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1. Hearn  EM, Dennis  JJ, Gray  MR, Foght  JM,     ( 2003 )

Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a.

Journal of bacteriology 185 (21)
PMID : 14563857  :   DOI  :   10.1128/jb.185.21.6233-6240.2003     PMC  :   PMC219392    
Abstract >>
The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.
KeywordMeSH Terms
2. Schreuder  HA, van der Laan  JM, Swarte  MB, Kalk  KH, Hol  WG, Drenth  J,     ( 1992 )

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

Proteins 14 (2)
PMID : 1409567  :   DOI  :   10.1002/prot.340140205    
Abstract >>
The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.
KeywordMeSH Terms
3. Hong  KH, Jang  WH, Choi  KD, Yoo  OJ,     ( 1991 )

Characterization of Pseudomonas fluorescens carboxylesterase: cloning and expression of the esterase gene in Escherichia coli.

Agricultural and biological chemistry 55 (11)
PMID : 1368750  :  
Abstract >>
The Pseudomonas fluorescens gene (estB) that encodes a novel esterase (esterase II) was cloned into Escherichia coli JM83. DNA sequencing found a single open reading frame of 654 nucleotides. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the esterase protein. A potential Shine-Dalgarno sequence is followed by the coding sequence of the estB gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterases. The enzyme expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. Homogeneity of the purified enzyme was confirmed using SDS-polyacrylamide gel electrophoresis. The native enzyme exists as a dimer consisting of two identical subunits, each with a molecular weight of 23,000. The results of the experiments for identifying substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3.1.1.1) and a serine residue is present at the active site of the esterase, as in the esterases of animal tissues.
KeywordMeSH Terms
4. Gimmestad  M, Sletta  H, Ertesvåg  H, Bakkevig  K, Jain  S, Suh  SJ, Skjåk-Braek  G, Ellingsen  TE, Ohman  DE, Valla  S,     ( 2003 )

The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.

Journal of bacteriology 185 (12)
PMID : 12775688  :   DOI  :   10.1128/jb.185.12.3515-3523.2003     PMC  :   PMC156231    
Abstract >>
Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.
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5. Chung  GH, Lee  YP, Jeohn  GH, Yoo  OJ, Rhee  JS,     ( 1991 )

Cloning and nucleotide sequence of thermostable lipase gene from Pseudomonas fluorescens SIK W1.

Agricultural and biological chemistry 55 (9)
PMID : 1368740  :  
Abstract >>
A gene coding for a thermostable lipase of Pseudomonas fluorescens SIK W1 was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. Twenty colonies with esterase activity on the tributyrin agar plate were isolated by screening the constructed Pseudomonas fluorescens genomic library. Only one out of the esterase positive 20 colonies had lipase activity on the agar plate containing olive oil and Rhodamine-B. The complete nucleotide sequence of the lipase gene was identified. The lipase gene consists of an open reading frame, 1347bp long, commencing with an ATG start codon encoding a polypeptide of 449 amino acid residues and a TGA stop codon. Comparison of this lipase amino acid sequence with those from another organisms sequenced to data showed the presence of the short homologous region Gly-X-Ser-X-Gly.
KeywordMeSH Terms
6. El-Sayed  AK, Hothersall  J, Cooper  SM, Stephens  E, Simpson  TJ, Thomas  CM,     ( 2003 )

Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586.

Chemistry & biology 10 (5)
PMID : 12770824  :  
Abstract >>
The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
7. Braker  G, Tiedje  JM,     ( 2003 )

Nitric oxide reductase (norB) genes from pure cultures and environmental samples.

Applied and environmental microbiology 69 (6)
PMID : 12788753  :   DOI  :   10.1128/aem.69.6.3476-3483.2003     PMC  :   PMC161466    
Abstract >>
A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria. norB database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB). The latter oxidizes cytochrome c, and the gene is localized adjacent to norC. While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains. These primer sets also specifically amplified norB from freshwater and marine sediments. Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB. Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set. Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms. cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.
KeywordMeSH Terms
8. Choi  KD, Jeohn  GH, Rhee  JS, Yoo  OJ,     ( 1990 )

Cloning and nucleotide sequence of an esterase gene from Pseudomonas fluorescens and expression of the gene in Escherichia coli.

Agricultural and biological chemistry 54 (8)
PMID : 1368608  :  
Abstract >>
A gene coding for a novel esterase of Pseudomonas fluorescens was cloned in this study. DNA sequencing showed that the open reading frame is comprised of 708 nucleotides. The coding sequence of the gene is preceded by a potential Shine-Dalgarno sequence and by a promoter-like structure. Following the stop codon a structure reminiscent of the E. coli rho-independent terminator is present. The enzyme expressed in an E. coli clone was mostly in the periplasmic space, released to the outside of the cell by osmotic shock and purified to homogeneity by QAE-Sephadex A-50 and DEAE-Sepharose columns. The native form of the enzyme consisted of two identical subunits, each with a molecular weight of 27,000. By studying the properties and substrate specificity, the enzyme was classified as an arylesterase (EC 3.1.1.2).
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9. Gilmartin  N, Ryan  D, Sherlock  O, Dowling  D,     ( 2003 )

BphK shows dechlorination activity against 4-chlorobenzoate, an end product of bph-promoted degradation of PCBs.

FEMS microbiology letters 222 (2)
PMID : 12770715  :   DOI  :   10.1016/S0378-1097(03)00309-4    
Abstract >>
A bphK gene encoding glutathione S-transferase (GST) activity is located in the bph operon in Burkholderia sp. strain LB400 but its role in polychlorinated biphenyl (PCB) metabolism is unknown. This gene was over-expressed in Escherichia coli and an in vivo assay based on growth of E. coli containing GST activity was used to identify potential novel substrates for this enzyme. Using this assay, 4-chlorobenzoate (4-CBA) was identified as a substrate for the BphK enzyme. High pressure liquid chromatography analysis and chloride ion detection showed removal of 4-CBA and an equivalent increase of chloride in cell extracts when incubated with this enzyme. These results would indicate that this BphK enzyme has dechlorination activity in relation to 4-CBA and may have a role in protection of other Bph enzymes against certain chlorinated metabolites of PCB degradation.
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10. Yanagisawa  T, Kawakami  M,     ( 2003 )

How does Pseudomonas fluorescens avoid suicide from its antibiotic pseudomonic acid?: Evidence for two evolutionarily distinct isoleucyl-tRNA synthetases conferring self-defense.

The Journal of biological chemistry 278 (28)
PMID : 12672810  :   DOI  :   10.1074/jbc.M302633200    
Abstract >>
Two isoleucyl-tRNA synthetases (IleRSs) encoded by two distinct genes (ileS1 and ileS2) were identified in pseudomonic acid (mupirocin)-producing Pseudomonas fluorescens. The most striking difference between the two IleRSs (IleRS-R1 and IleRS-R2) is the difference in their abilities to resist pseudomonic acid. Purified IleRS-R2 showed no sensitivity to pseudomonic acid even at a concentration of 5 mm, 105 times higher than the Ki value of IleRS-R1. The amino acid sequence of IleRS-R2 exhibits eukaryotic features that are originally found in eukaryotic proteins. Escherichia coli cells transformed with the ileS2 gene exerted pseudomonic acid resistance more than did those transformed with ileS1. Cells transformed with both genes became almost as resistant as P. fluorescens. These results suggest that the presence of IleRS-R2 could be the major reason why P. fluorescens is intrinsically resistant to the antibiotic. Here we suggest that the evolutionary scenario of the eukaryotic ileS2 gene can be explained by gene acquisition and that the pseudomonic acid producer may have maintained the ileS2 gene to protect itself from pseudomonic acid.
KeywordMeSH Terms
Bacterial Physiological Phenomena
11. Iwamoto  R, Amano  C, Ikehara  K, Ushida  N,     ( 2003 )

The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity.

Biochimica et biophysica acta 1647 (1��2��)
PMID : 12686150  :   DOI  :   10.1016/s1570-9639(03)00079-7    
Abstract >>
The complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P. fluorescens as a template. The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa. The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%). The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E. coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity. The TrxR from E. coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.
KeywordMeSH Terms
12. Rangaswamy  V, Hernández-Guzmán  G, Shufran  KA, Bender  CL,     ( 2002 )

Analysis of rILERS, an isoleucyl-tRNA synthetase gene associated with mupirocin production by Pseudomonas fluorescens NCIMB 10586.

DNA sequence : the journal of DNA sequencing and mapping 13 (6)
PMID : 12652905  :  
Abstract >>
Some strains of Pseudomonas fluorescens produce the antibiotic mupirocin, which functions as a competitive inhibitor of isoleucyl-tRNA synthetase (ILERS). Mupirocin-producing strains of P. fluorescens must overcome the inhibitory effects of the antibiotic to avoid self-suicide. However, it is not clear how P. fluorescens protects itself from the toxic effects of mupirocin. In this report, we describe a second gene encoding isoleucyl-tRNA synthetase (rILERS) in P. fluorescens that is associated with the mupirocin biosynthetic gene cluster. Random mutagenesis of the mupirocin-producing strain, P. fluorescens 10586, resulted in a mupirocin-defective mutant disrupted in a region with similarity to ILERS, the target site for mupirocin. The ILERS gene described in the present study was sequenced and shown to be encoded by a 3093 bp ORF, which is 264 bp larger than the ILERS gene previously identified in P. fluorescens 10586. rILERS from P. fluorescens is most closely related to prokaryotic or eukaryotic sources of ILERS that are resistant to mupirocin. Interestingly, the relatedness between rILERS and the ILERS previously described in P. fluorescens 10586 was low (24% similarity), which indicates that P. fluorescens contains two isoforms of isoleucyl-tRNA synthetase.
KeywordMeSH Terms
13. Picard  C, Bosco  M,     ( 2003 )

Genetic diversity of phlD gene from 2,4-diacetylphloroglucinol-producing Pseudomonas spp. strains from the maize rhizosphere.

FEMS microbiology letters 219 (2)
PMID : 12620616  :   DOI  :   10.1016/S0378-1097(03)00027-2    
Abstract >>
In biocontrol Pseudomonads, phlD is an essential gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG). HaeIII restriction of amplified phlD gene, previously proposed as the most discriminant analysis, showed no polymorphism among 144 Pseudomonas strains isolated from maize roots. However, these strains fell into three statistically significant DAPG production level groups. phlD sequences of 13 strains belonging to the three DAPG groups revealed a KspI restriction site only in good DAPG-producing strains. This result was confirmed on the 144 strains, 82 of which were identified as good-DAPG producers by both biochemical and amplified phlD KspI restriction analysis. They are candidates as potential biocontrol agents.
KeywordMeSH Terms
Genetic Variation
14. Muraki  T, Taki  M, Hasegawa  Y, Iwaki  H, Lau  PC,     ( 2003 )

Prokaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase in the 2-nitrobenzoate degradation pathway of Pseudomonas fluorescens strain KU-7.

Applied and environmental microbiology 69 (3)
PMID : 12620844  :   DOI  :   10.1128/aem.69.3.1564-1572.2003     PMC  :   PMC150085    
Abstract >>
The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes. The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe(2+) but is inhibited by other metal ions, such as Zn(2+), Cu(2+), and Cd(2+). The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.
KeywordMeSH Terms
Dioxygenases
15. 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
16. Kamerbeek  NM, Olsthoorn  AJ, Fraaije  MW, Janssen  DB,     ( 2003 )

Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase.

Applied and environmental microbiology 69 (1)
PMID : 12514023  :   DOI  :   10.1128/aem.69.1.419-426.2003     PMC  :   PMC152415    
Abstract >>
The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.
KeywordMeSH Terms
17. de Weert  S, Vermeiren  H, Mulders  IH, Kuiper  I, Hendrickx  N, Bloemberg  GV, Vanderleyden  J, De Mot  R, Lugtenberg  BJ,     ( 2002 )

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens.

Molecular plant-microbe interactions : MPMI 15 (11)
PMID : 12423023  :   DOI  :   10.1094/MPMI.2002.15.11.1173    
Abstract >>
Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.
KeywordMeSH Terms
18. Wei  B, Huang  T, Dalwadi  H, Sutton  CL, Bruckner  D, Braun  J,     ( 2002 )

Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen.

Infection and immunity 70 (12)
PMID : 12438326  :   DOI  :   10.1128/iai.70.12.6567-6575.2002     PMC  :   PMC133002    
Abstract >>
Commensal bacteria have emerged as an important disease factor in human Crohn's disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome. PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species. Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene. CD4(+) T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885. pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for pfiT and P. fluorescens in the pathogenesis of Crohn's disease.
KeywordMeSH Terms
19. 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
20. Fushinobu  S, Saku  T, Hidaka  M, Jun  SY, Nojiri  H, Yamane  H, Shoun  H, Omori  T, Wakagi  T,     ( 2002 )

Crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with cleavage products.

Protein science : a publication of the Protein Society 11 (9)
PMID : 12192074  :   DOI  :   10.1110/ps.0209602     PMC  :   PMC2373588    
Abstract >>
2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 A resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues. The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.
KeywordMeSH Terms
Protein Conformation
21. Kavanagh  KL, Klimacek  M, Nidetzky  B, Wilson  DK,     ( 2002 )

Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes. Specificity and catalytic mechanism.

