the infections by the P. putida/fluorescens group. MBLs were detected in only 0.3% of P. aeruginosa infections (11 patients) during the same period. PFGE revealed four P. aeruginosa clones: one producing bla(VIM-13) (two patients); and three producing bla(VIM-2) (two patients, six patients and one patient, respectively). MLST indicated that the VIM-13 clone was the internationally spread sequence type (ST)235, while the major VIM-2 lineage corresponded to ST179, which is associated with chronic respiratory infections. The VIM-1 integron was shown to have both plasmid and chromosomal location, while the VIM-13 integron was only chromosomal. The VIM-2 integron was located in the same transposon (Tn402/Tn5053-like) in all P. aeruginosa and P. putida isolates, suggesting its crucial role in the dissemination of VIM-2. The high diversity and proportion of MBL-positive P. putida suggests an environmental reservoir of these resistance determinants. Dissemination of these multidrug resistance elements to successful P. aeruginosa clones presents a major epidemiological and clinical threat.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Gene Transfer, Horizontal
290. Bruce  NC, Wilmot  CJ, Jordan  KN, Stephens  LD, Lowe  CR,     ( 1991 )

Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10.

The Biochemical journal 274 (Pt 3) (N/A)
PMID : 2012614  :   DOI  :   10.1042/bj2740875     PMC  :   PMC1149991    
Abstract >>
The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.
KeywordMeSH Terms
291. Harayama  S, Polissi  A, Rekik  M,     ( 1991 )

Divergent evolution of chloroplast-type ferredoxins.

FEBS letters 285 (1)
PMID : 2065785  :   DOI  :   10.1016/0014-5793(91)80730-q    
Abstract >>
The TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to Krebs cycle intermediates. The structural genes for these enzymes are encoded in two operons which comprise the xylCMABN and xylXYZLTEGFJQKIH genes, respectively. The function of the xylT gene has not yet been identified. The nucleotide sequence of xylT was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast-type ferredoxins. The nahT gene, the homologue of xylT, present on NAH plasmid NAH7 encoding naphthalene-degrading enzymes, was also sequenced. The sequence conservation between xylT and nahT strongly suggests that both gene products have some physiological function. Chloroplast-type ferredoxins have been discovered in photosynthetic organisms (plants, algae, cyanobacteria and Rhodobacter) and Halobacterium species. Furthermore, chloroplast-type ferredoxin-like sequences have been found in the electron-transfer components of some oxygenases. The sequences of XylT and NahT were compared with those of the previously identified chloroplast-type ferredoxins, in order to examine their evolutionary relationships.
KeywordMeSH Terms
Bacterial Proteins
Biological Evolution
Gene Frequency
292. Steiner  RA, Janssen  HJ, Roversi  P, Oakley  AJ, Fetzner  S,     ( 2010 )

Structural basis for cofactor-independent dioxygenation of N-heteroaromatic compounds at the alpha/beta-hydrolase fold.

Proceedings of the National Academy of Sciences of the United States of America 107 (2)
PMID : 20080731  :   DOI  :   10.1073/pnas.0909033107     PMC  :   PMC2818892    
Abstract >>
Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus R?61a and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the alpha/beta-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1-H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical alpha/beta-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The "oxyanion hole" of the alpha/beta-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions.
KeywordMeSH Terms
293. Suzuki  M, Hayakawa  T, Shaw  JP, Rekik  M, Harayama  S,     ( 1991 )

Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system.

Journal of bacteriology 173 (5)
PMID : 1999388  :   DOI  :   10.1128/jb.173.5.1690-1695.1991     PMC  :   PMC207319    
Abstract >>
Xylene monooxygenase, encoded by the TOL plasmid of Pseudomonas putida, catalyzes the oxidation of toluene and xylenes and consists of two different subunits encoded by xylA and xylM. In this study, the complete nucleotide sequences of these genes were determined and the amino acid sequences of the xylA and xylM products were deduced. The XylM sequence had a 25% homology with alkane hydroxylase, which catalyzes the omega-hydroxylation of fatty acids and the terminal hydroxylation of alkanes. The sequence of the first 90 amino acids of XylA exhibited a strong similarity to the sequence of chloroplast-type ferredoxins, whereas the rest of the XylA sequence resembled that of ferredoxin-NADP+ reductases. Based on this information, the structure and function of xylene monooxygenase were deduced. XylM may be a catalytic component for the hydroxylation of the carbon side chain of toluene and xylenes and, as is the alkane hydroxylase protein, may be a membrane-bound protein containing ferrous ion as a prosthetic group. XylA may have two domains consisting of an N-terminal region similar to chloroplast-type ferredoxins and a C-terminal region similar to ferredoxin-NADP+ reductases. The ferredoxin portion of XylA may contain a [2Fe-2S] cluster and reduce the oxidized form of the XylM hydroxylase. The activity determined by the C-terminal region of the XylA sequence may be the reduction of the oxidized form of ferredoxin by concomitant oxidation of NADH.
KeywordMeSH Terms
294. Chalmers  RM, Keen  JN, Fewson  CA,     ( 1991 )

Comparison of benzyl alcohol dehydrogenases and benzaldehyde dehydrogenases from the benzyl alcohol and mandelate pathways in Acinetobacter calcoaceticus and from the TOL-plasmid-encoded toluene pathway in Pseudomonas putida. N-terminal amino acid sequences, amino acid compositions and immunological cross-reactions.

The Biochemical journal 273(Pt 1) (N/A)
PMID : 1989592  :   DOI  :   10.1042/bj2730099     PMC  :   PMC1149885    
Abstract >>
1. N-Terminal sequences were determined for benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus N.C.I.B. 8250, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 in Pseudomonas putida MT53 and yeast K(+)-activated aldehyde dehydrogenase. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50161 (5 pages) at the British Library Document Supply Centre, Boston Spa. Wetherby. West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273. 5. The extent of sequence similarity suggests that the benzyl alcohol dehydrogenases are related to each other and also to established members of the family of long-chain Zn2(+)-dependent alcohol dehydrogenases. Benzaldehyde dehydrogenase II from Acinetobacter appears to be related to the Pseudomonas TOL-plasmid-encoded benzaldehyde dehydrogenase. The yeast K(+)-activated aldehyde dehydrogenase has similarity of sequence with the mammalian liver cytoplasmic class of aldehyde dehydrogenases but not with any of the Acinetobacter or Pseudomonas enzymes. 2. Antisera were raised in rabbits against the three Acinetobacter enzymes and both of the Pseudomonas enzymes, and the extents of the cross-reactions were determined by immunoprecipitation assays with native antigens and by immunoblotting with SDS-denatured antigens. Cross-reactions were detected between the alcohol dehydrogenases and also among the aldehyde dehydrogenases. This confirms the interpretation of the N-terminal sequence comparisons and also indicates that benzaldehyde dehydrogenase I from Acinetobacter may be related to the other two benzaldehyde dehydrogenases. 3. The amino acid compositions of the Acinetobacter and the Pseudomonas enzymes were determined and the numbers of amino acid residues per subunit were calculated to be: benzyl alcohol dehydrogenase and TOL-plasmid-encoded benzyl alcohol dehydrogenase, 381; benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II, 525; TOL-plasmid-encoded benzaldehyde dehydrogenase, 538.
KeywordMeSH Terms
295. You  IS, Ghosal  D, Gunsalus  IC,     ( 1991 )

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region.

Biochemistry 30 (6)
PMID : 1993181  :   DOI  :   10.1021/bi00220a028    
Abstract >>
Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.
KeywordMeSH Terms
Genes, Bacterial
Open Reading Frames
296. Gursky  LJ, Nikodinovic-Runic  J, Feenstra  KA, O'Connor  KE,     ( 2010 )

In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis.

Applied microbiology and biotechnology 85 (4)
PMID : 19568744  :   DOI  :   10.1007/s00253-009-2096-3    
Abstract >>
The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.
KeywordMeSH Terms
297. Mehnaz  S, Weselowski  B, Aftab  F, Zahid  S, Lazarovits  G, Iqbal  J,     ( 2009 )

Isolation, characterization, and effect of fluorescent pseudomonads on micropropagated sugarcane.

Canadian journal of microbiology 55 (8)
PMID : 19898541  :   DOI  :   10.1139/w09-050    
Abstract >>
In this study, we report on the isolation, identification, and characterization of seven fluorescent pseudomonads isolated from the roots, shoots, and rhizosphere soil of sugarcane and their impacts on the growth of sugarcane plantlets. 16S rRNA gene sequence of five isolates showed close homology with Pseudomonas putida, one with Pseudomonas graminis, and one with Pseudomonas fluorescens. Physiological and biochemical characterizations were determined using API50CH and QTS24 identification kits. The isolates were also subjected to tests for various known growth promoting properties including production of indole acetic acid, the ability to fix nitrogen via the presence of the nifH gene, and ability to solubilize phosphate. Biological control potential was determined from agar diffusion assays of HCN production and production of antifungal compounds against local isolates of Colletotrichum falcatum (that induces red-rot disease of sugarcane). Direct plant growth promoting effects were tested on sugarcane plantlets in tissue culture under gnotobiotic conditions. All seven isolates provided significant increases in fresh and dry masses but only five strains increased shoot height.
KeywordMeSH Terms
298. Shintani  M, Takahashi  Y, Tokumaru  H, Kadota  K, Hara  H, Miyakoshi  M, Naito  K, Yamane  H, Nishida  H, Nojiri  H,     ( 2010 )

Response of the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7 plasmid pCAR1.

Environmental microbiology 12 (6)
PMID : 19930443  :   DOI  :   10.1111/j.1462-2920.2009.02110.x    
Abstract >>
Plasmid carriage requires appropriate expression of the genes on the plasmid or host chromosome through cooperative transcriptional regulation. To clarify the impact of plasmid carriage on the host chromosome, we compared the chromosomal RNA maps of plasmid-free and plasmid-containing host strains using the incompatibility group P-7 archetype plasmid pCAR1, which is involved in carbazole degradation, and three distinct Pseudomonas strains. The possession of pCAR1 altered gene expression related to the iron acquisition systems in each host. Expression of the major siderophore pyoverdine was greater in plasmid-containing P. putida KT2440 and P. aeruginosa PAO1 than in the plasmid-free host strains, in part due to the expression of carbazole-degradative genes on pCAR1. The mexEFoprN operon encoding an efflux pump of the resistance-nodulation-cell division family was specifically upregulated by the carriage of pCAR1 in P. putida KT2440, whereas the expression of orthologous genes in the other species remained unaltered. Induction of the mexEFoprN genes increased the resistance of pCAR1-containing KT2440 to chloramphenicol compared with pCAR1-free KT2440. Our findings indicate that the possession of pCAR1 altered the growth rate of the host via the expression of genes on pCAR1 and the host chromosomes.
KeywordMeSH Terms
Gene Expression Profiling
Pseudomonas
299. Hume  AR, Nikodinovic-Runic  J, O'Connor  KE,     ( 2009 )

FadD from Pseudomonas putida CA-3 is a true long-chain fatty acyl coenzyme A synthetase that activates phenylalkanoic and alkanoic acids.