The Journal of biological chemistry 277 (45)
PMID : 12196534  :   DOI  :   10.1074/jbc.M206914200    
Abstract >>
Long-chain mannitol dehydrogenases are secondary alcohol dehydrogenases that are of wide interest because of their involvement in metabolism and potential applications in agriculture, medicine, and industry. They differ from other alcohol and polyol dehydrogenases because they do not contain a conserved tyrosine and are not dependent on Zn(2+) or other metal cofactors. The structures of the long-chain mannitol 2-dehydrogenase (54 kDa) from Pseudomonas fluorescens in a binary complex with NAD(+) and ternary complex with NAD(+) and d-mannitol have been determined to resolutions of 1.7 and 1.8 A and R-factors of 0.171 and 0.176, respectively. These results show an N-terminal domain that includes a typical Rossmann fold. The C-terminal domain is primarily alpha-helical and mediates mannitol binding. The electron lone pair of Lys-295 is steered by hydrogen-bonding interactions with the amide oxygen of Asn-300 and the main-chain carbonyl oxygen of Val-229 to act as the general base. Asn-191 and Asn-300 are involved in a web of hydrogen bonding, which precisely orients the mannitol O2 proton for abstraction. These residues also aid in stabilizing a negative charge in the intermediate state and in preventing the formation of nonproductive complexes with the substrate. The catalytic lysine may be returned to its unprotonated state using a rectifying proton tunnel driven by Glu-292 oscillating among different environments. Despite low sequence homology, the closest structural neighbors are glycerol-3-phosphate dehydrogenase, N-(1-d-carboxylethyl)-l-norvaline dehydrogenase, UDP-glucose dehydrogenase, and 6-phosphogluconate dehydrogenase, indicating a possible evolutionary relationship among these enzymes.
KeywordMeSH Terms
22. Mavrodi  DV, Mavrodi  OV, McSpadden-Gardener  BB, Landa  BB, Weller  DM, Thomashow  LS,     ( 2002 )

Identification of differences in genome content among phlD-positive Pseudomonas fluorescens strains by using PCR-based subtractive hybridization.

Applied and environmental microbiology 68 (10)
PMID : 12324371  :   DOI  :   10.1128/aem.68.10.5170-5176.2002     PMC  :   PMC126409    
Abstract >>
Certain 2,4-diacetylphloroglucinol-producing strains of Pseudomonas fluorescens colonize roots and suppress soilborne diseases more effectively than others from which they are otherwise phenotypically almost indistinguishable. We recovered DNA fragments present in the superior colonizer P. fluorescens Q8r1-96 but not in the less rhizosphere-competent strain Q2-87. Of the open reading frames in 32 independent Q8r1-96-specific clones, 1 was similar to colicin M from Escherichia coli, 3 resembled known regulatory proteins, and 28 had no significant match with sequences of known function. Seven clones hybridized preferentially to DNA from strains with superior rhizosphere competence, and sequences in two others were highly expressed in vitro and in the rhizosphere.
KeywordMeSH Terms
Genome, Bacterial
23. Mossialos  D, Ochsner  U, Baysse  C, Chablain  P, Pirnay  JP, Koedam  N, Budzikiewicz  H, Fernández  DU, Schäfer  M, Ravel  J, Cornelis  P,     ( 2002 )

Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine.

Molecular microbiology 45 (6)
PMID : 12354233  :   DOI  :   10.1046/j.1365-2958.2002.03120.x    
Abstract >>
Pyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore. While it has been shown that non-ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo-phore part. We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine. Transcriptional lacZ fusions showed that pvdL is co-transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron-regulated via PvdS. Similarly, RT-PCR analysis revealed that expression of pvsA is repressed by iron. Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N-terminus starts with an acyl-CoA ligase module, followed by three amino acid activation domains. Computer modelling of these domains suggests that PvsA in P. fluorescens and PvdL in P. aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore.
KeywordMeSH Terms
Oligopeptides
24. 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
25. Camacho Carvajal  MM, Wijfjes  AH, Mulders  IH, Lugtenberg  BJ, Bloemberg  GV,     ( 2002 )

Characterization of NADH dehydrogenases of Pseudomonas fluorescens WCS365 and their role in competitive root colonization.

Molecular plant-microbe interactions : MPMI 15 (7)
PMID : 12118882  :   DOI  :   10.1094/MPMI.2002.15.7.662    
Abstract >>
The excellent-root-colonizing Pseudomonas fluorescens WCS365 was selected previously as the parental strain for the isolation of mutants impaired in root colonization. Transposon mutagenesis of WCS365 and testing for root colonization resulted in the isolation of mutant strain PCL1201, which is approximately 100-fold impaired in competitive tomato root colonization. In this manuscript, we provide evidence that shows that the lack of NADH dehydrogenase I, an enzyme of the aerobic respiratory chain encoded by the nuo operon, is responsible for the impaired root-colonization ability of PCL1201. The complete sequence of the nuo operon (ranging from nuoA to nuoN) of P. fluorescens WCS365 was identified, including the promoter region and a transcriptional terminator consensus sequence downstream of nuoN. It was shown biochemically that PCL1201 is lacking NADH dehydrogenase I activity. In addition, the presence and activity of a second NADH dehydrogenase, encoded by the ndh gene, was identified to our knowledge for the first time in the genus Pseudomonas. Since it was assumed that low-oxygen conditions were present in the rhizosphere, we analyzed the activity of the nuo and the ndh promoters at different oxygen tensions. The results showed that both promoters are up-regulated by low concentrations of oxygen and that their levels of expression vary during growth. By using lacZ as a marker, it was shown that both the nuo operon and the ndh gene are expressed in the tomato rhizosphere. In contrast to the nuo mutant PCL1201, an ndh mutant of WCS365 appeared not to be impaired in competitive root tip colonization.
KeywordMeSH Terms
26. Hildebrandt  P, Musidlowska  A, Bornscheuer  UT, Altenbuchner  J,     ( 2002 )

Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106.

Applied microbiology and biotechnology 59 (4��5��)
PMID : 12172614  :   DOI  :   10.1007/s00253-002-1036-2    
Abstract >>
Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects. The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa. The gene was functionally expressed in E. coli using a rhamnose inducible expression system. The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C. This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH. The highest activity was found for cyclohexanone. The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure (R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%).
KeywordMeSH Terms
Cloning, Molecular
27. Heeb  S, Blumer  C, Haas  D,     ( 2002 )

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

Journal of bacteriology 184 (4)
PMID : 11807065  :   DOI  :   10.1128/jb.184.4.1046-1056.2002     PMC  :   PMC134805    
Abstract >>
In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.
KeywordMeSH Terms
Genes, Regulator
28. Spiers  AJ, Kahn  SG, Bohannon  J, Travisano  M, Rainey  PB,     ( 2002 )

Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness.

Genetics 161 (1)
PMID : 12019221  :   PMC  :   PMC1462107    
Abstract >>
A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known. When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge. Among the divergent morphs is a mutant type termed "wrinkly spreader" (WS) that colonizes a new niche through the formation of self-supporting biofilms. Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS(-)) mutants. Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP). WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS. A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation. Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.
KeywordMeSH Terms
Adaptation, Physiological
Evolution, Molecular
29. Abbas  A, Morrissey  JP, Marquez  PC, Sheehan  MM, Delany  IR, O'Gara  F,     ( 2002 )

Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113.

Journal of bacteriology 184 (11)
PMID : 12003942  :   DOI  :   10.1128/jb.184.11.3008-3016.2002     PMC  :   PMC135055    
Abstract >>
The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile. Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P. fluorescens F113 phlF mutant. The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a sigma(70) RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene. The repression by PhlF is due to its interaction with an inverted repeated sequence, phO, located downstream of the phlA transcriptional start site. Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind phO as a dimer or higher-order complex. Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to phO. PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-phO complex. Conversely, the PhlF-phO complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of phlA expression.
KeywordMeSH Terms
30. Morea  A, Mathee  K, Franklin  MJ, Giacomini  A, O'Regan  M, Ohman  DE,     ( 2001 )

Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens.

Gene 278 (1��2��)
PMID : 11707327  :   DOI  :   10.1016/s0378-1119(01)00685-0    
Abstract >>
The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn501) in the alginate biosynthetic operon that rendered it non-mucoid. Phenotypic complementation in this heterologous host was observed, and a complementing clone containing 32 kb of P. fluorescens DNA was obtained. Southern hybridization studies showed that genes involved in alginate biosynthesis (e.g. algD, algG, and algA) were approximately in the same order and position as in P. aeruginosa. When the clone was mobilized to a P. aeruginosa algG mutant that produced alginate as polymannuronate due to its C5-epimerase defect, complementation was observed and the alginate from the recombinant strain contained L-guluronate as determined by proton nuclear magnetic resonance spectroscopy. A sequence analysis of the P. fluorescens DNA containing algG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence of P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aeruginosa, AlgG from P. fluorescens appeared to have a signal sequence that would localize it to the periplasm where AlgG presumably acts as a C5-epimerase at the polymer level. Non-polar algG knockout mutants of P. fluorescens were defective in alginate production, suggesting a potential role for this protein in polymer formation.
KeywordMeSH Terms
31. Schnider-Keel  U, Lejbølle  KB, Baehler  E, Haas  D, Keel  C,     ( 2001 )

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

Applied and environmental microbiology 67 (12)
PMID : 11722923  :   DOI  :   10.1128/AEM.67.12.5683-5693.2001     PMC  :   PMC93360    
Abstract >>
A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.
KeywordMeSH Terms
Desiccation
Sigma Factor
32. Sánchez-Contreras  M, Martín  M, Villacieros  M, O'Gara  F, Bonilla  I, Rivilla  R,     ( 2002 )

Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113.

Journal of bacteriology 184 (6)
PMID : 11872710  :   DOI  :   10.1128/jb.184.6.1587-1596.2002     PMC  :   PMC134892    
Abstract >>
During colonization of the alfalfa rhizosphere, Pseudomonas fluorescens F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology. Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology. Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant. The motility behavior correlated with overproduction of the fliC-encoded protein flagellin but not with hyperflagellation. Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level. Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned gacA. Sequence analysis of the gacA alelle in variant F suggested selection of the phenotype in the rhizosphere. Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the gacA or gacS gene. Mutation of the F113 sss gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization.
KeywordMeSH Terms
Pseudomonas fluorescens
33. 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
34. Preston  GM, Bertrand  N, Rainey  PB,     ( 2001 )

Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25.

Molecular microbiology 41 (5)
PMID : 11555282  :   DOI  :   10.1046/j.1365-2958.2001.02560.x    
Abstract >>
In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Plant Development
35. 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
36. Smits  TH, Balada  SB, Witholt  B, van Beilen  JB,     ( 2002 )

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria.

Journal of bacteriology 184 (6)
PMID : 11872725  :   DOI  :   10.1128/jb.184.6.1733-1742.2002     PMC  :   PMC134907    
Abstract >>
We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.
KeywordMeSH Terms
37. Bellingham  NF, Morgan  JA, Saunders  JR, Winstanley  C,     ( 2001 )

Flagellin gene sequence variation in the genus Pseudomonas.

Systematic and applied microbiology 24 (2)
PMID : 11518318  :   DOI  :   10.1078/0723-2020-00031    
Abstract >>
Flagellin gene (fliC) sequences from 18 strains of Pseudomonas sensu stricto representing 8 different species, and 9 representative fliC sequences from other members of the gamma sub-division of proteobacteria, were compared. Analysis was performed on N-terminal, C-terminal and whole fliC sequences. The fliC analyses confirmed the inferred relationship between P. mendocina, P. oleovorans and P. aeruginosa based on 16S rRNA sequence comparisons. In addition, the analyses indicated that P. putida PRS2000 was closely related to P. fluorescens SBW25 and P. fluorescens NCIMB 9046T, but suggested that P. putida PaW8 and P. putida PRS2000 were more closely related to other Pseudomonas spp. than they were to each other. There were a number of inconsistencies in inferred evolutionary relationships between strains, depending on the analysis performed. In particular, whole flagellin gene comparisons often differed from those obtained using N- and C-terminal sequences. However, there were also inconsistencies between the terminal region analyses, suggesting that phylogenetic relationships inferred on the basis of fliC sequence should be treated with caution. Although the central domain of fliC is highly variable between Pseudomonas strains, there was evidence of sequence similarities between the central domains of different Pseudomonas fliC sequences. This indicates the possibility of recombination in the central domain of fliC genes within Pseudomonas species, and between these genes and those from other bacteria.
KeywordMeSH Terms
Genetic Variation
Sequence Analysis, DNA
38. Peters  M, Jõgi  E, Suitso  I, Punnisk  T, Nurk  A,     ( 2001 )

Features of the replicon of plasmid pAM10.6 of Pseudomonas fluorescens.

Plasmid 46 (1)
PMID : 11535033  :   DOI  :   10.1006/plas.2001.1524    
Abstract >>
We describe features of the basic replicon of the 10.6-kb medium-copy-number plasmid pAM10.6. pAM10.6 was able to replicate in various Pseudomonas strains but was maintained in Escherichia coli only after the p15A origin of replication was inserted. Deletion analysis suggests that the pAM10.6 origin of replication is located in a 0.5-kb region that includes inverted and direct repeats upstream of the repA gene. RepA (204 aa) has a clear homology to plasmid replication proteins of some other gram-negative bacteria. The pas (plasmid addiction system) (genes encoded in the region of 480-bp) stabilizes plasmid maintenance in P. putida cells under nonselective conditions for at least 200 generations. A 3.75-kb PstI fragment of pAM10.6 joined to a Km(r) gene was shown to be a minimal plasmid unit maintained in P. putida as a monomer. Further deletions of this 3.75-kb fragment caused a drive to form stable head-to-tail dimeric plasmids in P. putida.
KeywordMeSH Terms
DNA, Bacterial
Plasmids
Replicon
39. Kuiper  I, Bloemberg  GV, Noreen  S, Thomas-Oates  JE, Lugtenberg  BJ,     ( 2001 )

Increased uptake of putrescine in the rhizosphere inhibits competitive root colonization by Pseudomonas fluorescens strain WCS365.

Molecular plant-microbe interactions : MPMI 14 (9)
PMID : 11551074  :   DOI  :   10.1094/MPMI.2001.14.9.1096    
Abstract >>
Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.
KeywordMeSH Terms
40. Peters  M, Heinaru  A, Nurk  A,     ( 2001 )

Plasmid-encoded catalase KatA, the main catalase of Pseudomonas fluorescens strain Cb36.

FEMS microbiology letters 200 (2)
PMID : 11425481  :   DOI  :   10.1111/j.1574-6968.2001.tb10721.x    
Abstract >>
The plasmid-state catalase gene katA of the phenol gradative Pseudomonas fluorescens isolate Cb36 has been characterized and shown to be the major catalase of this strain. The predicted amino acid sequence of KatA revealed significant similarity with the catalase sequence from Neisseria meningitidis and has probably the non-pseudomonad origin. The specific activity of catalase was investigated and elevated catalase activity was found in stationary phase cells. The consensus sequence for promoters recognized by the stationary phase sigma factor sigma(s) was found 212 bp upstream of the putative ATG start codon. The ability of KatA to detoxify a high concentration of hydrogen peroxide and protect Pseudomonas putida and Escherichia coli cells was shown.
KeywordMeSH Terms
41. Koch  B, Jensen  LE, Nybroe  O,     ( 2001 )

A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site.

Journal of microbiological methods 45 (3)
PMID : 11348676  :  
Abstract >>
The use of Tn7-based systems for site-specific insertion of DNA into the chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn7 delivery vectors. In one group of vectors, the miniTn7 element, which is inserted into the chromosome, contains a multiple cloning site (MCS) and the kanamycin, streptomycin or gentamicin resistance markers. Another group of vectors intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter PA1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn7 into a specific, neutral intergenic region immediately downstream of the gene encoding glucosamine-6-phosphate synthetase (GlmS) in the tested fluorescent Pseudomonas strains. The gfp-tagging vector containing a gentamicin-resistance marker is useful for tagging strains carrying a Tn5 transposon. Tn5 transposons often carry kanamycin-resistance-encoding genes and are frequently used to generate bacterial mutants and to deliver reporter constructions in gene expression studies. To demonstrate the utility of a dual marker/reporter system, the Tn7-gfp marker system was combined with a Tn5-delivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn5-tagged locus could be determined by measuring bioluminescence.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Vectors
42. Beven  CA, Dieckelmann  M, Beacham  IR,     ( 2001 )

A strain of Pseudomonas fluorescens with two lipase-encoding genes, one of which possibly encodes cytoplasmic lipolytic activity.

Journal of applied microbiology 90 (6)
PMID : 11412328  :  
Abstract >>
A lipase-encoding gene (lipA) from a psychrotrophic strain of Pseudomonas fluorescens C9 has previously been characterized. It was also shown that when this gene was insertionally-inactivated, lipase activity was retained, suggesting that a second lipase may be present in this strain. The aim of this study was to determine whether this was the case. Using molecular cloning, chromosomal mutagenesis and enzymatic analysis, the presence of a second lipase-encoding gene (lipB) has been confirmed. The molecular weights of the putative products of lipA and lipB are 33 and 64.5 kDa, respectively, and their sequences are quite dissimilar (< 10% sequence identity). The lipB gene encodes a secreted lipase and is solely responsible for the 'lipolytic phenotype' of Ps. fluorescens C9. Expression of the lipA gene can be detected when expressed using an expression vector, but activity was only detected intracellularly in Ps. fluorescens C9, and not in the culture medium. Pseudomonas fluorescens C9 contains two dissimilar lipases. One (LipB) is secreted and responsible for the lipolytic phenotype; the evidence suggests that the other (LipA) could be intracellular, but it could be secreted and not detectable. Bacteria may contain more than one lipase activity. Ascribing phenotypes to particular enzymes therefore requires mutational analysis. The notion of an intracellular lipase activity is novel, and, if further substantiated, begs the question as to its normal substrate and physiological role.
KeywordMeSH Terms
Genes, Bacterial
43. Kulakova  AN, Kulakov  LA, Akulenko  NV, Ksenzenko  VN, Hamilton  JT, Quinn  JP,     ( 2001 )

Structural and functional analysis of the phosphonoacetate hydrolase (phnA) gene region in Pseudomonas fluorescens 23F.

Journal of bacteriology 183 (11)
PMID : 11344133  :   DOI  :   10.1128/JB.183.11.3268-3275.2001     PMC  :   PMC99623    
Abstract >>
The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene (phnA) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (phnA) indicated the presence of five open reading frames (ORFs). These include one (phnR) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (phnB) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB and phnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5' ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of phnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either Pseudomonas putida or Escherichia coli cells.
KeywordMeSH Terms
44. Ramette  A, Moënne-Loccoz  Y, Défago  G,     ( 2001 )

Polymorphism of the polyketide synthase gene phID in biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and comparison of PhID with plant polyketide synthases.

Molecular plant-microbe interactions : MPMI 14 (5)
PMID : 11332728  :   DOI  :   10.1094/MPMI.2001.14.5.639    
Abstract >>
Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.
KeywordMeSH Terms
45. Kamerbeek  NM, Moonen  MJ, Van Der Ven  JG, Van Berkel  WJ, Fraaije  MW, Janssen  DB,     ( 2001 )

4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds.

European journal of biochemistry 268 (9)
PMID : 11322873  :   DOI  :   10.1046/j.1432-1327.2001.02137.x    
Abstract >>
A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximately 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximately 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
KeywordMeSH Terms
46. Philippot  L, Mirleau  P, Mazurier  S, Siblot  S, Hartmann  A, Lemanceau  P, Germon  JC,     ( 2001 )

Characterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor and nos genes.

Biochimica et biophysica acta 1517 (3)
PMID : 11342223  :   DOI  :   10.1016/s0167-4781(00)00286-4    
Abstract >>
In this study, we report the cloning and characterization of denitrifying gene clusters of Pseudomonas fluorescens C7R12 containing the narXLDKGHJI, nirPOQSM, norCB and nosRZDFYL genes. While consensus sequences for Fnr-like protein binding sites were identified in the promoter regions of the nar, nir, nor and nos genes, consensus sequences corresponding to the NarL binding sites were identified only upstream the nar genes. Monitoring by mRNA analysis the expression of the narG, nirS, norB and nosZ structural genes suggests a sequential induction of the denitrification system in P. fluorescens.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
47. Woods  RG, Burger  M, Beven  CA, Beacham  IR,     ( 2001 )

The aprX-lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production.

Microbiology (Reading, England) 147 (Pt 2)
PMID : 11158351  :   DOI  :   10.1099/00221287-147-2-345    
Abstract >>
Extracellular protease and lipase production by psychrotrophic strains of Pseudomonas fluorescens is repressed by iron and regulated by temperature. The regulation of protease and lipase has been investigated in P. fluorescens B52. Whereas lipase production is increased below the optimum growth temperature ('low-temperature regulation'), protease production was relatively constant and only decreased above the optimum growth temperature. The genes encoding protease (aprX) and lipase (lipA) are encoded at opposite ends of a contiguous set of genes which also includes protease inhibitor, Type I secretion functions and two autotransporter proteins. Evidence is presented indicating that these genes constitute an operon, with a promoter adjacent to aprX which has been identified by S1 nuclease analysis. The regulation of aprX and lipA has been investigated at the RNA level and using lacZ fusion strains. Whereas the data are consistent with iron regulation at the transcriptional level, a lipA'-'lacZ fusion is not regulated by temperature, suggesting that temperature regulation is post-transcriptional or post-translational. The possibility of regulation at the level of mRNA decay is discussed.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
48. Mercado-Blanco  J, van der Drift  KM, Olsson  PE, Thomas-Oates  JE, van Loon  LC, Bakker  PA,     ( 2001 )

Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.

Journal of bacteriology 183 (6)
PMID : 11222588  :   DOI  :   10.1128/JB.183.6.1909-1920.2001     PMC  :   PMC95085    
Abstract >>
Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.
KeywordMeSH Terms
Benzamides
Genes, Bacterial
49. Matthijs  S, Koedam  N, Cornelis  P, De Greve  H,     ( 2000 )

The trehalose operon of Pseudomonas fluorescens ATCC 17400.

Research in microbiology 151 (10)
PMID : 11191810  :  
Abstract >>
The trehalose operon of Pseudomonas fluorescens ATCC 17400 consists of treP, treA and treR. The gene treP codes for a putative enzyme II subunit of the phosphotransferase system that catalyzes the phosphorylation of trehalose together with its translocation across the cell membrane and treA encodes a putative phosphotrehalase, which hydrolyzes the incoming trehalose-6-phosphate into glucose and glucose-6-phosphate. Both genes are negatively regulated by TreR, a repressor of the FadR-GntR family of transcription regulators. The operon that is induced by trehalose present in the medium shows a high similarity both in the function of genes and in the regulation with the trehalose operon of Bacillus subtilis.
KeywordMeSH Terms
Genes, Bacterial
50. Burger  M, Woods  RG, McCarthy  C, Beacham  IR,     ( 2000 )

Temperature regulation of protease in Pseudomonas fluorescens LS107d2 by an ECF sigma factor and a transmembrane activator.

Microbiology (Reading, England) 146 Pt 12 (N/A)
PMID : 11101673  :   DOI  :   10.1099/00221287-146-12-3149    
Abstract >>
The production of extracellular enzymes by Pseudomonas fluorescens is important with respect to phytopathogenesis and, in the case of psychrotrophic strains, food spoilage. The production of extracellular protease has been previously reported to be dependent on temperature in psychrotrophic strains of P. fluorescens; production is decreased above the optimum growth temperature with a relatively small change in growth rate. In this work, a transposon mutant of P. fluorescens LS107d2 has been isolated which, in contrast to the wild-type strain, is completely protease deficient at 29 degrees C, above the optimum growth temperature of 25 degrees C, but which produces protease at 23 degrees C. Further analysis revealed that this mutation is in a gene (prtR) which is part of a dicistronic operon, prtIR, in which the two genes are translationally coupled. Evidence is presented that prtI encodes a sigma factor related to others involved in extracytoplasmic functions (ECF sigma factors) and that prtR encodes a novel transmembrane activator of PrtI. PrtI, like PrtR, is also required for protease production at 29 degrees C but not at 23 degrees C. Analysis of the amino acid sequence of PrtR indicates that it is functionally related to a group of membrane-associated anti-sigma factors and a few transmembrane regulators, but is not significantly sequence related. Complementation analysis indicates that PrtR may also interact with sigma factors other than PrtI. The promoter region of the protease-encoding gene (aprX) in LS107d2 has been identified and has sequence features which could indicate interaction with either an ECF sigma factor or a primary sigma factor.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
51. Dalwadi  H, Wei  B, Landers  C, Yamane  A, Kim  J, Sutton  CL,     ( 2000 )

Identification of a novel bacterial sequence associated with Crohn's disease.

Gastroenterology 119 (1)
PMID : 10889151  :   DOI  :   10.1053/gast.2000.8519    
Abstract >>
Enteric microorganisms are implicated in the pathogenesis of Crohn's disease (CD), but no clear bacterial or viral species has been identified. In this study, representational difference analysis (RDA) was used to isolate DNA segments preferentially abundant in lamina propria mononuclear cells of lesional mucosa vs. adjacent uninvolved mucosa. Two RDA-derived microbial sequences were isolated (I1 and I2) and identified as novel homologues of the ptxR and tetR bacterial transcription-factor families. Quantitative competitive polymerase chain reaction of paraffin-embedded intestinal specimens from 212 patients showed that I2 DNA was present in many CD colonic lesions (43%), but was infrequent in other colonic specimens (9% of ulcerative colitis lesions and 5% of non-inflammatory bowel disease diseases; P<0.0001). I2 was prevalent in ileal specimens, regardless of disease status (43%-54%). Enzyme-linked immunosorbent assay analysis of 150 individuals with an I2 glutathione-S-transferase fusion protein showed frequent immunoglobulin A seroreactivity in CD (54% of patients), but infrequent seroreactivity in patients with ulcerative colitis, other inflammatory enteric diseases, or normals (10%, 19%, and 4%, respectively; P<0.001 to 0.00001). These findings relate CD to a novel lesion-localized and immunologically associated bacterial sequence, suggesting that the microorganism expressing the I2 gene product may be related to CD pathogenesis.
KeywordMeSH Terms
52. Laue  BE, Jiang  Y, Chhabra  SR, Jacob  S, Stewart  GS, Hardman  A, Downie  JA, O'Gara  F, Williams  P,     ( 2000 )

The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase.