Journal of bacteriology 191 (24)
PMID : 19820085  :   DOI  :   10.1128/JB.01016-09     PMC  :   PMC2786608    
Abstract >>
A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k(cat) and K(m) values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3DeltafadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3DeltafadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3DeltafadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longer-monomer units into the PHA polymer.
KeywordMeSH Terms
300. Cheng  Z, Duan  J, Hao  Y, McConkey  BJ, Glick  BR,     ( 2009 )

Identification of bacterial proteins mediating the interactions between Pseudomonas putida UW4 and Brassica napus (Canola).

Molecular plant-microbe interactions : MPMI 22 (6)
PMID : 19445593  :   DOI  :   10.1094/MPMI-22-6-0686    
Abstract >>
The influence of canola root exudates on the proteome of Pseudomonas putida UW4 and the mutant strain P. putida UW4/AcdS(-), which lacks a functional 1-aminocyclopropane-1-carboxylate deaminase gene, was examined using two-dimensional difference in-gel electrophoresis. Seventy-two proteins with significantly altered expression levels in the presence of canola root exudates were identified by mass spectrometry. Many of these proteins are involved in nutrient transport and utilization, cell envelope synthesis, and transcriptional or translational regulation and, hence, may play important roles in plant-bacterial interactions. Four proteins showing large changes in expression in response to canola root exudates in both the wild-type and mutant strains of P. putida UW4 (i.e., outer membrane protein F, peptide deformylase, transcription regulator Fis family protein, and a previously uncharacterized protein) were both overexpressed and disrupted in P. putida UW4 in an effort to better understand their functions. Functional studies of these modified strains revealed significantly enhanced or inhibited plant-growth-promoting abilities compared with the wild-type P. putida UW4, in agreement with the suggested involvement of three of these four proteins in plant-bacterial interactions. The work reported here suggests strategies to both identify potential antibacterial agents and develop bacterial strains that might be useful adjuncts to agriculture. This approach may be an effective means of identifying key proteins mediating the interactions of bacteria with their rhizosphere environment.
KeywordMeSH Terms
301. Kunze  M, Zerlin  KF, Retzlaff  A, Pohl  JO, Schmidt  E, Janssen  DB, Vilchez-Vargas  R, Pieper  DH, Reineke  W,     ( 2009 )

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome.

Microbiology (Reading, England) 155 (Pt 12)
PMID : 19744988  :   DOI  :   10.1099/mic.0.032110-0    
Abstract >>
Pseudomonas putida GJ31 has been reported to grow on chlorobenzene using a meta-cleavage pathway with chlorocatechol 2,3-dioxygenase (CbzE) as a key enzyme. The CbzE-encoding gene was found to be localized on the 180 kb plasmid pKW1 in a cbzTEXGS cluster, which is flanked by transposases and encodes only a partial (chloro)catechol meta-cleavage pathway comprising ferredoxin reductase, chlorocatechol 2,3-dioxygenase, an unknown protein, 2-hydroxymuconic semialdehyde dehydrogenase and glutathione S-transferase. Downstream of cbzTEXGS are located cbzJ, encoding a novel type of 2-hydroxypent-2,4-dienoate hydratase, and a transposon region highly similar to Tn5501. Upstream of cbzTEXGS, traNEOFG transfer genes were found. The search for gene clusters possibly completing the (chloro)catechol metabolic pathway of GJ31 revealed the presence of two additional catabolic gene clusters on pKW1. The mhpRBCDFETP cluster encodes enzymes for the dissimilation of 2,3-dihydroxyphenylpropionate in a novel arrangement characterized by the absence of a gene encoding 3-(3-hydroxyphenyl)propionate monooxygenase and the presence of a GntR-type regulator, whereas the nahINLOMKJ cluster encodes part of the naphthalene metabolic pathway. Transcription studies supported their possible involvement in chlorobenzene degradation. The upper pathway cluster, comprising genes encoding a chlorobenzene dioxygenase and a chlorobenzene dihydrodiol dehydrogenase, was localized on the chromosome. A high level of transcription in response to chlorobenzene revealed it to be crucial for chlorobenzene degradation. The chlorobenzene degradation pathway in strain GJ31 is thus a mosaic encoded by four gene clusters.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
302. Hill  KE, Weightman  AJ,     ( 2003 )

Horizontal transfer of dehalogenase genes on IncP1beta plasmids during bacterial adaptation to degrade alpha-halocarboxylic acids.

FEMS microbiology ecology 45 (3)
PMID : 19719596  :   DOI  :   10.1016/S0168-6496(03)00158-2    
Abstract >>
The diversity of bacterial alpha-halocarboxylic acid (alphaHA) dehalogenases from a polluted soil was investigated. Polymerase chain reaction (PCR) primers designed to amplify group I and group II dehalogenase (deh) gene sequences were used to screen bacterial isolates, nine beta-Proteobacteria and one gamma-Proteobacterium, from soil enrichments. Primers successfully amplified deh sequences from all 10 alphaHA-utilising isolates. Bacteria isolated at 15 or 30 degrees C on chloroacetic acid or 2-chloropropionic acid from the same polluted soil were shown to contain up to four plasmids, some of these common between isolates. Analysis of deletion mutants and Southern hybridisation showed that each isolate contained an apparently identical IncP1beta plasmid c. 80 kb in size, carrying group I deh genes in addition to an associated insertion sequence element. Moreover, an identical conjugative catabolic plasmid was isolated exogenously in several transconjugants independently selected from biparental matings between Ralstonia eutropha JMP222 and enrichment samples. PCR cloning and sequencing of deh genes directly from enrichment cultures inoculated with the same soil revealed that an identical deh gene was present in both primary, secondary and tertiary enrichment cultures, although this deh could not be amplified directly from soil. Two alphaHA-utilising bacteria isolated at lower temperature were found also to contain group II deh genes. Transfer of the deh catabolic phenotype to R. eutropha strain JMP222 occurred at high frequencies for four strains tested, a result that was consistent with assignment of the plasmids to the IncP1 incompatibility group. The promiscuous nature and broad host range of IncP plasmids make them likely to be involved in horizontal gene transfer during adaptation of bacteria to degrade organohalogens.
KeywordMeSH Terms
303. Pudney  CR, McGrory  T, Lafite  P, Pang  J, Hay  S, Leys  D, Sutcliffe  MJ, Scrutton  NS,     ( 2009 )

Parallel pathways and free-energy landscapes for enzymatic hydride transfer probed by hydrostatic pressure.

Chembiochem : a European journal of chemical biology 10 (8)
PMID : 19405065  :   DOI  :   10.1002/cbic.200900071    
Abstract >>
Mutation of an active-site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor-acceptor distance in the wild-type enzyme, increased conformational freedom "dampens" its effect in the mutant.We show that hydride transfer from NADH to FMN catalysed by the N189A mutant of morphinone reductase occurs along parallel "chemical" pathways in a conformationally rich free-energy landscape. We have developed experimental kinetic and spectroscopic tools by using hydrostatic pressure to explore this free-energy landscape. The crystal structure of the N189A mutant enzyme in complex with the unreactive coenzyme analogue NADH(4) indicates that the nicotinamide moiety of the analogue is conformationally less restrained than the corresponding structure of the wild-type NADH(4) complex. This increased degree of conformational freedom in the N189A enzyme gives rise to the concept of multiple reactive configurations (MRCs), and we show that the relative population of these states across the free-energy landscape can be perturbed experimentally as a function of pressure. Specifically, the amplitudes of individual kinetic phases that were observed in stopped-flow studies of the hydride transfer reaction are sensitive to pressure; this indicates that pressure drives an altered distribution across the energy landscape. We show by absorbance spectroscopy that the loss of charge-transfer character of the enzyme-coenzyme complex is attributed to the altered population of MRCs on the landscape. The existence of a conformationally rich landscape in the N189A mutant is supported by molecular dynamics simulations at low and high pressure. The work provides firm experimental and computational support for the existence of parallel pathways arising from multiple conformational states of the enzyme-coenzyme complex. Hydrostatic pressure is a powerful and general probe of multidimensional energy landscapes that can be used to analyse experimentally parallel pathways for enzyme-catalysed reactions. We suggest that this is especially the case following directed mutation of a protein, which can lead to increased population of reactant states that are essentially inaccessible in the free-energy landscape of wild-type enzyme.
KeywordMeSH Terms
304. Promden  W, Vangnai  AS, Toyama  H, Matsushita  K, Pongsawasdi  P,     ( 2009 )

Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5.

Microbiology (Reading, England) 155 (Pt 2)
PMID : 19202108  :   DOI  :   10.1099/mic.0.021956-0    
Abstract >>
The transcriptional regulation of three distinct alcohol oxidation systems, alcohol dehydrogenase (ADH)-I, ADH-IIB and ADH-IIG, in Pseudomonas putida HK5 was investigated under various induction conditions. The promoter activities of the genes involved in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC, encoding ADH-I and a cytochrome c, respectively. Primary and secondary C3 and C4 alcohols and butyraldehyde specifically induced the divergent promoters of qbdBA and aldA, encoding ADH-IIB and an NAD-dependent aldehyde dehydrogenase, respectively. The qgdA promoter of ADH-IIG responded well to (S)-(+)-1,2-propanediol induction. In addition, the roles of genes encoding the response regulators exaE and agmR, located downstream of qedA, were inferred from the properties of exaE- or agmR-disrupted mutants and gene complementation tests. The gene products of both exaE and agmR were strictly necessary for qedA transcription. The mutation and complementation studies also suggested a role for AgmR, but not ExaE, in the transcriptional regulation of qbdBA (ADH-IIB) and qgdA (AGH-IIG). A hypothetical scheme describing a regulatory network, which directs expression of the three distinct alcohol oxidation systems in P. putida HK5, was derived.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic
305. Rehdorf  J, Zimmer  CL, Bornscheuer  UT,     ( 2009 )

Cloning, expression, characterization, and biocatalytic investigation of the 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1.

Applied and environmental microbiology 75 (10)
PMID : 19251889  :   DOI  :   10.1128/AEM.02707-08     PMC  :   PMC2681629    
Abstract >>
While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E >>100).
KeywordMeSH Terms
306. Wang  PF, Yep  A, Kenyon  GL, McLeish  MJ,     ( 2009 )

Using directed evolution to probe the substrate specificity of mandelamide hydrolase.

Protein engineering, design & selection : PEDS 22 (2)
PMID : 19074156  :   DOI  :   10.1093/protein/gzn073    
Abstract >>
Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliphatic substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homology model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly202 appears to control the preference for aromatic substrates as the G202A variant showed three orders of magnitude decrease in k(cat)/K(m) for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant.
KeywordMeSH Terms
Directed Molecular Evolution
307. Landro  JA, Kallarakal  AT, Ransom  SC, Gerlt  JA, Kozarich  JW, Neidhart  DJ, Kenyon  GL,     ( 1991 )

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant.

Biochemistry 30 (38)
PMID : 1909893  :   DOI  :   10.1021/bi00102a020    
Abstract >>
The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)
KeywordMeSH Terms
308. Miyakoshi  M, Nishida  H, Shintani  M, Yamane  H, Nojiri  H,     ( 2009 )

High-resolution mapping of plasmid transcriptomes in different host bacteria.

BMC genomics 10 (N/A)
PMID : 19134166  :   DOI  :   10.1186/1471-2164-10-12     PMC  :   PMC2642839    
Abstract >>
Plasmids are extrachromosomal elements that replicate autonomously, and many can be transmitted between bacterial cells through conjugation. Although the transcription pattern of genes on a plasmid can be altered by a change in host background, the expression range of plasmid genes that will result in phenotypic variation has not been quantitatively investigated. Using a microarray with evenly tiled probes at a density of 9 bp, we mapped and quantified the transcripts of the carbazole catabolic plasmid pCAR1 in its original host Pseudomonas resinovorans CA10 and the transconjugant P. putida KT2440(pCAR1) during growth on either carbazole or succinate as the sole carbon source. We identified the operons in pCAR1, which consisted of nearly identical transcription units despite the difference in host background during growth on the same carbon source. In accordance with previous studies, the catabolic operons for carbazole degradation were upregulated during growth on carbazole in both hosts. However, our tiling array results also showed that several operons flanking the transfer gene cluster were transcribed at significantly higher levels in the transconjugant than in the original host. The number of transcripts and the positions of the transcription start sites agreed with our quantitative RT-PCR and primer extension results. Our tiling array results indicate that the levels of transcription for the operons on a plasmid can vary by host background. High-resolution mapping using an unbiased tiling array is a valuable tool for the simultaneous identification and quantification of prokaryotic transcriptomes including polycistronic operons and non-coding RNAs.
KeywordMeSH Terms
Chromosome Mapping
Gene Expression Profiling
309. Takahashi  Y, Shintani  M, Yamane  H, Nojiri  H,     ( 2009 )

The complete nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally identical IncP-7 carbazole degradative plasmids.

Bioscience, biotechnology, and biochemistry 73 (3)
PMID : 19270415  :   DOI  :   10.1271/bbb.80665    
Abstract >>
pCAR1 and pCAR2 are IncP-7 self-transmissible carbazole degradative plasmids. Their respective hosts showed clearly different conjugative host ranges. Their complete nucleotide sequences were virtually the same, and can be regarded as structurally the same plasmid, indicating that the difference in the conjugative host range was caused by host cell backgrounds.
KeywordMeSH Terms
310. Patzer  JA, Walsh  TR, Weeks  J, Dzierzanowska  D, Toleman  MA,     ( 2009 )

Emergence and persistence of integron structures harbouring VIM genes in the Children's Memorial Health Institute, Warsaw, Poland, 1998-2006.

The Journal of antimicrobial chemotherapy 63 (2)
PMID : 19095681  :   DOI  :   10.1093/jac/dkn512    
Abstract >>
The aim was to perform a genetically detailed study of the emergence of metallo-beta-lactamase (MBL) genes in Pseudomonas spp. in the Children's Memorial Health Institute over a 9 year period. Carbapenem-resistant Pseudomonas spp. isolates were collected from 1998 to 2006 and screened for MBL production. MBL-positive isolates were further investigated by a combination of genetic techniques including PCR, genomic location experiments using pulsed-field gel electrophoresis (PFGE) of I-Ceu1, S1 and SpeI digests, and sequencing. Of the 20 MBL-containing Pseudomonas isolates collected from 1998 to 2006, 16 Pseudomonas aeruginosa isolates contained an identical class 1 integron structure. Two P. aeruginosa isolates contained the bla(VIM-2) gene, and two Pseudomonas putida isolates harboured the bla(VIM-4) gene cassette in different integron structures. PFGE analysis indicated that all bla(VIM-4)-containing P. aeruginosa isolates were closely related, whereas the P. putida isolates were not. All MBL genes in this study were chromosomally encoded, and all isolates harboured only one class 1 integron. The bla(VIM-2) isolates were clonal, and the genetic structure supporting the bla(VIM-2) gene was found in an identical chromosomal position. MBL gene emergence in this hospital has paralleled a 6-fold increase in carbapenem usage. We have found an increase in MBL gene diversity, MBL host organisms and MBL genetic support structures in the hospital over the 9 year study period. There is also compelling evidence of the persistence of individual strains in the hospital throughout the study period. This suggests that once MBL genes have emerged in a hospital environment, they are difficult to remove.
KeywordMeSH Terms
Integrons
311. Menn  FM, Zylstra  GJ, Gibson  DT,     ( 1991 )

Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1.

Gene 104 (1)
PMID : 1916282  :   DOI  :   10.1016/0378-1119(91)90470-v    
Abstract >>
The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes. The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation. The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753. The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.
KeywordMeSH Terms
Genes, Bacterial
312. Tang  H, Wang  L, Meng  X, Ma  L, Wang  S, He  X, Wu  G, Xu  P,     ( 2009 )

Novel nicotine oxidoreductase-encoding gene involved in nicotine degradation by Pseudomonas putida strain S16.

Applied and environmental microbiology 75 (3)
PMID : 19060159  :   DOI  :   10.1128/AEM.02300-08     PMC  :   PMC2632140    
Abstract >>
There are quite a few ongoing biochemical investigations of nicotine degradation in different organisms. In this work, we identified and sequenced a gene (designated nicA) involved in nicotine degradation by Pseudomonas putida strain S16. The gene product, NicA, was heterologously expressed and characterized as a nicotine oxidoreductase catalyzing the initial steps of nicotine metabolism. Biochemical analyses using resting cells and the purified enzyme suggested that nicA encodes an oxidoreductase, which converts nicotine to 3-succinoylpyridine through pseudooxynicotine. Based on enzymatic reactions and direct evidence obtained using H(2)(18)O labeling, the process may consist of enzyme-catalyzed dehydrogenation, followed by spontaneous hydrolysis and then repetition of the dehydrogenation and hydrolysis steps. Sequence comparisons revealed that the gene showed 40% similarity to genes encoding NADH dehydrogenase subunit I and cytochrome c oxidase subunit I in eukaryotes. Our findings demonstrate that the molecular mechanism for nicotine degradation in strain S16 involves the pyrrolidine pathway and is similar to the mechanism in mammals, in which pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen, is produced.
KeywordMeSH Terms
313. Palmer  JA, Hatter  K, Sokatch  JR,     ( 1991 )

Cloning and sequence analysis of the LPD-glc structural gene of Pseudomonas putida.

Journal of bacteriology 173 (10)
PMID : 1902462  :   DOI  :   10.1128/jb.173.10.3109-3116.1991     PMC  :   PMC207904    
Abstract >>
Pseudomonas putida is able to produce three lipoamide dehydrogenases: (i) LPD-glc, which is the E3 component of the pyruvate and 2-ketoglutarate dehydrogenase complexes and the L-factor for the glycine oxidation system; (ii) LPD-val, which is the specific E3 component of the branched-chain keto acid dehydrogenase complex and is induced by growth on leucine, isoleucine, or valine; and (iii) LPD-3, which was discovered in a lpdG mutant and whose role is unknown. Southern hybridization with an oligonucleotide probe encoding the highly conserved redox-active site produced three bands corresponding to the genes encoding these three lipoamide dehydrogenases. The complete structural gene for LPD-glc, lpdG, was isolated, and its nucleotide sequence was determined. The latter consists of 476 codons plus a stop codon, TAA. The structural gene for LPD-glc is preceded by a partial open reading frame with strong similarity to the E2 component of 2-ketoglutarate dehydrogenase of Escherichia coli. This suggests that lpdG is part of the 2-ketoglutarate dehydrogenase operon. LPD-glc was expressed in Pseudomonas putida JS348 from pHP4 which contains a partial open reading frame corresponding to the E2 component, 94 bases of noncoding DNA, and the structural gene for lpdG. This result indicates that lpdG can be expressed separately from the other genes of the operon.
KeywordMeSH Terms
Genes, Bacterial
314. Dubern  JF, Coppoolse  ER, Stiekema  WJ, Bloemberg  GV,     ( 2008 )

Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445.

Microbiology (Reading, England) 154 (Pt 7)
PMID : 18599835  :   DOI  :   10.1099/mic.0.2008/016444-0    
Abstract >>
Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the d form of the amino acid residues while the two following modules recognize the l form and the last module the l or d form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Multigene Family
315. Sevastsyanovich  YR, Krasowiak  R, Bingle  LE, Haines  AS, Sokolov  SL, Kosheleva  IA, Leuchuk  AA, Titok  MA, Smalla  K, Thomas  CM,     ( 2008 )

Diversity of IncP-9 plasmids of Pseudomonas.