Microbiology (Reading, England) 146 (Pt 10) (N/A)
PMID : 11021923  :   DOI  :   10.1099/00221287-146-10-2469    
Abstract >>
Several different species of Pseudomonas: produce N:-acylhomoserine lactones (AHLs), quorum-sensing signal molecules which are involved in the cell-density-dependent control of secondary metabolite and virulence gene expression. When Pseudomonas fluorescens F113 was cross-streaked against AHL biosensors capable of sensitively detecting either short (C(4)-C(8)) or long (C(10)-C(14)) acyl chain AHLs, no activity was detectable. However, by extracting cell-free stationary-phase culture supernatants with dichloromethane followed by reverse-phase HPLC, three distinct fractions were obtained capable of activating the AHL biosensors. Three AHLs were subsequently characterized using high-resolution MS and chemical synthesis. These were (i) N:-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH, C(14:1)-HSL), a molecule previously known as the Rhizobium leguminosarum small bacteriocin as a consequence of its growth inhibitory properties, (ii) N:-decanoylhomoserine lactone (C(10)-HSL) and (iii) N:-hexanoylhomoserine lactone (C(6)-HSL). A gene (hdtS) capable of directing synthesis of all three P. fluorescens AHLs in Escherichia coli was cloned and sequenced. In vitro transcription/translation of hdtS yielded a protein of approximately 33 kDa capable of directing the synthesis of 3OH, C(14:1)-HSL, C(10)-HSL and C(6)-HSL in E. coli. HdtS does not belong to either of the known AHL synthase families (LuxI or LuxM) and is related to the lysophosphatidic acid acyltransferase family. HdtS may therefore constitute a member of a third protein family capable of AHL biosynthesis.
KeywordMeSH Terms
53. Mirleau  P, Delorme  S, Philippot  L, Meyer  J, Mazurier  S, Lemanceau  P,     ( 2000 )

Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake.

FEMS microbiology ecology 34 (1)
PMID : 11053734  :   DOI  :   10.1111/j.1574-6941.2000.tb00752.x    
Abstract >>
Fluorescent pseudomonads have evolved an efficient strategy of iron uptake based on the synthesis of the siderophore pyoverdine and its relevant outer membrane receptor. The possible implication of pyoverdine synthesis and uptake on the ecological competence of a model strain (Pseudomonas fluorescens C7R12) in soil habitats was evaluated using a pyoverdine minus mutant (PL1) obtained by random insertion of the transposon Tn5. The Tn5 flanking DNA was amplified by inverse PCR and sequenced. The nucleotide sequence was found to show a high level of identity with pvsB, a pyoverdine synthetase. As expected, the mutant PL1 was significantly more susceptible to iron starvation than the wild-type strain despite its ability to produce another unknown siderophore. As with the wild-type strain, the mutant PL1 was able to incorporate the wild-type pyoverdine and five pyoverdines of foreign origin, but at a significantly lower rate despite the similarity of the outer membrane protein patterns of the two strains. The survival kinetics of the wild-type and of the pyoverdine minus mutant, in bulk and rhizosphere soil, were compared under gnotobiotic and non-gnotobiotic conditions. In gnotobiotic model systems, both strains, when inoculated separately, showed a similar survival in soil and rhizosphere, suggesting that iron was not a limiting factor. In contrast, when inoculated together, the bacterial competition was favorable to the pyoverdine producer C7R12. The efficient fitness of PL1 in the presence of the indigenous microflora, even when coinoculated with C7R12, is assumed to be related to its ability to uptake heterologous pyoverdines. Altogether, these results suggest that pyoverdine-mediated iron uptake is involved in the ecological competence of the strain P. fluorescens C7R12.
KeywordMeSH Terms
54. Stockwell  VO, Whistler  CA,     ( 2000 )

Lon protease influences antibiotic production and UV tolerance of Pseudomonas fluorescens Pf-5.

Applied and environmental microbiology 66 (7)
PMID : 10877760  :   DOI  :   10.1128/aem.66.7.2718-2725.2000     PMC  :   PMC92065    
Abstract >>
Pseudomonas fluorescens Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor sigma(S) and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5. lon mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The lon gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor sigma(32) (sigma(H)) of Escherichia coli, and Lon accumulation by Pf-5 increased after heat shock. Therefore, sigma(H) represents the third sigma factor (with sigma(S) and sigma(70)) implicated in the regulation of antibiotic production by P. fluorescens. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator sigma(S) or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.
KeywordMeSH Terms
Bacterial Proteins
Escherichia coli Proteins
Protease La
Sigma Factor
Ultraviolet Rays
55. Blumer  C,     ( 2000 )

Multicopy suppression of a gacA mutation by the infC operon in Pseudomonas fluorescens CHA0: competition with the global translational regulator RsmA.

FEMS microbiology letters 187 (1)
PMID : 10828400  :   DOI  :   10.1111/j.1574-6968.2000.tb09136.x    
Abstract >>
The gacA gene of the biocontrol strain Pseudomonas fluorescens CHA0 codes for a response regulator which, together with the sensor kinase GacS (=LemA), is required for the production of exoenzymes and secondary metabolites involved in biocontrol, including hydrogen cyanide (HCN). A gacA multicopy suppressor was isolated from a cosmid library of strain CHA0 and identified as the infC-rpmI-rplT operon, which encodes the translation initiation factor IF3 and the ribosomal proteins L35 and L20. The efficiency of suppression was about 30%, as determined by the use of a GacA-controlled reporter construct, i.e. a translational hcnA'-'lacZ fusion. Overexpression of the rsmA gene (coding for a global translational repressor) reversed the suppressive effect of the amplified infC operon. This finding suggests that some product(s) of the infC operon can compete with RsmA at the level of translation in P. fluorescens CHA0 and that important biocontrol traits can be regulated at this level.
KeywordMeSH Terms
RNA-Binding Proteins
Suppression, Genetic
56. Barghini  P, Roncetti  AR, Civolani  C,     ( 2000 )

Bioconversion of ferulic acid into vanillic acid by means of a vanillate-negative mutant of Pseudomonas fluorescens strain BF13.

Applied and environmental microbiology 66 (6)
PMID : 10831404  :   DOI  :   10.1128/aem.66.6.2311-2317.2000     PMC  :   PMC110519    
Abstract >>
From a ferulic-acid-degrading Pseudomonas fluorescens strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing p-coumaric acid (0.1% [wt/vol]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon's site of insertion was cloned and sequenced revealing two open reading frames of 1,062 (vanA) and 954 (vanB) bp, respectively. The van genes are organized in a cluster and encode the subunits of the vanillate-O-demethylase, which catalyzes the first step of the vanillate catabolism. Amino acid sequences deduced from vanA and vanB genes were shown to have high identity with known VanAs and VanBs from Pseudomonas and Acinetobacter spp. Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-O-demethylase components from P. fluorescens BF13. The terminal oxygenase VanA is characterized by a conserved Rieske-type [2Fe-2S](R) ligand center. The reductase VanB contains a plant-type ferredoxin [2Fe-2S](Fd), flavin mononucleotide, and NAD-ribose binding domains which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-type vanAB genes to BF13-97 complemented this mutant, which recovered its ability to grow on either vanillic or ferulic acid.
KeywordMeSH Terms
Mutation
57. 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
58. Sheehan  MM, Fenton  A, Delany  I,     ( 2000 )

Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor.

Microbiology (Reading, England) 146 (Pt 2) (N/A)
PMID : 10708392  :   DOI  :   10.1099/00221287-146-2-537    
Abstract >>
The antifungal metabolite 2,4-diacetylphloroglucinol plays a major role in the biocontrol capabilities of Pseudomonas fluorescens. The phloroglucinol biosynthetic locus of P. fluorescens F113 has been isolated previously. From nucleotide sequence data, a putative regulator gene (phlF) was identified upstream and divergently transcribed from the phlACBD phloroglucinol biosynthetic genes. PhlF shows similarity to various transcriptional repressors in the EMBL database and exhibits a helix-turn-helix motif in its amino acid sequence. phlF was cloned into an expression vector and the PhlF protein product was purified. Gel retardation experiments demonstrated PhlF to be a DNA-binding protein and showed that it binds to the phlA-phlF intergenic region. Introduction of phlF into P. fluorescens F113 in multiple copies resulted in repression of phloroglucinol production in this strain. This effect was mediated at the transcription level since the expression of a phloroglucinol biosynthetic gene fusion in this background was equally repressed. Furthermore, the inactivation of phlF results in derepression of phloroglucinol production in this strain.
KeywordMeSH Terms
Bacterial Proteins
59. 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
60. Gigot-Bonnefoy  C, Reimmann  C, Duffy  B, Blumer  C, Maurhofer  M, Seematter  A, Schnider-Keel  U,     ( 2000 )

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

Journal of bacteriology 182 (5)
PMID : 10671440  :   DOI  :   10.1128/jb.182.5.1215-1225.2000     PMC  :   PMC94405    
Abstract >>
The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.
KeywordMeSH Terms
61. Johnson  Z, Ochsner  UA,     ( 2000 )

Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa.

Microbiology (Reading, England) 146 (Pt 1) (N/A)
PMID : 10658665  :   DOI  :   10.1099/00221287-146-1-185    
Abstract >>
A gene cluster similar to haem iron uptake loci of bacterial pathogens was identified in Pseudomonas aeruginosa. This phu locus ('Pseudomonas haem uptake') consisted of the phuR receptor gene and the phuSTUVW operon encoding a typical ABC transporter. Expression of phuR and phuSTUVW from mapped transcriptional-start sites occurred under iron-restricted growth conditions and was directly controlled by the Fur protein. Binding of Fur was demonstrated by DNase footprinting of two adjacent 'Fur boxes' that overlapped both the phuR and phuSTUVW promoters. Two tandem repeats of 154 bp were identified downstream of the phuSTUVW operon, each of which contained a strong Fur-dependent promoter driving expression of iron-regulated RNAs antisense to phuSTUVW. Mutant strains with deletions in phuR and phuSTUV showed greatly reduced growth with either haem or haemoglobin as the only iron source: the defects were complemented by plasmids harbouring the phuR or the phuSTUV genes, respectively. Deletions of phuW or of the tandem repeats had only minor effects on haem utilization. The remaining haem and haemoglobin uptake still observed in the deltaphuR or deltaphuSTUV deletion mutants was due to a second haem-acquisition system, has, which was also under the direct control of Fur. This second haem-receptor gene, hasR, was identified upstream of and in an operon with hasA, encoding a haem-binding extracellular protein. A deltahasR mutant also exhibited decreased utilization of haem and haemoglobin, and a deltaphuR deltahasR double mutant was virtually unable to take up either compound. Both the PhuR and HasR proteins were detected in the outer-membrane fraction of P. aeruginosa grown in low-iron media. Taken together, the evidence suggests that the phu and has loci encode two distinct systems required for the acquisition of haem and haemoglobin in P. aeruginosa.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
Sigma Factor
62. Kawai  E, Idei  A,     ( 1999 )

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA.

Journal of bacteriology 181 (24)
PMID : 10601212  :   PMC  :   PMC94212    
Abstract >>
Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.
KeywordMeSH Terms
Carrier Proteins
63. Burd  W, Hill  DS, Hammer  PE,     ( 1999 )

Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains.

FEMS microbiology letters 180 (1)
PMID : 10547442  :   DOI  :   10.1111/j.1574-6968.1999.tb08775.x    
Abstract >>
The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.
KeywordMeSH Terms
Genes, Bacterial
64. Heeb  S, Blumer  C,     ( 1999 )

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

Proceedings of the National Academy of Sciences of the United States of America 96 (24)
PMID : 10570200  :   DOI  :   10.1073/pnas.96.24.14073     PMC  :   PMC24192    
Abstract >>
The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.
KeywordMeSH Terms
RNA-Binding Proteins
65. 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
66. Overkamp  KM, Schreuder  HA, Eppink  MH,     ( 1999 )

Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.

Journal of molecular biology 292 (1)
PMID : 10493859  :   DOI  :   10.1006/jmbi.1999.3015    
Abstract >>
p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase.
KeywordMeSH Terms
67. Kumura  H, Shimazaki  K, Idei  A, Kawai  E,     ( 1999 )

The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33.

Biochimica et biophysica acta 1446 (3)
PMID : 10524213  :   DOI  :   10.1016/s0167-4781(99)00094-9    
Abstract >>
In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.
KeywordMeSH Terms
Multigene Family
68. Schoofs  G, Hancock  RE, De Mot  R,     ( 1999 )

Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.