Microbiology (Reading, England) 154 (Pt 10)
PMID : 18832300  :   DOI  :   10.1099/mic.0.2008/017939-0     PMC  :   PMC2885752    
Abstract >>
IncP-9 plasmids are important vehicles for degradation and resistance genes that contribute to the adaptability of Pseudomonas species in a variety of natural habitats. The three completely sequenced IncP-9 plasmids, pWW0, pDTG1 and NAH7, show extensive homology in replication, partitioning and transfer loci (an approximately 25 kb region) and to a lesser extent in the remaining backbone segments. We used PCR, DNA sequencing, hybridization and phylogenetic analyses to investigate the genetic diversity of 30 IncP-9 plasmids as well as the possibility of recombination between plasmids belonging to this family. Phylogenetic analysis of rep and oriV sequences revealed nine plasmid subgroups with 7-35 % divergence between them. Only one phenotypic character was normally associated with each subgroup, except for the IncP-9beta cluster, which included naphthalene- and toluene-degradation plasmids. The PCR and hybridization analysis using pWW0- and pDTG1-specific primers and probes targeting selected backbone loci showed that members of different IncP-9 subgroups have considerable similarity in their overall organization, supporting the existence of a conserved ancestral IncP-9 sequence. The results suggested that some IncP-9 plasmids are the product of recombination between plasmids of different IncP-9 subgroups but demonstrated clearly that insertion of degradative transposons has occurred on multiple occasions, indicating that association of this phenotype with these plasmids is not simply the result of divergent evolution from a single successful ancestral degradative plasmid.
KeywordMeSH Terms
Genetic Variation
316. Neidhart  DJ, Howell  PL, Petsko  GA, Powers  VM, Li  RS, Kenyon  GL, Gerlt  JA,     ( 1991 )

Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-A resolution: identification of the active site and possible catalytic residues.

Biochemistry 30 (38)
PMID : 1892834  :   DOI  :   10.1021/bi00102a019    
Abstract >>
The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS)
KeywordMeSH Terms
317. Banerjee  A, Dubey  S, Kaul  P, Barse  B, Piotrowski  M, Banerjee  UC,     ( 2009 )

Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies.

Molecular biotechnology 41 (1)
PMID : 18704770  :   DOI  :   10.1007/s12033-008-9094-z    
Abstract >>
Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(-)-mandelic acid.
KeywordMeSH Terms
Bioreactors
318. Nichols  NN, Mertens  JA,     ( 2008 )

Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism.

FEMS microbiology letters 284 (1)
PMID : 18492059  :   DOI  :   10.1111/j.1574-6968.2008.01196.x    
Abstract >>
Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid (FA) and coenzyme A intermediates. Two P. putida transposon mutants were isolated that had impaired growth on furfural and FA, and DNA flanking the transposon insertion site was cloned from both mutants. The transposons disrupted psfB, a LysR-family regulatory gene in mutant PSF2 and psfF, a GcvR-type regulatory gene in PSF9. Disruption of two genes adjacent to psfB demonstrated that both are important for growth on FA, and ORFs in the proximity of psfB and psfF were transcriptionally activated during growth of P. putida on FA. Transcript levels increased in response to FA by 10-fold (a putative permease gene) to >1000-fold (a putative decarboxylase gene). The LysR-family gene appears to act positively, and the GcvR-family gene negatively, in regulating expression of neighboring genes in response to FA.
KeywordMeSH Terms
Gene Expression Profiling
Gene Expression Regulation, Bacterial
319. Mathews  FS, Chen  ZW, Bellamy  HD, McIntire  WS,     ( 1991 )

Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution.

Biochemistry 30 (1)
PMID : 1846290  :   DOI  :   10.1021/bi00215a034    
Abstract >>
p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000. It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller. Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third. An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives. The phases were improved with the B.C. Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis. The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system. The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system. The flavoprotein subunit consists of three domains in close contact. The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site. The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side. The flavin ring lies at the juncture of the first two domains. The third domain lies against the first domain and helps cover the rest of the FAD chain. The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit. The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart. The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.
KeywordMeSH Terms
320. Halouska  S, Zhou  Y, Becker  DF, Powers  R,     ( 2009 )

Solution structure of the Pseudomonas putida protein PpPutA45 and its DNA complex.

Proteins 75 (1)
PMID : 18767154  :   DOI  :   10.1002/prot.22217     PMC  :   PMC2650008    
Abstract >>
Proline utilization A (PutA) is a membrane-associated multifunctional enzyme that catalyzes the oxidation of proline to glutamate in a two-step process. In certain, gram-negative bacteria such as Pseudomonas putida, PutA also acts as an auto repressor in the cytoplasm, when an insufficient concentration of proline is available. Here, the N-terminal residues 1-45 of PutA from P. putida (PpPutA45) are shown to be responsible for DNA binding and dimerization. The solution structure of PpPutA45 was determined using NMR methods, where the protein is shown to be a symmetrical homodimer (12 kDa) consisting of two ribbon-helix-helix (RHH) structures. DNA sequence recognition by PpPutA45 was determined using DNA gel mobility shift assays and NMR chemical shift perturbations (CSPs). PpPutA45 was shown to bind a 14 base-pair DNA oligomer (5'-GCGGTTGCACCTTT-3'). A model of the PpPutA45-DNA oligomer complex was generated using Haddock 2.1. The antiparallel beta-sheet that results from PpPutA45 dimerization serves as the DNA recognition binding site by inserting into the DNA major groove. The dimeric core of four alpha-helices provides a structural scaffold for the beta-sheet from which residues Thr5, Gly7, and Lys9 make sequence-specific contacts with the DNA. The structural model implies flexibility of Lys9 which can make hydrogen bond contacts with either guanine or thymine. The high sequence and structure conservation of the PutA RHH domain suggest interdomain interactions play an important role in the evolution of the protein.
KeywordMeSH Terms
321. Vo  MT, Lee  KW, Jung  YM, Lee  YH,     ( 2008 )

Comparative effect of overexpressed phaJ and fabG genes supplementing (R)-3-hydroxyalkanoate monomer units on biosynthesis of mcl-polyhydroxyalkanoate in Pseudomonas putida KCTC1639.

Journal of bioscience and bioengineering 106 (1)
PMID : 18691538  :   DOI  :   10.1263/jbb.106.95    
Abstract >>
phaJ and fabG genes encoding (R)-specific 2-enoyl-CoA hydratase and 3-ketoacyl-acyl carrier protein reductase, respectively, were cloned from Pseudomonas putida KCTC1639 and amplified in the parent strain to achieve oversupplementation of (R)-3-hydroxyalkanoate monomer units for the biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The comparative effect of the overexpressed fabG and phaJ genes in P. putida KCTC1639 on the biosynthesis of mcl-PHA and the cell growth were elucidated. Overexpression of phaJ enhanced the biosynthesis of mcl-PHA, increasing its content and concentration from 18% to 27% and 0.38 to 0.51 g/l, respectively. Conversely, fabG overexpression tended to depress the biosynthesis of mcl-PHA, possibly due to the reversible conversion of (R)-3-hydroxyalkanoate monomer units into 3-ketoacyl-CoA.
KeywordMeSH Terms
322. Brown  EN, Friemann  R, Karlsson  A, Parales  JV, Couture  MM, Eltis  LD, Ramaswamy  S,     ( 2008 )

Determining Rieske cluster reduction potentials.

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 13 (8)
PMID : 18719951  :   DOI  :   10.1007/s00775-008-0413-4    
Abstract >>
The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.
KeywordMeSH Terms
323. Cheng  Z, Duncker  BP, McConkey  BJ, Glick  BR,     ( 2008 )

Transcriptional regulation of ACC deaminase gene expression in Pseudomonas putida UW4.

Canadian journal of microbiology 54 (2)
PMID : 18388982  :   DOI  :   10.1139/w07-128    
Abstract >>
One of the major mechanisms that plant growth-promoting bacteria use to facilitate plant growth is through the lowering of plant ethylene levels by the bacterial enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Many of the bacterial ACC deaminase genes (acdS) that have been examined to date are under the transcriptional control of a leucine-responsive regulatory protein, Lrp, encoded by acdR and referred to here as AcdR. The work presented here is focused on how AcdR and the newly discovered AcdB protein from Pseudomonas putida UW4 are involved in the regulation of acdS expression. First, the results of gel retardation experiments showed that AcdR binds to the acdS regulatory region, and this binding activity in vitro is not affected by the addition of 2 mmol x L-1 ACC but can be eliminated by addition of 20 microg x mL-1 leucine. Second, a potential regulatory protein, AcdB, involved in the regulation of acdS expression, was identified through both yeast 2-hybrid screen and coimmunoprecipitation based on its ability to bind to AcdR; subsequently, its binding to the acdS regulatory region in the presence of ACC was shown by gel retardation experiments. The data are interpreted in terms of a model in which AcdR and AcdB co-regulate the expression of the acdS gene.
KeywordMeSH Terms
Gene Expression Regulation
324. Schmidberger  JW, Wilce  JA, Weightman  AJ, Whisstock  JC, Wilce  MC,     ( 2008 )

The crystal structure of DehI reveals a new alpha-haloacid dehalogenase fold and active-site mechanism.

Journal of molecular biology 378 (1)
PMID : 18353360  :   DOI  :   10.1016/j.jmb.2008.02.035    
Abstract >>
Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I alpha-haloacid dehalogenase that can process both L- and D-substrates. The structure shows that the DehI monomer consists of two domains of approximately 130 amino acids that have approximately 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both L- and D-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to D-only enzymes. These details will assist with future bioengineering of dehalogenases.
KeywordMeSH Terms
325. Grueninger  D, Treiber  N, Ziegler  MO, Koetter  JW, Schulze  MS, Schulz  GE,     ( 2008 )

Designed protein-protein association.

Science (New York, N.Y.) 319 (5860)
PMID : 18187656  :   DOI  :   10.1126/science.1150421    
Abstract >>
The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures.
KeywordMeSH Terms
Protein Engineering
326. Tang  H, Wang  S, Ma  L, Meng  X, Deng  Z, Zhang  D, Ma  C, Xu  P,     ( 2008 )

A novel gene, encoding 6-hydroxy-3-succinoylpyridine hydroxylase, involved in nicotine degradation by Pseudomonas putida strain S16.

Applied and environmental microbiology 74 (5)
PMID : 18203859  :   DOI  :   10.1128/AEM.02529-07     PMC  :   PMC2258644    
Abstract >>
Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a way similar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine (HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are two important precursors for drug syntheses. However, there is little information on the molecular mechanism for nicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bp gene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine, HSP, and DHP were identified from resting cell reactions of the transformant containing the gene cluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonas putida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directly to DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa) is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acid shows no similarity with any protein of known function.
KeywordMeSH Terms
327. Promden  W, Vangnai  AS, Pongsawasdi  P, Adachi  O, Matsushita  K, Toyama  H,     ( 2008 )

Disruption of quinoprotein ethanol dehydrogenase gene and adjacent genes in Pseudomonas putida HK5.

FEMS microbiology letters 280 (2)
PMID : 18218017  :   DOI  :   10.1111/j.1574-6968.2008.01060.x    
Abstract >>
Pseudomonas putida HK5 produces three different quinoprotein alcohol dehydrogenases: ADH-I, ADH-IIB and ADH-IIG. Gene organization of qedA, the gene for ADH-I, and other 10 genes in the cluster was related to the genome sequences of five other Pseudomonas strains. Insertion mutations in either qedA, exaE or agmR eliminated ADH-I activity, although the mutants were still able to grow on ethanol but more slowly than the wild-type strain. Mutant analysis demonstrated the requirement of agmR and exaE in ADH-I expression, and the tentative involvement of agmR, but not exaE, in the induction of ADH-IIB and ADH-IIG activities.
KeywordMeSH Terms
Genes, Bacterial
328. Bogaerts  P, Huang  TD, Rodriguez-Villalobos  H, Bauraing  C, Deplano  A, Struelens  MJ, Glupczynski  Y,     ( 2008 )

Nosocomial infections caused by multidrug-resistant Pseudomonas putida isolates producing VIM-2 and VIM-4 metallo-beta-lactamases.

The Journal of antimicrobial chemotherapy 61 (3)
PMID : 18238886  :   DOI  :   10.1093/jac/dkm529    
Abstract >>
N/A
KeywordMeSH Terms
329. Takano  T, Higuchi  W, Zaraket  H, Otsuka  T, Baranovich  T, Enany  S, Saito  K, Isobe  H, Dohmae  S, Ozaki  K, Takano  M, Iwao  Y, Shibuya  M, Okubo  T, Yabe  S, Shi  D, Reva  I, Teng  LJ, Yamamoto  T,     ( 2008 )

Novel characteristics of community-acquired methicillin-resistant Staphylococcus aureus strains belonging to multilocus sequence type 59 in Taiwan.

Antimicrobial agents and chemotherapy 52 (3)
PMID : 18086843  :   DOI  :   10.1128/AAC.01001-07     PMC  :   PMC2258532    
Abstract >>
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains, which often produce Panton-Valentine leucocidin (PVL), are increasingly noted worldwide. In this study, we examined 42 MRSA strains (25 PVL-positive [PVL+] strains and 17 PVL-negative [PVL(-)] strains) isolated in Taiwan for their molecular characteristics. The PVL+ MRSA strains included CA-MRSA strains with multilocus sequence type (ST) 59 (major PVL+ MRSA in Taiwan), its variants, and worldwide CA-MRSA ST30 strains. The PVL(-) MRSA strains included the pandemic Hungarian MRSA ST239 strain, the Hungarian MRSA ST239 variant, MRSA ST59 (largely hospital-acquired MRSA strains) and its variants, the pandemic New York/Japan MRSA ST5 strain (Japanese type), and the MRSA ST8 strain. The major PVL+ CA-MRSA ST59 strain possessed a tetracycline resistance-conferring (tetK positive) penicillinase plasmid and a drug resistance gene cluster (a possible composite transposon) for multidrug resistance. Moreover, it carried a novel staphylococcal cassette chromosome mec (SCCmec) with two distinct ccrC genes (ccrC2-C8). This SCCmec (previously named SCCmec type V(T)) was tentatively designated SCCmec type VII. Sequencing of the PVL genes revealed the polymorphisms, and the PVL+ CA-MRSA ST59 strain possessed the ST59-specific PVL gene sequence. The data suggest that a significant amount of clonal spread is occurring in Taiwan and that the major PVL+ CA-MRSA ST59 Taiwan strain exhibits unique genetic characteristics, such as a novel SCCmec type and an ST59-specific PVL gene sequence.
KeywordMeSH Terms
Methicillin Resistance
Polymorphism, Genetic
Staphylococcus aureus
330. Leveau  JH, Gerards  S,     ( 2008 )

Discovery of a bacterial gene cluster for catabolism of the plant hormone indole 3-acetic acid.

FEMS microbiology ecology 65 (2)
PMID : 18205812  :   DOI  :   10.1111/j.1574-6941.2008.00436.x    
Abstract >>
The isolation and annotation of an 8994-bp DNA fragment from Pseudomonas putida 1290, which conferred upon P. putida KT2440 the ability to utilize the plant hormone indole 3-acetic acid (IAA) as a sole source of carbon and energy, is described. This iac locus (for indole 3-acetic acid catabolism) was identified through analysis of a plasposon mutant of P. putida 1290 that was no longer able to grow on IAA or indole 3-acetaldehyde and was unable to protect radish roots from stunting by exogenously added IAA. The iac locus consisted of 10 genes with coding similarity to enzymes acting on indole or amidated aromatics and to proteins with regulatory or unknown function. Highly similar iac gene clusters were identified in the genomes of 22 bacterial species. Five of these, i.e. P. putida GB-1, Marinomonas sp. MWYL1, Burkholderia sp. 383, Sphingomonas wittichii RW1 and Rhodococcus sp. RHA1, were tested to confirm that bacteria with IAA-degrading ability have representatives in the Alpha-, Beta- and Gammaproteobacteria and in the Actinobacteria. In P. putida 1290, cat and pca genes were found to be essential to IAA-degradation, suggesting that IAA is channeled via catechol into the beta-ketoadipate pathway. Also contributing to the IAA degrading phenotype were genes involved in tricarboxylate cycling, gluconeogenesis, and carbon/nitrogen sensing.
KeywordMeSH Terms
Multigene Family
331. Yamaguchi  Y, Jin  W, Matsunaga  K, Ikemizu  S, Yamagata  Y, Wachino  J, Shibata  N, Arakawa  Y, Kurosaki  H,     ( 2007 )

Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor.

Journal of medicinal chemistry 50 (26)
PMID : 18052313  :   DOI  :   10.1021/jm701031n    
Abstract >>
The VIM-2 metallo-beta-lactamase enzyme from Pseudomonas aeruginosa catalyzes the hydrolysis of most beta-lactam antibiotics including carbapenems, and there are currently no potent inhibitors of such enzymes. We found rac-2-omega-phenylpropyl-3-mercaptopropionic acid, phenylC3SH, to be a potent inhibitor of VIM-2. The structure of the VIM-2-phenylC3SH complex was determined by X-ray crystallography to 2.3 A. The structure revealed that the thiol group of phenylC3SH bridged to the two zinc(II) ions and the phenyl group interacted with Tyr67(47) on loop1 near the active site, by pi-pi stacking interactions. The methylene group interacted with Phe61(42) located at the bottom of loop1 through CH-pi interactions. Dynamic movements were observed in Arg228(185) and Asn233(190) on loop2, compared with the native structure (PDB code: 1KO3). These results suggest that the above-mentioned four residues play important roles in the binding and recognition of inhibitors or substrates and in stabilizing a loop in the VIM-2 enzyme.
KeywordMeSH Terms
beta-Lactamase Inhibitors
332. Pudney  CR, Hay  S, Pang  J, Costello  C, Leys  D, Sutcliffe  MJ, Scrutton  NS,     ( 2007 )

Mutagenesis of morphinone reductase induces multiple reactive configurations and identifies potential ambiguity in kinetic analysis of enzyme tunneling mechanisms.

Journal of the American Chemical Society 129 (45)
PMID : 17939663  :   DOI  :   10.1021/ja074463h    
Abstract >>
We have identified multiple reactive configurations (MRCs) of an enzyme-coenzyme complex that have measurably different kinetic properties. In the complex formed between morphinone reductase (MR) and the NADH analogue 1,4,5,6-tetrahydro-NADH (NADH4) the nicotinamide moiety is restrained close to the FMN isoalloxazine ring by hydrogen bonds from Asn-189 and His-186 as determined from the X-ray crystal structure. Molecular dynamic simulations indicate that removal of one of these hydrogen bonds in the N189A MR mutant allows the nicotinamide moiety to occupy a region of configurational space not accessible in wild-type enzyme. Using stopped-flow spectroscopy, we show that reduction of the FMN cofactor by NADH in N189A MR is multiphasic, identifying at least four different reactive configurations of the MR-NADH complex. This contrasts with wild-type MR in which hydride transfer occurs by environmentally coupled tunneling in a single kinetic phase [Pudney et al. J. Am. Chem. Soc. 2006, 128, 14053-14058]. Values for primary and alpha-secondary kinetic isotope effects, and their temperature dependence, for three of the kinetic phases in the N189A MR are consistent with hydride transfer by tunneling. Our analysis enables derivation of mechanistic information concerning different reactive configurations of the same enzyme-coenzyme complex using ensemble stopped-flow methods. Implications for the interpretation from kinetic data of tunneling mechanisms in enzymes are discussed.
KeywordMeSH Terms
333. Paithankar  KS, Feller  C, Kuettner  EB, Keim  A, Grunow  M, Sträter  N,     ( 2007 )

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida.

The FEBS journal 274 (21)
PMID : 17958702  :   DOI  :   10.1111/j.1742-4658.2007.06102.x    
Abstract >>
D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.
KeywordMeSH Terms
334. Horn  JM, Harayama  S, Timmis  KN,     ( 1991 )

DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism.

Molecular microbiology 5 (10)
PMID : 1791759  :   DOI  :   10.1111/j.1365-2958.1991.tb02091.x    
Abstract >>
The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)
KeywordMeSH Terms
Aldehyde Oxidoreductases
Biological Evolution
Genes, Bacterial
Multigene Family
Plasmids
Proteins
335. Jurenaite-Urbanaviciene  S, Serksnaite  J, Kriukiene  E, Giedriene  J, Venclovas  C, Lubys  A,     ( 2007 )

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains.

Proceedings of the National Academy of Sciences of the United States of America 104 (25)
PMID : 17553965  :   DOI  :   10.1073/pnas.0610365104     PMC  :   PMC1965518    
Abstract >>
Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.
KeywordMeSH Terms
DNA Cleavage
336. Ahn  SJ, Yang  CH, Cooksey  DA,     ( 2007 )

Pseudomonas putida 06909 genes expressed during colonization on mycelial surfaces and phenotypic characterization of mutants.