Journal of bacteriology 181 (16)
PMID : 10438740  :   PMC  :   PMC93957    
Abstract >>
The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.
KeywordMeSH Terms
Bacterial Proteins
Gene Expression Regulation, Bacterial
69. 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
70. Thomashow  LS,     ( 1999 )

Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87.

Journal of bacteriology 181 (10)
PMID : 10322017  :   PMC  :   PMC93771    
Abstract >>
The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.
KeywordMeSH Terms
Operon
71. Loper  JE, Chaney  N,     ( 1999 )

Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5.

Journal of bacteriology 181 (7)
PMID : 10094695  :   PMC  :   PMC93630    
Abstract >>
Ten genes (plt) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of Pseudomonas fluorescens Pf-5. The deduced amino acid sequences of eight plt genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (pltB, pltC), an acyl coenzyme A (acyl-CoA) dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and three halogenases (pltA, pltD, and pltM). Insertions of the transposon Tn5 or Tn3-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5. The presumed functions of the eight plt products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors. Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. The deduced amino acid sequence of the product of another plt gene, pltR, is similar to those of members of the LysR family of transcriptional activators. pltR and pltM are transcribed divergently from the pltLABCDEFG gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating pltRM from pltLABCDEFG. Transcription of the pyoluteorin biosynthesis genes pltB, pltE, and pltF, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a pltR mutant of Pf-5. Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
72. Huang  YP,     ( 1999 )

DNA polymerase C of the thermophilic bacterium Thermus aquaticus: classification and phylogenetic analysis of the family C DNA polymerases.

Journal of molecular evolution 48 (6)
PMID : 10229580  :  
Abstract >>
Bacterial family C DNA polymerases (DNA pol IIIs), the major chromosomal replicative enzymes, have been provisionally classified based on primary sequences and domain structures into three classes: class I (Escherichia coli DNA pol C-type), class II (Bacillus subtilis DNA pol C-type), and class III (cyanobacterial DNA pol C-type), respectively. We have sequenced the structural gene encoding the DNA pol C catalytic subunit of the thermophilic bacterium Thermus aquaticus. This gene, designated the Taq DNA pol C gene, contains a 3660-bp open reading frame which specifies a polypeptide of molecular weight of 137,388 daltons. Comparative sequence analyses revealed that Taq DNA pol C is a class I family C DNA polymerase. The Taq DNA pol C is most closely related to the Deinococcus radiodurans DNA pol C. Although a phylogenetic tree based on the class I family C DNA pols is still in the provisional stage, some important conclusion can be drawn. First, the high-G+C and the low-G+C Gram-positive bacteria are not monophyletic. Second, the low-G+C Gram-positive bacteria contain multigenes of family C DNA pols (classes I and II). Third, the cyanobacterial family C DNA pol, classified as class III because it is encoded by a split gene, forms a group with the high-G+C Gram-positive bacteria.
KeywordMeSH Terms
Phylogeny
73. Hallin  S, Lindgren  PE,     ( 1999 )

PCR detection of genes encoding nitrite reductase in denitrifying bacteria.

Applied and environmental microbiology 65 (4)
PMID : 10103263  :   PMC  :   PMC91233    
Abstract >>
Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.
KeywordMeSH Terms
Genes, Bacterial
74. Pan  JG, Rhee  JS,     ( 1999 )

Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1.

Journal of bacteriology 181 (6)
PMID : 10074078  :   PMC  :   PMC93584    
Abstract >>
Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.
KeywordMeSH Terms
75. Eppink  MH, Bunthol  C, Schreuder  HA,     ( 1999 )

Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications for NADPH recognition and structural stability.

FEBS letters 443 (3)
PMID : 10025942  :   DOI  :   10.1016/s0014-5793(98)01726-8    
Abstract >>
Phe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 A is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 A revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.
KeywordMeSH Terms
Amino Acid Substitution
76. Nagasawa  T, Yoshida  T, Yamane  T, Kawai  T, Hurh  B,     ( 1999 )

Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.

European journal of biochemistry 260 (1)
PMID : 10091591  :   DOI  :   10.1046/j.1432-1327.1999.00124.x    
Abstract >>
6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.
KeywordMeSH Terms
77. Bellingham  NF, Morgan  JA, Saunders  JR, Hart  CA,     ( 1999 )

Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates.

Applied and environmental microbiology 65 (3)
PMID : 10049879  :   PMC  :   PMC91160    
Abstract >>
Pseudomonas aeruginosa, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains. While P. aeruginosa was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples. A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical P. aeruginosa strains are not readily distinguishable. The variation in the central regions of the flagellin genes of seven of the isolates was investigated further. The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp). The route by which flagellin gene variation has occurred in P. aeruginosa is discussed.
KeywordMeSH Terms
Environmental Microbiology
78. Lejon  DP, Nowak  V, Bouko  S, Pascault  N, Mougel  C, Martins  JM, Ranjard  L,     ( 2007 )

Fingerprinting and diversity of bacterial copA genes in response to soil types, soil organic status and copper contamination.

FEMS microbiology ecology 61 (3)
PMID : 17696885  :   DOI  :   10.1111/j.1574-6941.2007.00365.x    
Abstract >>
A molecular fingerprinting assay was developed to assess the diversity of copA genes, one of the genetic determinants involved in bacterial resistance to copper. Consensus primers of the copA genes were deduced from an alignment of sequences from proteobacterial strains. A PCR detection procedure was optimized for bacterial strains and allowed the description of a novel copA genetic determinant in Pseudomonas fluorescens. The copA DNA fingerprinting procedure was optimized for DNA directly extracted from soils differing in their physico-chemical characteristics and in their organic status (SOS). Particular copA genetic structures were obtained for each studied soil and a coinertia analysis with soil physico-chemical characteristics revealed the strong influence of pH, soil texture and the quality of soil organic matter. The molecular phylogeny of copA gene confirmed that specific copA genes clusters are specific for each SOS. Furthermore, this study demonstrates that this approach was sensitive to short-term responses of copA gene diversity to copper additions to soil samples, suggesting that community adaptation is preferentially controlled by the diversity of the innate copA genes rather than by the bioavailability of the metal.
KeywordMeSH Terms
Soil Microbiology
79. Nian  H, Zhang  J, Song  F, Fan  L, Huang  D,     ( 2007 )

Isolation of transposon mutants and characterization of genes involved in biofilm formation by Pseudomonas fluorescens TC222.

Archives of microbiology 188 (3)
PMID : 17453174  :   DOI  :   10.1007/s00203-007-0235-8    
Abstract >>
Biofilm formation mutants are often found to have defective or altered motility. The motility phenotype was exploited to identify Pseudomonas fluorescens biofilm formation mutants. Fourteen motility mutants were obtained from P. fluorescens isolate TC222 and eight stable mutants were studied further. The eight transposon insertion mutants showed altered ability to form biofilm compared with the parent. Five Tn5-inserted genes from these mutants were cloned and sequenced. Genetic analysis showed that two insertions were located in genes affecting multiple cell surface characteristics, including lipopolysaccharide (rfbD) and polar flagella (fliR). Three genes encoding for a putative Mig-14 family protein (epsB), a probable bacteriophage signal peptide protein (bspA) and a soluble pyridine nucleotide transhydrogenase (pyrA) were reported for the first time to be involved in biofilm formation. Complementation experiments of rfbD and epsB genes proved that biofilm formation of the corresponding mutants could be restored. Further semi-quantitative reverse transcription-PCR analysis showed that both rfbD and epsB can express their transcripts much higher in the complemented strains than that in wild-type strains. The transcripts of both genes in their mutants could not be detected.
KeywordMeSH Terms
80. Saravanakumar  D, Samiyappan  R,     ( 2007 )

ACC deaminase from Pseudomonas fluorescens mediated saline resistance in groundnut (Arachis hypogea) plants.

Journal of applied microbiology 102 (5)
PMID : 17448163  :   DOI  :   10.1111/j.1365-2672.2006.03179.x    
Abstract >>
To study the effect of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase from Pseudomonas fluorescens against saline stress under in vitro and field conditions in groundnut (Arachis hypogea) plants. Four plant growth-promoting rhizobacteria (PGPR) strains were used in this study to evaluate their efficacy in groundnut plants against saline stress under in vitro. Among the four PGPR strains used, Ps. fluorescens strain TDK1 showed greater performance in improving the plant growth parameters of groundnut seedlings in vitro. PCR amplification using Pseudomonas-specific 16S-23S rRNA internal transcribed spacers (ITS) primers revealed that all the four strains belonged to the group of fluorescent pseudomonads. ITS region of Ps. fluorescens strain TDK1 was cloned and sequenced. ACC deaminase activity using biochemical and molecular (PCR) analysis revealed that among all the four strains, Ps. fluorescens strain TDK1 showed greater amount of ACC deaminase activity and positive reaction to PCR amplification. ACC deaminase gene from Ps. fluorescens strain TDK1 was isolated, cloned and sequenced. Pseudomonas bioformulations were developed and they were tested in groundnut plants under saline-affected soils. The results indicated the superior performance by Ps. fluorescens strain TDK1 possessing ACC deaminase activity in improving yield parameters in groundnut plants despite salinity. Pseudomonas fluorescens strain TDK1 possessing ACC deaminase activity enhanced the saline resistance in groundnut plants, which in turn resulted in increased yield when compared with the groundnuts treated with Pseudomonas strains not having ACC deaminase activity. The promising role of ACC deaminase from Ps. fluorescens strain TDK1 in alleviating saline stress has been concluded in groundnut plants. This study will be useful for exploiting the activity of ACC deaminase from microbial strains against various biotic and abiotic stresses wherever ACC accumulated as precursor for ethylene biosynthesis.
KeywordMeSH Terms
Carbon-Carbon Lyases
Soil Microbiology
81. Iwaki  H, Muraki  T, Ishihara  S, Hasegawa  Y, Rankin  KN, Sulea  T, Boyd  J, Lau  PC,     ( 2007 )

Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis.

Journal of bacteriology 189 (9)
PMID : 17277060  :   DOI  :   10.1128/JB.01098-06     PMC  :   PMC1855914    
Abstract >>
Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the beta5-alpha2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.
KeywordMeSH Terms
82. Di Gennaro  P, Ferrara  S, Ronco  I, Galli  E, Sello  G, Papacchini  M, Bestetti  G,     ( 2007 )

Styrene lower catabolic pathway in Pseudomonas fluorescens ST: identification and characterization of genes for phenylacetic acid degradation.

Archives of microbiology 188 (2)
PMID : 17377771  :   DOI  :   10.1007/s00203-007-0226-9    
Abstract >>
Pseudomonas fluorescens ST is a styrene degrading microorganism that, by the sequential oxidation of the vinyl side chain, converts styrene to phenylacetic acid. The cluster of styrene upper pathway catabolic genes (sty genes) has been previously localized on a chromosomal region. This report describes the isolation, sequencing and analysis of a new chromosomal fragment deriving from the ST strain genomic bank that contains the styrene lower degradative pathway genes (paa genes), involved in the metabolism of phenylacetic acid. Analysis of the paa gene cluster led to the description of 14 putative genes: a gene encoding a phenylacetyl-CoA ligase (paaF), the enzyme required for the activation of phenylacetic acid; five ORFs encoding the subunits of a ring hydroxylation multienzymatic system (paaGHIJK); the gene paaW encoding a membrane protein of unknown function; five genes for a beta-oxidation-like system (paaABCDE), involved in the steps following the aromatic ring cleavage; a gene encoding a putative permease (paaL) and a gene (paaN) probably involved in the aromatic ring cleavage. The function of some of the isolated genes has been proved by means of biotransformation experiments.
KeywordMeSH Terms
Genes, Bacterial
83. de Bruijn  I, de Kock  MJ, Yang  M, de Waard  P, van Beek  TA, Raaijmakers  JM,     ( 2007 )

Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species.

Molecular microbiology 63 (2)
PMID : 17241198  :   DOI  :   10.1111/j.1365-2958.2006.05525.x    
Abstract >>
Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.
KeywordMeSH Terms
Genome, Bacterial
84. 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
85. Leonard  PM, Brzozowski  AM, Lebedev  A, Marshall  CM, Smith  DJ, Verma  CS, Walton  NJ, Grogan  G,     ( 2006 )

The 1.8 A resolution structure of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL) from Pseudomonas fluorescens, an enzyme that catalyses the transformation of feruloyl-coenzyme A to vanillin.

Acta crystallographica. Section D, Biological crystallography 62 (Pt 12)
PMID : 17139085  :   DOI  :   10.1107/S0907444906039199    
Abstract >>
The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 A resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate.
KeywordMeSH Terms
86. Jaouen  T, Coquet  L, Marvin-Guy  L, Orange  N, Chevalier  S, Dé  E,     ( 2006 )

Functional characterization of Pseudomonas fluorescens OprE and OprQ membrane proteins.