Journal of applied microbiology 103 (1)
PMID : 17584458  :   DOI  :   10.1111/j.1365-2672.2006.03232.x    
Abstract >>
The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas-Phytophthora interaction as a biological control mechanism, and to understand the roles of these genes. Sixteen Ps. putida genes with increased expression on Phytophthora mycelial surfaces were identified using in vivo expression technology (IVET) screening. Sequence analysis of these Phytophthora mycelium-induced (pmi) genes revealed that many of them display similarity to genes known or predicted to be involved in carbohydrate catabolism, energy metabolism, amino acid/nucleotide metabolism, and membrane transport processes. Disruption of three pmi genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde-3-phosphate dehydrogenase showed significant phenotypic differences involved in the colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these pmi genes were induced by glycogen and other substances, such as organic acids and amino acids utilized by Ps. putida. The IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provide insight into the possible mechanisms of mycelial colonization by this bacterium. The IVET screening through a bacterial genome library might be a huge task. However, because the genes involved in direct interaction with Phytophthora and in bacterial adaptation can be identified, the IVET system will be a valuable tool in studying biocontrol bacteria at the molecular and ecological levels.
KeywordMeSH Terms
Genes, Bacterial
337. Ransom  SC, Gerlt  JA, Powers  VM, Kenyon  GL,     ( 1988 )

Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida.

Biochemistry 27 (2)
PMID : 2831968  :   DOI  :   10.1021/bi00402a006    
Abstract >>
The gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) was cloned in Pseudomonas aeruginosa (ATCC 15692). The selection for the cloned gene was based upon the inability of P. aeruginosa to grow on (R)-mandelate as sole carbon source by virtue of the absence of mandelate racemase in its mandelate pathway. Fragments of P. putida DNA obtained by digestion of chromosomal DNA with Sau3A were ligated into the BamHI site of the Gram-negative vector pKT230 and transformed into the P. aeruginosa host. A transformant able to utilize (R)-mandelate as sole carbon source was characterized, and the plasmid was found to contain approximately five kilobase pairs of P. putida DNA. Subcloning of this DNA revealed the position of the gene for the racemase within the cloned DNA from P. putida. The dideoxy-DNA sequencing procedure was used to determine the sequence of the gene and its translated sequence. The amino acid sequence and molecular weight for mandelate racemase deduced from the gene sequence (38 570) are in excellent agreement with amino acid composition and molecular weight data for the polypeptide recently determined with enzyme isolated from P. putida; these recent determinations of the polypeptide molecular weight differ significantly from the originally reported value of 69,500 [Fee, Judith A., Hegeman, G.D., & Kenyon, G.L. (1974) Biochemistry 13,2528], which was used to demonstrate that alpha-phenylglycidate, an active site directed irreversible inhibitor, binds to the enzyme with a stoichiometry of 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)
KeywordMeSH Terms
Cloning, Molecular
Genes
Genes, Bacterial
Transcription, Genetic
338. Ghequire  MG, Kemland  L, De Mot  R,     ( 2017 )

Novel Immunity Proteins Associated with Colicin M-like Bacteriocins Exhibit Promiscuous Protection in Pseudomonas.

Frontiers in microbiology 8 (N/A)
PMID : 28194143  :   DOI  :   10.3389/fmicb.2017.00093     PMC  :   PMC5277000    
Abstract >>
Bacteriocins related to colicin M, acting via cleavage of the cell wall precursor lipid II, have been characterized in �^- and �]-proteobacteria. Depending on the species, immunity is provided by either an inner membrane-anchored periplasmic protein or by an integral membrane protein. In Pseudomonas however, the immunity partner of colicin M-like bacteriocins remains unknown. Based on an in silico analysis in pseudomonad genomes, we here identify a gene encoding a putative immunity partner that represents a novel type of integral membrane protein (PmiA, Pseudomonas colicin M-like immunity type A). By heterologous expression of pmiA genes in susceptible strains, we show that immunity to colicin M-like bacteriocins is indeed provided by the cognate PmiA. Sequence homology among PmiA proteins is essentially absent, except for a short motif with a conserved periplasm-exposed aspartate residue. However, PmiA's protective function is not abolished by changing this acidic residue to the uncharged alanine. Immunity by PmiAs appears promiscuous to the extent that PmiA homologs from a clade sharing <40% pairwise amino acid identity, equally provide protection against the bacteriocin linked to the original PmiA. This study shows that multiple immunity factors have evolved independently to silence lipid II-targeting enzymatic bacteriocins. Their relaxed bacteriocin immunization capacity contrasts to the strict specificity of immunity proteins shielding the enzymatic domain of nuclease bacteriocins. The nature of associated immune functions needs consideration when using such natural protein antibiotics or designing novel variants.
KeywordMeSH Terms
PmiA
PmiB
colicin M
lipid II
peptidoglycan
pyocin
339. Ghosal  D, You  IS,     ( 1988 )

Nucleotide homology and organization of chlorocatechol oxidation genes of plasmids pJP4 and pAC27.

Molecular & general genetics : MGG 211 (1)
PMID : 2830460  :   DOI  :   10.1007/bf00338401    
Abstract >>
The 2,4-dichlorophenoxyacetate (2,4-D) catabolic plasmid pJP4 of Alcaligenes eutrophus JMP134 contains two sets of nonidentical chlorocatechol oxidation gene sequences physically separated by a 7 kb DNA region. We determined the nucleotide sequence of the 1.6 kb HindIII fragment containing the known genes tfdC and tfdD (Don et al. 1985) which encode pyrocatechase and cycloisomerase, respectively. The 1.3 kb BglII-HindIII segment of recombinant plasmid pDC25 containing at least three chlorocatechol (clc) oxidation genes of the pAC27 plasmid in Pseudomonas putida AC867 (Ghosal et al. 1985a; Frantz and Chakrabarty 1986), was also sequenced. When the tfdC gene of the pJP4 plasmid was compared with gene clcA of plasmid pAC27, which encodes the chlorocatechol specific pyrocatechase (pyrocatechase II), the two genes showed 63% nucleotide sequence homology with 60% homology in their amino acid sequences. In both plasmid pJP4 and pAC27, the two genes encoding the pyrocatechase and the cycloisomerase showed a 4 bp overlap spanning the initiation codon of the cycloisomerase gene and the termination codon of the pyrocatechase gene. The sizes of the polypeptides encoded by the isofunctional genes tfdC and clcA are very similar and thus reflect their functional homology.
KeywordMeSH Terms
Dioxygenases
Genes, Bacterial
Plasmids
340. Maroniche  GA, Rubio  EJ, Consiglio  A, Perticari  A,     ( 2016 )

Plant-associated fluorescent Pseudomonas from red lateritic soil: Beneficial characteristics and their impact on lettuce growth.

The Journal of general and applied microbiology 62 (5)
PMID : 27725403  :   DOI  :   10.2323/jgam.2016.04.006    
Abstract >>
Fluorescent Pseudomonas are ubiquitous soil bacteria that usually establish mutualistic associations with plants, promoting their growth and health by several mechanisms. This makes them interesting candidates for the development of crop bio-inoculants. In this work, we isolated phosphate-solubilizing fluorescent Pseudomonas from the rhizosphere and inner tissues of different plant species growing in red soil from Misiones, Argentina. Seven isolates displaying strong phosphate solubilization were selected for further studies. Molecular identification by rpoD genotyping indicated that they belong to different species within the P. fluorescens and P. putida phylogenetic groups. Screening for in vitro traits such as phosphate solubilization, growth regulators synthesis or degradation, motility and antagonism against phytopathogens or other bacteria, revealed a unique profile of characteristics for each strain. Their plant growth-promoting potential was assayed using lettuce as a model for inoculation under controlled and greenhouse conditions. Five of the strains increased the growth of lettuce plants. Overall, the strongest lettuce growth promoter under both conditions was strain ZME4, isolated from inner tissues of maize. No clear association between lettuce growth promotion and in vitro beneficial traits was detected. In conclusion, several phosphate solubilizing pseudomonads from red soil were isolated that display a rich array of plant growth promotion traits, thus showing a potential for the development of new inoculants.
KeywordMeSH Terms
Soil Microbiology
341. Blaschke  L, Wagner  W, Werkmeister  C, Wild  M, Gihring  A, Rupp  S, Zibek  S,     ( 2017 )

Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3.

Journal of biotechnology 241 (N/A)
PMID : 27836796  :   DOI  :   10.1016/j.jbiotec.2016.11.007    
Abstract >>
Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling.
KeywordMeSH Terms
Biomethanol
Cofactor regeneration
Escherichia coli rhamnose promoter
Formaldehyde dismutase
Oxidoreductase
Pseudomonas putida
342. Borodin  AM, Danilkovich  AV, Allikmets  RL,     ( 1989 )

[Nucleotide sequence of the rplL gene coding for ribosomal protein L7/L12 of Pseudomonas putida].

Bioorganicheskaia khimiia 15 (4)
PMID : 2751714  :  
Abstract >>
The Pseudomonas putida rpl L gene coding for ribosomal protein L7/L12 was cloned and sequenced. Although Asp55 residue in L7/L12 was previously shown to be conservative in ten different organisms, the Pseudomonas putida L7/L12 proved to contain Asn55, thus showing that Asp55 is not invariant.
KeywordMeSH Terms
343. Schell  MA, Sukordhaman  M,     ( 1989 )

Evidence that the transcription activator encoded by the Pseudomonas putida nahR gene is evolutionarily related to the transcription activators encoded by the Rhizobium nodD genes.

Journal of bacteriology 171 (4)
PMID : 2703465  :   DOI  :   10.1128/jb.171.4.1952-1959.1989     PMC  :   PMC209844    
Abstract >>
The nahR gene of the 83-kilobase naphthalene degradation plasmid NAH7 of Pseudomonas putida encodes a 34-kilodalton polypeptide which binds to the nah and sal promoters to activate transcription of the degradation genes in response to the inducer salicylate. The DNA sequence of the nahR gene was determined, and a derived amino acid sequence of the NahR protein was obtained. A computer search for homologous proteins showed that within the first 124 amino-terminal residues, NahR has approximately 35% identity with the transcriptional activator proteins encoded by the nodD genes of Rhizobium species. Allowing for ultraconservative amino acid substitutions, greater than 47% overall similarity was found between NahR and NodD, while 32% similarity was found between NahR and another transcription activator, LysR of Escherichia coli. The region of greatest similarity among all three proteins contained a probable helix-turn-helix DNA-binding motif as suggested by homology with the proposed consensus sequence for Cro-like DNA-binding domains. The high level of amino acid identity between NahR and NodD, in conjunction with the observations that nahR and nodD are 45% homologous in DNA sequence, are divergently transcribed from homologous promoters near the structural genes they control, and have similar DNA-binding sites, strongly suggests that these two genes evolved from a common ancestor.
KeywordMeSH Terms
Genes, Bacterial
344. Coitinho  JB, Pereira  MS, Costa  DM, Guimarães  SL, Araújo  SS, Hengge  AC, Brandão  TA, Nagem  RA,     ( 2016 )

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

Biochemistry 55 (38)
PMID : 27580341  :   DOI  :   10.1021/acs.biochem.6b00614    
Abstract >>
The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows �\/�] folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.
KeywordMeSH Terms
345. Felux  AK, Franchini  P, Schleheck  D,     ( 2015 )

Permanent draft genome sequence of sulfoquinovose-degrading Pseudomonas putida strain SQ1.

Standards in genomic sciences 10 (N/A)
PMID : 27408681  :   DOI  :   10.1186/s40793-015-0033-x     PMC  :   PMC4940961    
Abstract >>
Pseudomonas putida SQ1 was isolated for its ability to utilize the plant sugar sulfoquinovose (6-deoxy-6-sulfoglucose) for growth, in order to define its SQ-degradation pathway and the enzymes and genes involved. Here we describe the features of the organism, together with its draft genome sequence and annotation. The draft genome comprises 5,328,888 bp and is predicted to encode 5,824 protein-coding genes; the overall G + C content is 61.58 %. The genome annotation is being used for identification of proteins that might be involved in SQ degradation by peptide fingerprinting-mass spectrometry.
KeywordMeSH Terms
Gram-negative
Pseudomonadaceae
Pseudomonas putida SQ1
aerobic
organosulfonate
plant sulfolipid
sulfoquinovose biodegradation
346. Guimarães  SL, Coitinho  JB, Costa  DM, Araújo  SS, Whitman  CP, Nagem  RA,     ( 2016 )

Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous �]-Keto Acid.

Biochemistry 55 (18)
PMID : 27082660  :   DOI  :   10.1021/acs.biochem.6b00050     PMC  :   PMC4862935    
Abstract >>
The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent �]-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous �]-keto acid decarboxylase and the first crystal structure of a 4-OD.
KeywordMeSH Terms
347. Tacão  M, Correia  A, Henriques  IS,     ( 2015 )

Low Prevalence of Carbapenem-Resistant Bacteria in River Water: Resistance Is Mostly Related to Intrinsic Mechanisms.

Microbial drug resistance (Larchmont, N.Y.) 21 (5)
PMID : 26430939  :   DOI  :   10.1089/mdr.2015.0072    
Abstract >>
Carbapenems are last-resort antibiotics to handle serious infections caused by multiresistant bacteria. The incidence of resistance to these antibiotics has been increasing and new resistance mechanisms have emerged. The dissemination of carbapenem resistance in the environment has been overlooked. The main goal of this research was to assess the prevalence and diversity of carbapenem-resistant bacteria in riverine ecosystems. The presence of frequently reported carbapenemase-encoding genes was inspected. The proportion of imipenem-resistant bacteria was on average 2.24 CFU/ml. Imipenem-resistant strains (n=110) were identified as Pseudomonas spp., Stenotrophomonas maltophilia, Aeromonas spp., Chromobacterium haemolyticum, Shewanella xiamenensis, and members of Enterobacteriaceae. Carbapenem-resistant bacteria were highly resistant to other beta-lactams such as quinolones, aminoglycosides, chloramphenicol, tetracyclines, and sulfamethoxazole/trimethoprim. Carbapenem resistance was mostly associated with intrinsically resistant bacteria. As intrinsic resistance mechanisms, we have identified the blaCphA gene in 77.3% of Aeromonas spp., blaL1 in all S. maltophilia, and blaOXA-48-like in all S. xiamenensis. As acquired resistance mechanisms, we have detected the blaVIM-2 gene in six Pseudomonas spp. (5.45%). Integrons with gene cassettes encoding resistance to aminoglycosides (aacA and aacC genes), trimethoprim (dfrB1b), and carbapenems (blaVIM-2) were found in Pseudomonas spp. Results suggest that carbapenem resistance dissemination in riverine ecosystems is still at an early stage. Nevertheless, monitoring these aquatic compartments for the presence of resistance genes and its host organisms is essential to outline strategies to minimize resistance dissemination.
KeywordMeSH Terms
Water Microbiology
348. Harayama  S, Rekik  M,     ( 1989 )

Bacterial aromatic ring-cleavage enzymes are classified into two different gene families.

The Journal of biological chemistry 264 (26)
PMID : 2670937  :  
Abstract >>
Dioxygenases that catalyze the cleavage of the aromatic ring are classified into two groups according to their mode of ring fission. Substrates of ring-cleavage dioxygenases usually contain hydroxyl groups on adjacent aromatic carbons, and intradiol enzymes cleave the ring between these two hydroxyl groups. Extradiol enzymes in contrast cleave the ring between one hydroxylated carbon and its adjacent nonhydroxylated carbon. In this study, we determined the complete nucleotide sequence of nahC, the structural gene for 1,2-dihydroxynaphthalene dioxygenase encoded in the NAH7 plasmid of Pseudomonas putida. This enzyme is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene. The amino acid sequence of the dioxygenase deduced from the DNA sequence demonstrated that the molecular weight of the enzyme is 33,882. This result was in agreement with those of maxicell analyses that showed that the nahC product was a 36-kDa protein. Interestingly, the amino acid sequence of 1,2-dihydroxynaphthalene dioxygenase was 50% homologous with that of 2,3-dihydroxybiphenyl dioxygenase, which catalyzes extradiol cleavage of the first ring of 2,3-dihydroxybiphenyl (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429). The amino acid sequence similarity of 1,2-dihydroxynaphthalene dioxygenase with catechol 2,3-dioxygenase, which is an authentic extradiol dioxygenase, was rather low (16%). However, a statistical analysis by the method of S. B. Needleman and C. D. Wunsch [1970) J. Mol. Biol. 48, 443-453) clearly showed that these two dioxygenases are evolutionarily related. Therefore, these extradiol enzymes are considered as products of the same gene superfamily. From the significant sequence similarity between intradiol enzymes, it has been shown (Neidle, E. L., Harnett, C., Bonitz, S., and Ornston, L. N. (1988) J. Bacteriol. 170, 4874-4880) that intradiol enzymes evolved from a common ancestor. Comparison of the amino acid sequence of extradiol enzymes with those of intradiol dioxygenases did not show any significant global or localized similarity.
KeywordMeSH Terms
Dioxygenases
Genes
Genes, Bacterial
349. Boronin  AM, Tsoĭ  TV, Kosheleva  IA, Arinbasarov  MU, Adanin  VM,     ( 1989 )

[Cloning of Pseudomonas putida genes responsible for the primary stages of oxidation of naphthalene in Escherichia coli cells].

Genetika 25 (2)
PMID : 2661326  :  
Abstract >>
Data on cloning Pseudomonas putida D-plasmid pBS286 (IncP-9) genes which are responsible for primary stages of naphthalene oxidation as well as data on the expression of these genes in Escherichia coli cells are presented. Recombinant plasmid pBS959 containing the whole constitutive nahA locus encoding naphthalene dioxygenase, a key enzyme of the naphthalene oxidation pathway, has been constructed on the basis of the pUC19 vector. An evidence has been obtained that at least a portion of the sequence of structural nahB gene is cloned in the recombinant pBS959 plasmid. Constitutive expression of the nahA gene is controlled by its own promoter and leads to conversion of indole to indigo in E. coli cells, strain HB101. Plasmid pBS959 is characterized by high structural stability; some instability observed is due to segregation.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
350. Inouye  S, Yamada  M, Nakazawa  A, Nakazawa  T,     ( 1989 )

Cloning and sequence analysis of the ntrA (rpoN) gene of Pseudomonas putida.

Gene 85 (1)
PMID : 2695395  :   DOI  :   10.1016/0378-1119(89)90474-5    
Abstract >>
The gene encoding a sigma factor NtrA (RpoN) was cloned from Pseudomonas putida by cross-hybridization with a probe containing a part of the corresponding Escherichia coli gene. The cloned gene complemented an ntrA mutation of E. coli in activation of xyl genes on the TOL plasmid. The predicted amino acid (aa) sequence of P. putida NtrA (497 aa; Mr 56,215) is highly homologous to NtrA proteins from Azotobacter vinelandii (81.7%), Klebsiella pneumoniae (52.6%), and Rhizobium meliloti (36.1%). There are two other open reading frames (ORF1, ORF3) upstream and downstream from the ntrA gene, respectively. These ntrA-flanking ORFs are also highly homologous to the corresponding ORFs of K. pneumoniae, A. vinelandii, and R. meliloti.
KeywordMeSH Terms
Genes, Bacterial
351. Xia  Z, Zhang  W, Lei  L, Liu  X, Wei  HL,     ( 2015 )

Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis.

Applied microbiology and biotechnology 99 (15)
PMID : 25808517  :   DOI  :   10.1007/s00253-015-6529-x    
Abstract >>
Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.
KeywordMeSH Terms
Mutagenesis, Insertional
352. Isupov  MN, Schröder  E, Gibson  RP, Beecher  J, Donadio  G, Saneei  V, Dcunha  SA, McGhie  EJ, Sayer  C, Davenport  CF, Lau  PC, Hasegawa  Y, Iwaki  H, Kadow  M, Balke  K, Bornscheuer  UT, Bourenkov  G, Littlechild  JA,     ( 2015 )

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

Acta crystallographica. Section D, Biological crystallography 71 (Pt 11)
PMID : 26527149  :   DOI  :   10.1107/S1399004715017939     PMC  :   PMC4631483    
Abstract >>
The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 ? resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a �]-bulge at the C-terminus of �]-strand 3, which is a feature observed in many proteins of this superfamily.
KeywordMeSH Terms
FMN-dependent monooxygenase
industrial biocatalysis
protein structure
353. Ghosal  D, You  IS,     ( 1989 )

Operon structure and nucleotide homology of the chlorocatechol oxidation genes of plasmids pJP4 and pAC27.