Biochemical and biophysical research communications 346 (3)
PMID : 16777062  :   DOI  :   10.1016/j.bbrc.2006.06.013    
Abstract >>
Outer membrane (OM) proteins of the OprD family may enable bacteria of the genus Pseudomonas to adapt to various environments by modulating OM permeability. The OprE and OprQ porins from P. fluorescens strain MF0 were purified and identified by MALDI-TOF mass spectrometry and N-terminal and internal microsequencing. These proteins, when reconstituted in an artificial planar lipid bilayer, induced similar ion channels with low single-conductance values. Secondary structure prediction of both proteins showed similar folding patterns into a 16 transmembrane beta-strands barrel but a highly variable amino-acid composition and length for their putative external loops implicated in porin function. Both proteins were overexpressed under poor oxygenation conditions, but not by using several amino acids as sole carbon source, indicating a different specificity for these proteins compared to the paradigm of this protein family, OprD.
KeywordMeSH Terms
87. Denayer  S, Matthijs  S, Cornelis  P,     ( 2006 )

Resistance to vanadium in Pseudomonas fluorescens ATCC 17400 caused by mutations in TCA cycle enzymes.

FEMS microbiology letters 264 (1)
PMID : 17020548  :   DOI  :   10.1111/j.1574-6968.2006.00435.x    
Abstract >>
Vanadium inhibits the growth of Pseudomonas fluorescens ATCC 17400 in the low-iron casamino acids medium and even more when iron is added to the medium. Analysis of transposon mutants allowed the isolation of two mutants with increased resistance to vanadium. One mutant had an insertion in the idh gene coding for the tricarboxylic acid enzyme isocitrate dehydrogenase. The second mutant had the transposon inserted into acnD, one out of three genes coding for a 2-methyl-isocitrate dehydratase (aconitase). In this mutant, there was a higher level of acnB aconitase transcripts while the levels of acnA transcripts were unchanged. A nonpolar idh mutant was obtained, which showed the same level of resistance against vanadium as the original transposon mutant.
KeywordMeSH Terms
Mutation
88. Mavrodi  OV, Mavrodi  DV, Weller  DM, Thomashow  LS,     ( 2006 )

Role of ptsP, orfT, and sss recombinase genes in root colonization by Pseudomonas fluorescens Q8r1-96.

Applied and environmental microbiology 72 (11)
PMID : 16936061  :   DOI  :   10.1128/AEM.01215-06     PMC  :   PMC1636191    
Abstract >>
Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.
KeywordMeSH Terms
Pest Control, Biological
89. Blaha  D, Prigent-Combaret  C, Mirza  MS, Moënne-Loccoz  Y,     ( 2006 )

Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography.

FEMS microbiology ecology 56 (3)
PMID : 16689877  :   DOI  :   10.1111/j.1574-6941.2006.00082.x    
Abstract >>
Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in plant growth-promoting rhizobacteria (PGPR) and phytosymbiotic bacteria, but the diversity of the corresponding gene (acdS) is poorly documented. Here, acdS sequences were obtained by screening putative ACC deaminase sequences listed in databases, based on phylogenetic properties and key residues. In addition, acdS was sought in 71 proteobacterial strains by PCR amplification and/or hybridization using colony dot blots. The presence of acdS was confirmed in established AcdS+ bacteria and evidenced noticeably in Azospirillum (previously reported as AcdS-), in 10 species of Burkholderia and six Burkholderia cepacia genomovars (which included PGPR, phytopathogens and opportunistic human pathogens), and in five Agrobacterium genomovars. The occurrence of acdS in true and opportunistic pathogens raises new questions concerning their ecology in plant-associated habitats. Many (but not all) acdS+ bacteria displayed ACC deaminase activity in vitro, including two Burkholderia clinical isolates. Phylogenetic analysis of partial acdS and deduced AcdS sequences evidenced three main phylogenetic clusters, each gathering pathogens and plant-beneficial strains of contrasting geographic and habitat origins. The acdS phylogenetic tree was only partly congruent with the rrs tree. Two clusters gathered both Betaprotobacteria and Gammaproteobacteria, suggesting extensive horizontal transfers of acdS, noticeably between plant-associated Proteobacteria.
KeywordMeSH Terms
90. Martynowski  D, Eyobo  Y, Li  T, Yang  K, Liu  A, Zhang  H,     ( 2006 )

Crystal structure of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylation reaction.

Biochemistry 45 (35)
PMID : 16939194  :   DOI  :   10.1021/bi060903q    
Abstract >>
Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is a widespread enzyme found in many bacterial species and all currently sequenced eukaryotic organisms. It occupies a key position at the branching point of two metabolic pathways: the tryptophan to quinolinate pathway and the bacterial 2-nitrobenzoic acid degradation pathway. The activity of ACMSD determines whether the metabolites in both pathways are converted to quinolinic acid for NAD biosynthesis or to acetyl-CoA for the citric acid cycle. Here we report the first high-resolution crystal structure of ACMSD from Pseudomonas fluorescens which validates our previous predictions that this enzyme is a member of the metal-dependent amidohydrolase superfamily of the (beta/alpha)(8) TIM barrel fold. The structure of the enzyme in its native form, determined at 1.65 A resolution, reveals the precise spatial arrangement of the active site metal center and identifies a potential substrate-binding pocket. The identity of the native active site metal was determined to be Zn. Also determined was the structure of the enzyme complexed with cobalt at 2.50 A resolution. The hydrogen bonding network around the metal center suggests that Arg51 and His228 may play important roles in catalysis. The metal center configuration of PfACMSD is very similar to that of Zn-dependent adenosine deaminase and Fe-dependent cytosine deaminase, suggesting that ACMSD may share certain similarities in its catalytic mechanism with these enzymes. These data enable us to propose possible catalytic mechanisms for ACMSD which appear to be unprecedented among all currently characterized decarboxylases.
KeywordMeSH Terms
91. O'Hare  HM, Huang  F, Holding  A, Choroba  OW, Spencer  JB,     ( 2006 )

Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution.

FEBS letters 580 (14)
PMID : 16730004  :   DOI  :   10.1016/j.febslet.2006.05.018    
Abstract >>
Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.
KeywordMeSH Terms
Directed Molecular Evolution
92. Crozier  KR, Moran  GR,     ( 2007 )

Heterologous expression and purification of kynurenine-3-monooxygenase from Pseudomonas fluorescens strain 17400.

Protein expression and purification 51 (2)
PMID : 16973376  :   DOI  :   10.1016/j.pep.2006.07.024    
Abstract >>
Kynurenine 3-monooxygenase (KMO) is an NADPH-dependent flavoprotein hydroxylase that catalyzes the conversion of l-Kynurenine (L-Kyn) to 3-hydroxykynurenine (3OHKyn). The reaction is central to the tryptophan degradative pathway and takes place within microglial cells defining cellular concentrations of the N-methyl-d-aspatate (NMDA) receptor agonist quinolinate and antagonist kynurenate. The influence over the cellular concentrations of these NMDA receptor effectors makes KMO an attractive target for the treatment of ischemic stroke. Pseudomonas fluorescens str 17400, expresses five activities of tryptophan catabolism including that of KMO. The KMO gene from P. fluorescens was cloned into the pET-17b plasmid using incorporated NdeI and XhoI restriction sites. This construct yielded PfKMO to 20% of total cell protein after 12h of expression at 22 degrees C without induction by isopropyl-beta-thiogalactopyranoside (IPTG). The enzyme could be readily purified using ammonium sulfate fractionation and ion exchange chromatography, resulting in pure KMO with a turnover number of 5.0 s(-1). PfKMO activity was dependent on the reduction state of the enzyme. Preparation and storage benefited from the presence of a reductant such as dithiothreitol or beta-mercaptoethanol. The loss of activity was found to be directly related to the oxidation of thiols as measured by dinitrothiobenzoate assay. Steady-state assays monitoring the consumption of dioxygen were used to measure apparent kinetic parameters and ligand perturbation of flavin fluorescence was used to determine a Kd value for both L-Kyn and the inhibitor m-nitrobenzoylalanine. PfKMO is offered as prototypical bacterial form of the enzyme to serve as a viable platform on which to base future KMO studies.
KeywordMeSH Terms
93. Luo  Y, Zheng  Y, Jiang  Z, Ma  Y, Wei  D,     ( 2006 )

A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification.

Applied microbiology and biotechnology 73 (2)
PMID : 16724189  :   DOI  :   10.1007/s00253-006-0478-3    
Abstract >>
A lipase-producing bacterium strain B68 screened from soil samples of China was identified as Pseudomonas fluorescens. With GenomeWalker, the open reading frame of lipase gene lipB68, encoding 476 amino acids, was cloned and expressed in Escherichia coli BL21 (DE3). By affinity chromatography, the recombinant LipB68 protein was purified to the purity of 95%. As a member of lipase subfamily I.3, LipB68 has a unique optimum temperature of 20 degrees C, which was the lowest in this subfamily. In chiral resolution, LipB68 effectively catalyzed the transesterification of both alpha-phenylethanol and alpha-phenylpropanol at 20 degrees C, achieving E values greater than 100 and 60 after 120 h, respectively. Among all the known catalysts in biodiesel production, LipB68 produced biodiesel with a yield of 92% after 12 h, at the lowest temperature of 20 degrees C, and is the first one of the I.3 lipase subfamily reported to be capable of catalyzing the transesterification reaction of biodiesel production. Since lipase-mediated biodiesel production is normally carried out at 35-50 degrees C, the availability of a highly active lipase with a low optimal temperature can provide substantial savings in energy consumption. Thus, this novel psychrophilic lipase (LipB68) may represent a highly competitive energy-saving biocatalyst.
KeywordMeSH Terms
94. 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 (1��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
95. Hinsa  SM, O'Toole  GA,     ( 2006 )

Biofilm formation by Pseudomonas fluorescens WCS365: a role for LapD.

Microbiology (Reading, England) 152 (Pt 5)
PMID : 16622054  :   DOI  :   10.1099/mic.0.28696-0    
Abstract >>
A role for the outer-membrane-associated LapA protein in early biofilm formation by Pseudomonas fluorescens WCS365 has previously been shown. This paper reports that lapD, a gene located adjacent to the lapA gene, also plays a role in biofilm formation. A mutation in lapD results in a conditional biofilm defect in a static assay - this biofilm phenotype is exacerbated when biofilm formation is assayed in a flow-cell system. Furthermore, a lapD mutation shows a partial defect in the transition from reversible to irreversible attachment, consistent with an early role for the lapD gene product in biofilm formation. LapD is shown to be localized to the inner membrane of P. fluorescens. The data show decreased LapA associated with the cell surface, but no apparent change in cytoplasmic levels of this protein or lapA transcription, in a lapD mutant. A model is proposed wherein the role of LapD in biofilm formation is modulating the secretion of the LapA adhesin.
KeywordMeSH Terms
96. Goymer  P, Kahn  SG, Malone  JG, Gehrig  SM, Spiers  AJ, Rainey  PB,     ( 2006 )

Adaptive divergence in experimental populations of Pseudomonas fluorescens. II. Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype.

Genetics 173 (2)
PMID : 16624907  :   DOI  :   10.1534/genetics.106.055863     PMC  :   PMC1526540    
Abstract >>
Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations undergoing adaptive radiation. Previous work identified genes contributing to the evolutionary success of WS. Here we scrutinize the GGDEF response regulator protein WspR and show that it is both necessary and sufficient for WS. Activation of WspR occurs by phosphorylation and different levels of activation generate phenotypic differences among WS genotypes. Five alleles of wspR, each encoding a protein with a single amino acid substitution, were generated by mutagenesis. Two alleles are constitutively active and cause the ancestral genotype to develop a WS phenotype; the phenotypic effects are allele specific and independent of phosphorylation. Three alleles contain changes in the GGDEF domain and when overexpressed in WS cause reversion to the ancestral phenotype. Ability to mimic this effect by overexpression of a liberated N-terminal domain shows that in WS, regulatory components upstream of WspR are overactive. To connect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both structured and unstructured environments: alleles had adaptive and deleterious effects with trade-offs evident across environments. Despite the proclivity of mutations within wspR to generate WS, sequence analysis of wspR from 53 independently obtained WS showed no evidence of sequence change in this gene.
KeywordMeSH Terms
97. 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
98. Baehler  E, de Werra  P, Wick  LY, Péchy-Tarr  M, Mathys  S, Maurhofer  M, Keel  C,     ( 2006 )

Two novel MvaT-like global regulators control exoproduct formation and biocontrol activity in root-associated Pseudomonas fluorescens CHA0.

Molecular plant-microbe interactions : MPMI 19 (3)
PMID : 16570661  :   DOI  :   10.1094/MPMI-19-0313    
Abstract >>
Pseudomonas fluorescens CHA0 protects various crop plants against root diseases caused by pathogenic fungi. Among a range of exoproducts excreted by strain CHA0, the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) are particularly relevant to the strain's biocontrol potential. Here, we report on the characterization of MvaT and MvaV as novel regulators of biocontrol activity in strain CHA0. We establish the two proteins as further members of an emerging family of MvaT-like regulators in pseudomonads that are structurally and functionally related to the DNA-binding protein H-NS. In mvaT and mvaV in frame-deletion mutants of strain CHA0, PLT production was enhanced about four- and 1.5-fold, respectively, whereas DAPG production remained at wild-type levels. Remarkably, PLT production was increased up to 20-fold in an mvaT mvaV double mutant. DAPG biosynthesis was almost completely repressed in this mutant. The effects on antibiotic production could be confirmed by following expression of gfp-based reporter fusions to the corresponding biosynthetic genes. MvaT and MvaV also influenced levels of other exoproducts, motility, and physicochemical cell-surface properties to various extents. Compared with the wild type, mvaT and mvaV mutants had an about 20% reduced capacity (in terms of plant fresh weight) to protect cucumber from a root rot caused by Pythium ultimum. Biocontrol activity was nearly completely abolished in the double mutant Our findings indicate that MvaT and MvaV act together as further global regulatory elements in the complex network controlling expression of biocontrol traits in plant-beneficial pseudomonads.
KeywordMeSH Terms
99. De La Fuente  L, Mavrodi  DV, Landa  BB, Thomashow  LS, Weller  DM,     ( 2006 )

phlD-based genetic diversity and detection of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens.

FEMS microbiology ecology 56 (1)
PMID : 16542406  :   DOI  :   10.1111/j.1574-6941.2006.00074.x    
Abstract >>
Diversity within a worldwide collection of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains was assessed by sequencing the phlD gene. Phylogenetic analyses based on the phlD sequences of 70 isolates supported the previous classification into 18 BOX-PCR genotypes (A-Q and T). Exploiting polymorphisms within the sequence of phlD, we designed and used allele-specific PCR primers with a PCR-based dilution endpoint assay to quantify the population sizes of A-, B-, D-, K-, L- and P-genotype strains grown individually or in pairs in vitro, in the rhizosphere of wheat and in bulk soil. Except for P. fluorescens Q8r1-96, which strongly inhibited the growth of P. fluorescens Q2-87, inhibition between pairs of strains grown in vitro did not affect the accuracy of the method. The allele-specific primer-based technique is a rapid method for studies of the interactions between genotypes of 2,4-diacetylphloroglucinol producers in natural environments.
KeywordMeSH Terms
Soil Microbiology
100. Jun  SY, Fushinobu  S, Nojiri  H, Omori  T, Shoun  H, Wakagi  T,     ( 2006 )

Improving the catalytic efficiency of a meta-cleavage product hydrolase (CumD) from Pseudomonas fluorescens IP01.

Biochimica et biophysica acta 1764 (7)
PMID : 16844437  :   DOI  :   10.1016/j.bbapap.2006.05.010    
Abstract >>
The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate the substrate specificity and catalytic efficiency of CumD toward substrates derived from monocyclic aromatic compounds, we constructed the CumD mutants, A129V, I199V, and V227I, as well as four types of double and triple mutants. Toward substrates with smaller side chains (e.g. 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the k(cat)/K(m) values of the single mutants were 4.2-11 fold higher than that of the wild type enzyme and 1.8-4.7 fold higher than that of the meta-cleavage product hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the highest k(cat)/K(m) value for 2-hydroxy-6-oxohepta-2,4-dienoate (6-ethyl-HODA). The crystal structure of the A129V mutant was determined at 1.65 A resolution, enabling location of the Ogamma atom of the Ser103 side chain. A chloride ion was bound to the oxyanion hole of the active site, and mutant enzymes at the residues forming this site were also examined. The k(cat) values of Ser34 mutants were decreased 2.9-65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing the anionic oxygen of the proposed intermediate state (gem-diolate). This is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential "triad", being intact.
KeywordMeSH Terms
101. Mavrodi  OV, Mavrodi  DV, Park  AA, Weller  DM, Thomashow  LS,     ( 2006 )

The role of dsbA in colonization of the wheat rhizosphere by Pseudomonas fluorescens Q8r1-96.

Microbiology (Reading, England) 152 (Pt 3)
PMID : 16514165  :   DOI  :   10.1099/mic.0.28545-0    
Abstract >>
Certain well-conserved genes in fluorescent Pseudomonas spp. are involved in pathogenic interactions between the bacteria and evolutionarily diverse hosts including plants, insects and vertebrate animals. One such gene, dsbA, encodes a periplasmic disulfide-bond-forming enzyme implicated in the biogenesis of exported proteins and cell surface structures. This study focused on the role of dsbA in Pseudomonas fluorescens Q8r1-96, a biological control strain that produces the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and is known for its exceptional ability to colonize the roots of wheat and pea. The deduced DsbA protein from Q8r1-96 is similar to other predicted thiol : disulfide interchange proteins and contains a conserved DsbA catalytic site, a pattern associated with the thioredoxin family active site, and a signal peptide and cleavage site. A dsbA mutant of Q8r1-96 exhibited decreased motility and fluorescence, and altered colony morphology; however, it produced more 2,4-DAPG and total phloroglucinol-related compounds and was more inhibitory in vitro to the fungal root pathogen Gaeumannomyces graminis var. tritici than was the parental strain. When introduced separately into a natural soil, Q8r1-96 and the dsbA mutant did not differ in their ability to colonize the rhizosphere of wheat in greenhouse experiments lasting 12 weeks. However, when the two strains were co-inoculated, the parental strain consistently out-competed the dsbA mutant. It was concluded that dsbA does not contribute to the exceptional rhizosphere competence of Q8r1-96, although the dsbA mutation reduces competitiveness when the mutant competes with the parental strain in the same niche in the rhizosphere. The results also suggest that exoenzymes and multimeric cell surface structures are unlikely to have a critical role in root colonization by this strain.
KeywordMeSH Terms
102. Tan  Y, Miller  KJ,     ( 1992 )

Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52.

Applied and environmental microbiology 58 (4)
PMID : 1599260  :   PMC  :   PMC195611    
Abstract >>
In this study, we report the cloning and expression of lipase gene from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. Sequence analysis revealed one major open reading frame of 1,428 nucleotides that was predicted to encode a protein with a molecular weight of 50,241. The predicted enzyme was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date.
KeywordMeSH Terms
103. Landa  BB, Mavrodi  OV, Schroeder  KL, Allende-Molar  R, Weller  DM,     ( 2006 )

Enrichment and genotypic diversity of phlD-containing fluorescent Pseudomonas spp. in two soils after a century of wheat and flax monoculture.

FEMS microbiology ecology 55 (3)
PMID : 16466375  :   DOI  :   10.1111/j.1574-6941.2005.00038.x    
Abstract >>
Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root(-1) or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.
KeywordMeSH Terms
Genetic Variation
Soil Microbiology
104. 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
105. Baehler  E, Bottiglieri  M, Péchy-Tarr  M, Maurhofer  M, Keel  C,     ( 2005 )

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

Journal of applied microbiology 99 (1)
PMID : 15960662  :   DOI  :   10.1111/j.1365-2672.2005.02597.x    
Abstract >>
To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.
KeywordMeSH Terms
106. Retallack  DM, Thomas  TC, Shao  Y, Haney  KL, Resnick  SM, Lee  VD, Squires  CH,     ( 2006 )

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions.

Microbial cell factories 5 (N/A)
PMID : 16396686  :   DOI  :   10.1186/1475-2859-5-1     PMC  :   PMC1360089    
Abstract >>
In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.
KeywordMeSH Terms
107. Bottiglieri  M, Keel  C,     ( 2006 )

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

Applied and environmental microbiology 72 (1)
PMID : 16391073  :   DOI  :   10.1128/AEM.72.1.418-427.2006     PMC  :   PMC1352262    
Abstract >>
The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.
KeywordMeSH Terms
108. Lee  J, Zhao  H,     ( 2006 )

Mechanistic studies on the conversion of arylamines into arylnitro compounds by aminopyrrolnitrin oxygenase: identification of intermediates and kinetic studies.

Angewandte Chemie (International ed. in English) 45 (4)
PMID : 16342311  :   DOI  :   10.1002/anie.200502903    
Abstract >>
N/A
KeywordMeSH Terms
109. Zhang  XX, Lilley  AK, Bailey  MJ, Rainey  PB,     ( 2004 )

The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases.

FEMS microbiology ecology 51 (1)
PMID : 16329852  :   DOI  :   10.1016/j.femsec.2004.07.006    
Abstract >>
Plasmid pQBR103 (approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria.
KeywordMeSH Terms
110. Mosbacher  TG, Mueller  M, Schulz  GE,     ( 2005 )

Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens.

The FEBS journal 272 (23)
PMID : 16302970  :   DOI  :   10.1111/j.1742-4658.2005.04998.x    
Abstract >>
Pseudomonas fluorescens is able to grow on R-benzoin as the sole carbon and energy source because it harbours the enzyme benzaldehyde lyase that cleaves the acyloin linkage using thiamine diphosphate (ThDP) as a cofactor. In the reverse reaction, this lyase catalyses the carboligation of two aldehydes with high substrate and stereospecificity. The enzyme structure was determined by X-ray diffraction at 2.6 A resolution. A structure-based comparison with other proteins showed that benzaldehyde lyase belongs to a group of closely related ThDP-dependent enzymes. The ThDP cofactors of these enzymes are fixed at their two ends in separate domains, suspending a comparatively mobile thiazolium ring between them. While the residues binding the two ends of ThDP are well conserved, the lining of the active centre pocket around the thiazolium moiety varies greatly within the group. Accounting for the known reaction chemistry, the natural substrate R-benzoin was modelled unambiguously into the active centre of the reported benzaldehyde lyase. Due to its substrate spectrum and stereospecificity, the enzyme extends the synthetic potential for carboligations appreciably.
KeywordMeSH Terms
Protein Structure, Tertiary
111. Brodhagen  M, Paulsen  I, Loper  JE,     ( 2005 )

Reciprocal regulation of pyoluteorin production with membrane transporter gene expression in Pseudomonas fluorescens Pf-5.

Applied and environmental microbiology 71 (11)
PMID : 16269724  :   DOI  :   10.1128/AEM.71.11.6900-6909.2005     PMC  :   PMC1287665    
Abstract >>
Pyoluteorin is a chlorinated polyketide antibiotic secreted by the rhizosphere bacterium Pseudomonas fluorescens Pf-5. Genes encoding enzymes and transcriptional regulators involved in pyoluteorin production are clustered in the genome of Pf-5. Sequence analysis of genes adjacent to the known pyoluteorin biosynthetic gene cluster revealed the presence of an ABC transporter system. We disrupted two putative ABC transporter genes by inserting transcriptional fusions to an ice nucleation reporter gene. Mutations in pltI and pltJ, which are predicted to encode a membrane fusion protein and an ATP-binding cassette of the ABC transporter, respectively, greatly reduced pyoluteorin production by Pf-5. During the transition from exponential growth to stationary phase, populations of a pltI mutant were lower than those of a pltI+ strain in a culture medium containing pyoluteorin, suggesting a role for the transport system in efflux and the resistance of Pf-5 to the antibiotic. Although pltI or pltJ mutant strains displayed low pyoluteorin production, they did not accumulate proportionately more of the antibiotic intracellularly, indicating that pltI and pltJ do not encode an exclusive exporter for pyoluteorin. Transcription of the putative pyoluteorin efflux genes pltI and pltJ was enhanced by exogenous pyoluteorin. These new observations parallel an earlier finding that pyoluteorin enhances the transcription of pyoluteorin biosynthesis genes and pyoluteorin production in Pf-5. This report provides evidence of a coordination of pyoluteorin production and the transcription of genes encoding a linked transport apparatus, wherein each requires the other for optimal expression.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
112. Kiziak  C, Conradt  D, Stolz  A, Mattes  R, Klein  J,     ( 2005 )

Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme.

Microbiology (Reading, England) 151 (Pt 11)
PMID : 16272385  :   DOI  :   10.1099/mic.0.28246-0    
Abstract >>
The gene encoding an enantioselective arylacetonitrilase was identified on a 3.8 kb DNA fragment from the genomic DNA of Pseudomonas fluorescens EBC191. The gene was isolated, sequenced and cloned into the L-rhamnose-inducible expression vector pJOE2775. The nitrilase was produced in large quantities and purified as a histidine-tagged enzyme from crude extracts of L-rhamnose-induced cells of Escherichia coli JM109. The purified nitrilase was significantly stabilized during storage by the addition of 1 M ammonium sulfate. The temperature optimum (50 degrees C), pH optimum (pH 6.5), and specific activity of the recombinant nitrilase were similar to those of the native enzyme from P. fluorescens EBC191. The enzyme hydrolysed various phenylacetonitriles with different substituents in the 2-position and also heterocyclic and bicyclic arylacetonitriles to the corresponding carboxylic acids. The conversion of most arylacetonitriles was accompanied by the formation of different amounts of amides as by-products. The relative amounts of amides formed from different nitriles increased with an increasing negative inductive effect of the substituent in the 2-position. The acids and amides that were formed from chiral nitriles demonstrated in most cases opposite enantiomeric excesses. Thus mandelonitrile was converted by the nitrilase preferentially to R-mandelic acid and S-mandelic acid amide. The nitrilase gene is physically linked in the genome of P. fluorescens with genes encoding the degradative pathway for mandelic acid. This might suggest a natural function of the nitrilase in the degradation of mandelonitrile or similar naturally occurring hydroxynitriles.
KeywordMeSH Terms
Cloning, Molecular
Gene Expression Regulation, Bacterial
Recombinant Proteins
113. Kojima  Y, Kobayashi  M, Shimizu  S,     ( 2003 )

A novel lipase from Pseudomonas fluorescens HU380: gene cloning, overproduction, renaturation-activation, two-step purification, and characterization.