Gene 83 (2)
PMID : 2583528  :   DOI  :   10.1016/0378-1119(89)90108-x    
Abstract >>
Alcaligenes eutrophus harboring plasmid pJP4 (strain JMP134) is capable of growing on both 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba), while Pseudomonas putida carrying plasmid pAC27 (strain AC867) can utilize only 3-Cba as the sole carbon source. The tfdCDEF operon of the pJP4 plasmid and the clcABD operon of plasmid pAC27 each encode enzymes for the degradation of chlorocatechols (Clc), key intermediates in the catabolism of 2,4-D and 3-Cba. Similarities in the nucleotide (nt) sequences of genes tfdC and clcA, encoding pyrocatechases, were reported earlier [Ghosal and You, Mol. Gen. Genet. 211 (1988a) 113-120]. Genes tfdD and clcB, encoding Clc-specific cycloisomerases, have been completely sequenced. The tfdD gene (1107 bp) is slightly smaller than gene clcB (1113 bp). Comparison of the two cycloisomerase-encoding genes reveals that the nt sequences are 63% homologous with 62% homology in the deduced amino acid (aa) sequences of the polypeptides they encode. Genes tfdD and tfdE are contiguous in the tfdCDEF operon, whereas the corresponding genes, clcB and clcD, of the clcABD operon, are known to be separated by a long open reading frame of unknown function. The predicted N-terminal aa sequences of the two hydrolase-encoding genes, tfdE and clcD, also show homology. The structural and nt homologies between the two Clc operons, tfdCDEF and clcABD, suggest their relatedness.
KeywordMeSH Terms
Genes, Bacterial
Operon
Plasmids
354. Knaus  T, Paul  CE, Levy  CW, de Vries  S, Mutti  FG, Hollmann  F, Scrutton  NS,     ( 2016 )

Better than Nature: Nicotinamide Biomimetics That Outperform Natural Coenzymes.

Journal of the American Chemical Society 138 (3)
PMID : 26727612  :   DOI  :   10.1021/jacs.5b12252     PMC  :   PMC4731831    
Abstract >>
The search for affordable, green biocatalytic processes is a challenge for chemicals manufacture. Redox biotransformations are potentially attractive, but they rely on unstable and expensive nicotinamide coenzymes that have prevented their widespread exploitation. Stoichiometric use of natural coenzymes is not viable economically, and the instability of these molecules hinders catalytic processes that employ coenzyme recycling. Here, we investigate the efficiency of man-made synthetic biomimetics of the natural coenzymes NAD(P)H in redox biocatalysis. Extensive studies with a range of oxidoreductases belonging to the "ene" reductase family show that these biomimetics are excellent analogues of the natural coenzymes, revealed also in crystal structures of the ene reductase XenA with selected biomimetics. In selected cases, these biomimetics outperform the natural coenzymes. "Better-than-Nature" biomimetics should find widespread application in fine and specialty chemicals production by harnessing the power of high stereo-, regio-, and chemoselective redox biocatalysts and enabling reactions under mild conditions at low cost.
KeywordMeSH Terms
355. Uemura  T, Kita  A, Watanabe  Y, Adachi  M, Kuroki  R, Morimoto  Y,     ( 2016 )

The catalytic mechanism of decarboxylative hydroxylation of salicylate hydroxylase revealed by crystal structure analysis at 2.5 ? resolution.

Biochemical and biophysical research communications 469 (2)
PMID : 26616054  :   DOI  :   10.1016/j.bbrc.2015.11.087    
Abstract >>
The X-ray crystal structure of a salicylate hydroxylase from Pseudomonas putida S-1 complexed with coenzyme FAD has been determined to a resolution of 2.5 ?. Structural conservation with p- or m-hydroxybenzoate hydroxylase is very good throughout the topology, despite a low amino sequence identity of 20-40% between these three hydroxylases. Salicylate hydroxylase is composed of three distinct domains and includes FAD between domains I and II, which is accessible to solvent. In this study, which analyzes the tertiary structure of the enzyme, the unique reaction of salicylate, i.e. decarboxylative hydroxylation, and the structural roles of amino acids surrounding the substrate, are considered.
KeywordMeSH Terms
Decarboxylative hydroxylation
Enzyme
Flavin-dependent
Salicylate hydroxylase
X-ray analysis
Models, Chemical
Models, Molecular
356. Hui Bon Hoa  G, Di Primo  C, Dondaine  I, Sligar  SG, Gunsalus  IC, Douzou  P,     ( 1989 )

Conformational changes of cytochromes P-450cam and P-450lin induced by high pressure.

Biochemistry 28 (2)
PMID : 2578028  :   DOI  :   10.1021/bi00428a035    
Abstract >>
Absorbance and fluorescence spectra of bacterial cytochrome P-450cam and cytochrome P-450lin have been studied as a function of pressure. These pressure-induced spectral perturbations fall into two categories, which are interpreted as resulting from denaturation domains and are discussed in terms of protein structural dynamics. The results presented herein support a view that these two bacterial cytochromes have large structural differences and suggest a picture in which the gellike cortex of each protein may play an essential role in stability and function.
KeywordMeSH Terms
357. Chao  HJ, Chen  YF, Fang  T, Xu  Y, Huang  WE, Zhou  NY,     ( 2016 )

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866.

Applied and environmental microbiology 82 (2)
PMID : 26567311  :   DOI  :   10.1128/AEM.03105-15     PMC  :   PMC4711120    
Abstract >>
In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg(-1) and showed apparent Km values of 11.40 �� 3.05 �gM for 4-hydroxyisophthalate with NADPH and 11.23 �� 2.43 �gM with NADH and similar Km values for NADPH and NADH (64.31 �� 13.16 and 72.76 �� 12.06 �gM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.
KeywordMeSH Terms
358. Kok  M, Oldenhuis  R, van der Linden  MP, Meulenberg  CH, Kingma  J, Witholt  B,     ( 1989 )

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase.

The Journal of biological chemistry 264 (10)
PMID : 2647719  :  
Abstract >>
The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.
KeywordMeSH Terms
Genes
Genes, Bacterial
Operon
359. Kok  M, Oldenhuis  R, van der Linden  MP, Raatjes  P, Kingma  J, van Lelyveld  PH, Witholt  B,     ( 1989 )

The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression.

The Journal of biological chemistry 264 (10)
PMID : 2647718  :  
Abstract >>
We have identified and sequenced the Pseudomonas OCT plasmid-encoded alkane hydroxylase gene (alkB) and its promoter. The transcription initiation site of the alkBAC mRNA was determined by nuclease S1 mapping. A putative interaction site with RNA-polymerase was identified based on homology of the alk promoter with other Pseudomonas promoters. The alkB gene encodes a 401-amino acid polypeptide which, despite an unusual codon composition, can be expressed at high levels in Escherichia coli and Pseudomonas. The amino-terminal sequence of the purified cytoplasmic membrane alkane hydroxylase was determined and was found to be in agreement with the nucleotide sequence. The translation product of the alkB gene contains nine hydrophobic sequences of which eight are sufficiently long to be membrane-spanning segments. The amino-terminal sequence resembles that of several bacterial integral membrane proteins and is not cleaved off following translation.
KeywordMeSH Terms
Genes
Genes, Bacterial
Transcription, Genetic
360. Felux  AK, Spiteller  D, Klebensberger  J, Schleheck  D,     ( 2015 )

Entner-Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1.

Proceedings of the National Academy of Sciences of the United States of America 112 (31)
PMID : 26195800  :   DOI  :   10.1073/pnas.1507049112     PMC  :   PMC4534283    
Abstract >>
Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD(+)-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)(+)-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.
KeywordMeSH Terms
6-deoxy-6-sulfoglucose
bacterial biodegradation
organosulfonate
sulfolipid
sulfur cycle
Metabolic Networks and Pathways
361. Koga  H, Yamaguchi  E, Matsunaga  K, Aramaki  H, Horiuchi  T,     ( 1989 )

Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida.

Journal of biochemistry 106 (5)
PMID : 2613690  :   DOI  :   10.1093/oxfordjournals.jbchem.a122939    
Abstract >>
Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin. The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al. (1986) J. Bacteriol. 166, 1089-1095). Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB). These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.
KeywordMeSH Terms
362. Sheoran  N, Valiya Nadakkakath  A, Munjal  V, Kundu  A, Subaharan  K, Venugopal  V, Rajamma  S, Eapen  SJ, Kumar  A,     ( 2015 )

Genetic analysis of plant endophytic Pseudomonas putida BP25 and chemo-profiling of its antimicrobial volatile organic compounds.

Microbiological research 173 (N/A)
PMID : 25801973  :   DOI  :   10.1016/j.micres.2015.02.001    
Abstract >>
Black pepper associated bacterium BP25 was isolated from root endosphere of apparently healthy cultivar Panniyur-5 that protected black pepper against Phytophthora capsici and Radopholus similis - the major production constraints. The bacterium was characterized and mechanisms of its antagonistic action against major pathogens are elucidated. The polyphasic phenotypic analysis revealed its identity as Pseudomonas putida. Multi locus sequence typing revealed that the bacterium shared gene sequences with several other isolates representing diverse habitats. Tissue localization assays exploiting green fluorescence protein expression clearly indicated that PpBP25 endophytically colonized not only its host plant - black pepper, but also other distantly related plants such as ginger and arabidopsis. PpBP25 colonies could be enumerated from internal tissues of plants four weeks post inoculation indicated its stable establishment and persistence in the plant system. The bacterium inhibited broad range of pathogens such as Phytophthora capsici, Pythium myriotylum, Giberella moniliformis, Rhizoctonia solani, Athelia rolfsii, Colletotrichum gloeosporioides and plant parasitic nematode, Radopholus similis by its volatile substances. GC/MS based chemical profiling revealed presence of Heneicosane; Tetratetracontane; Pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); Tetracosyl heptafluorobutyrate; 1-3-Eicosene, (E)-; 1-Heneicosanol; Octadecyl trifluoroacetate and 1-Pentadecene in PpBP25 metabolite. Dynamic head space GC/MS analysis of airborne volatiles indicated the presence of aromatic compounds such as 1-Undecene;Disulfide dimethyl; Pyrazine, methyl-Pyrazine, 2,5-dimethyl-; Isoamyl alcohol; Pyrazine, methyl-; Dimethyl trisulfide, etc. The work paved way for profiling of broad spectrum antimicrobial VOCs in endophytic PpBP25 for crop protection.
KeywordMeSH Terms
Black pepper
Endophytic bacteria
Ginger
Pseudomonas putida
Volatile organic compounds
363. Jensen  JL, Balbo  A, Neau  DB, Chakravarthy  S, Zhao  H, Sinha  SC, Colbert  CL,   &