Journal of bioscience and bioengineering 96 (3)
PMID : 16233516  :  
Abstract >>
The extracellular lipase gene (lipA) from Pseudomonas fluorescens HU380 was cloned from a genomic library constructed in pBluescript SK+. Nucleotide sequence analysis revealed an open reading frame of 1854 by encoding the lipase. Its deduced amino acid sequence included internal amino acid sequences of the lipase from this strain: The lipase showed significant sequence similarity to lipases of Serratia marcescens strains and P. fluorescens strains. In Escherichia coli, lipA was expressed in the form of inclusion bodies, which were subsequently solubilized by urea followed by dialysis. The refolded protein was soluble and biologically active. The lipase purified from the E. coli transformant by this denaturation-renaturation procedure followed by only two steps of column chromatographs exhibited the same electrophoretic mobility as did the enzyme purified from P. fluorescens HU380, and both enzymes were quite similar in physicochemical properties such as specific activity, suggesting that the recombinant lipase protein has an intrinsic folding capability in vitro. The function of its C-terminal region is also discussed.
KeywordMeSH Terms
114. Dong  C, Flecks  S, Unversucht  S, Haupt  C, van Pée  KH, Naismith  JH,     ( 2005 )

Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination.

Science (New York, N.Y.) 309 (5744)
PMID : 16195462  :   DOI  :   10.1126/science.1116510     PMC  :   PMC3315827    
Abstract >>
Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.
KeywordMeSH Terms
115. Velasco  A, Acebo  P, Gomez  A, Schleissner  C, Rodríguez  P, Aparicio  T, Conde  S, Muñoz  R, de la Calle  F, Garcia  JL, Sánchez-Puelles  JM,     ( 2005 )

Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: designing new cytotoxic compounds.

Molecular microbiology 56 (1)
PMID : 15773985  :   DOI  :   10.1111/j.1365-2958.2004.04433.x    
Abstract >>
Safracin is an antibiotic with anti-tumour activity produced by Pseudomonas fluorescens A2-2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non-ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross-feeding experiments have confirmed that 3-hydroxy-5-methyl-O-methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N-methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti-cancer compounds.
KeywordMeSH Terms
Bacterial Proteins
Multigene Family
116. Bobrova  VK, Miliutina  IA, Troitskiĭ  AV,     ( N/A )

[Genetic diversity in pseudomonads associated with cereal cultures infected with basal bacteriosis].

Mikrobiologiia 74 (4)
PMID : 16211859  :  
Abstract >>
The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S-23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae ("Syringae" cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici ("Fluorescens" cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the "Fluorescens" cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.
KeywordMeSH Terms
117. Fushinobu  S, Jun  SY, Hidaka  M, Nojiri  H, Yamane  H, Shoun  H, Omori  T, Wakagi  T,     ( 2005 )

A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products.

Bioscience, biotechnology, and biochemistry 69 (3)
PMID : 15784976  :   DOI  :   10.1271/bbb.69.491    
Abstract >>
Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the k(cat) values of the corresponding substrates. The Ile139 and Trp143 residues on helix alpha4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.
KeywordMeSH Terms
118. Péchy-Tarr  M, Bottiglieri  M, Mathys  S, Lejbølle  KB, Schnider-Keel  U, Maurhofer  M, Keel  C,     ( 2005 )

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0.

Molecular plant-microbe interactions : MPMI 18 (3)
PMID : 15782640  :   DOI  :   10.1094/MPMI-18-0260    
Abstract >>
Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.
KeywordMeSH Terms
119. Dong  X, Fushinobu  S, Fukuda  E, Terada  T, Nakamura  S, Shimizu  K, Nojiri  H, Omori  T, Shoun  H, Wakagi  T,     ( 2005 )

Crystal structure of the terminal oxygenase component of cumene dioxygenase from Pseudomonas fluorescens IP01.

Journal of bacteriology 187 (7)
PMID : 15774891  :   DOI  :   10.1128/JB.187.7.2483-2490.2005     PMC  :   PMC1065230    
Abstract >>
The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 A by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.
KeywordMeSH Terms
120. Martínez-Granero  F, Capdevila  S, Sánchez-Contreras  M, Martín  M, Rivilla  R,     ( 2005 )

Two site-specific recombinases are implicated in phenotypic variation and competitive rhizosphere colonization in Pseudomonas fluorescens.

Microbiology (Reading, England) 151 (Pt 3)
PMID : 15758242  :   DOI  :   10.1099/mic.0.27583-0    
Abstract >>
The biocontrol agent Pseudomonas fluorescens F113 undergoes phenotypic variation during rhizosphere colonization, and this variation has been related to the activity of a site-specific recombinase encoded by the sss gene. Here, it is shown that a second recombinase encoded by the xerD gene is also implicated in phenotypic variation. A putative xerD gene from this strain was cloned, and sequence analysis confirmed that it encoded a site-specific recombinase of the lambda integrase family. Mutants affected in the sss or xerD genes produced a very low quantity of phenotypic variants compared to the wild-type strain, both under prolonged cultivation in the laboratory and after rhizosphere colonization, and they were severely impaired in competitive root colonization. Overexpression of the genes encoding either recombinase resulted in a substantial increment in the production of phenotypic variants under both culture and rhizosphere colonization conditions, implying that both site-specific recombinases are involved in phenotypic variation. Overexpression of the sss gene suppressed the phenotype of a xerD mutant, but overexpression of the xerD gene had no effect on the phenotype of an sss mutant. Genetic analysis of the phenotypic variants obtained after overexpression of the genes encoding both the recombinases showed that they carried mutations in the gacA/S genes, which are necessary to produce a variety of secondary metabolites. These results indicate that the Gac system is affected by the activity of the site-specific recombinases. Transcriptional fusions of the sss and xerD genes with a promoterless lacZ gene showed that both genes have a similar expression pattern, with maximal expression during stationary phase. Although the expression of both genes was independent of diffusible compounds present in root exudates, it was induced by the plant, since bacteria attached to the root showed enhanced expression.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
121. Milani  M, Leoni  L, Rampioni  G, Zennaro  E, Ascenzi  P, Bolognesi  M,     ( 2005 )

An active-like structure in the unphosphorylated StyR response regulator suggests a phosphorylation- dependent allosteric activation mechanism.

Structure (London, England : 1993) 13 (9)
PMID : 16154086  :   DOI  :   10.1016/j.str.2005.05.014    
Abstract >>
StyR belongs to the FixJ subfamily of signal transduction response regulators; it controls transcription of the styABCD operon coding for styrene catabolism in Pseudomonas fluorescens ST. The crystal structure of unphosphorylated StyR is reported at 2.2 A resolution. StyR is composed of an N-terminal regulatory domain (StyR-N) and a C-terminal DNA binding domain (StyR-C). The two domains are separated by an elongated linker alpha helix (34 residues), a new feature in known response regulator structures. StyR-C is structured similarly to the DNA binding domain of the response regulator NarL. StyR-N shows structural reorganization of the phosphate receiving region involved in activation/homodimerization: specific residues adopt an "active-like" conformation, and the alpha4 helix, involved in dimerization of the homologous FixJ response regulator, is trimmed to just one helical turn. Overall, structural considerations suggest that phosphorylation may act as an allosteric switch, shifting a preexisting StyR equilibrium toward the active, dimeric, DNA binding form.
KeywordMeSH Terms
122. Jiang  Z, Zheng  Y, Luo  Y, Wang  G, Wang  H, Ma  Y, Wei  D,     ( 2005 )

Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52.

Molecular biotechnology 31 (2)
PMID : 16170209  :   DOI  :   10.1385/MB:31:2:095    
Abstract >>
A novel lipase gene (lipB52) was isolated directly from the genomic DNA of Pseudomonas fluorescens B52 with the genome-walking method, an effective method for isolating lipase gene from bacteria. There was an open reading frame (ORF) of 1854 bp, which encoded 617 amino acids. The lipase gene (lipB52) was cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant Pichia pastoris were screened with a high throughput method. The recombinant was induced by methanol to secrete active lipase into the culture medium. The recombinant lipase LipB52 was also purified and characterized. The optimum temperature for the purified lipase LipB52 was 40 degrees C at pH 8.0. It exhibited better thermostability and pH stability than its homologs.
KeywordMeSH Terms
Gene Expression
123. Siddiqui  IA, Haas  D, Heeb  S,     ( 2005 )

Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita.

Applied and environmental microbiology 71 (9)
PMID : 16151170  :   DOI  :   10.1128/AEM.71.9.5646-5649.2005     PMC  :   PMC1214651    
Abstract >>
In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.
KeywordMeSH Terms
Pest Control, Biological
124. Cullinane  M, Baysse  C, Morrissey  JP, O'Gara  F,     ( 2005 )

Identification of two lysophosphatidic acid acyltransferase genes with overlapping function in Pseudomonas fluorescens.

Microbiology (Reading, England) 151 (Pt 9)
PMID : 16151217  :   DOI  :   10.1099/mic.0.27958-0    
Abstract >>
Phosphatidic acid (PA) is known to be a crucial phospholipid intermediate in cell membrane biosynthesis. In Escherichia coli, this molecule is produced from lysophosphatidic acid (LPA) by LPA acyltransferase (EC 2.3.1.51), encoded by plsC. E. coli possesses only one such LPA acyltransferase and a plsC mutant is non-permissive for growth at elevated temperatures. This study describes the identification and characterization of two genes from Pseudomonas fluorescens F113 that encode enzymes with LPA acyltransferase activity. One of the genes, hdtS, was previously described, whereas patB is a novel gene. In addition, a putative lyso-ornithine lipid acyltransferase was also identified. All three proteins possess conserved acyltransferase domains and are homologous to PlsC and to LPA acyltransferases identified in Neisseria meningitidis. Functional analysis determined that both HdtS and PatB are functional LPA acyltransferases, as both complemented an E. coli plsC mutant. Mutants lacking each of the putative acyltransferases were constructed and analysed. Growth defects were observed for hdtS and patB single mutants, and a double hdtSpatB mutant could not be constructed. To determine precise roles in phospholipid synthesis, fatty acid methyl ester analysis was carried out. The hdtS mutant displayed a profile consistent with a defect in LPA acyltransferase activity, whereas no such phenotype was observed in the patB mutant, indicating that hdtS encodes the primary LPA acyltransferase in the cell. The presence of at least two genes specifying LPA acyltransferase activity may have implications for the function and survival of P. fluorescens in diverse environments.
KeywordMeSH Terms
125. Lee  J, Simurdiak  M, Zhao  H,     ( 2005 )

Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation.

The Journal of biological chemistry 280 (44)
PMID : 16150698  :   DOI  :   10.1074/jbc.M505334200    
Abstract >>
Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.
KeywordMeSH Terms
126. Mindlin  S, Minakhin  L, Petrova  M, Kholodii  G, Minakhina  S, Gorlenko  Z, Nikiforov  V,     ( 2005 )

Present-day mercury resistance transposons are common in bacteria preserved in permafrost grounds since the Upper Pleistocene.

Research in microbiology 156 (10)
PMID : 16084067  :   DOI  :   10.1016/j.resmic.2005.05.011    
Abstract >>
Transposons closely related to mercury resistance transposons Tn5041, Tn5053, and Tn5056, which have been previously described in present-day bacteria, were detected in a survey of 12 mercury-resistant Pseudomonas strains isolated from permafrost samples aged 15-40 thousand years. In addition, Tn5042, a novel type of mercury resistance transposon, was revealed in the permafrost strain collection and its variants found to be common among present-day bacteria. The results reveal that no drastic changes in the distribution mode of the different types of mercury resistance transposons among environmental bacteria have taken place in the last 15-40 thousand years.
KeywordMeSH Terms
DNA Transposable Elements
Ice
127. van den Heuvel  RH, Tahallah  N, Kamerbeek  NM, Fraaije  MW, van Berkel  WJ, Janssen  DB, Heck  AJ,     ( 2005 )

Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase.

The Journal of biological chemistry 280 (37)
PMID : 16049018  :   DOI  :   10.1074/jbc.M503758200    
Abstract >>
The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP(+) only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP(+) interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP(+)) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.
KeywordMeSH Terms
128. Marek-Kozaczuk  M, Rogalski  J, Skorupska  A,     ( 2005 )

The nadA gene of Pseudomonas fluorescens PGPR strain 267.1.

Current microbiology 51 (1��2��)
PMID : 16049659  :   DOI  :   10.1007/s00284-005-4553-2    
Abstract >>
An insertion mutant of Pseudomonas fluorescens PGPR strain 267.1 was found to be auxotrophic for niacin (nicotinic acid) and could not synthesize quinolinic acid. The Tn5 interrupted gene was cloned and sequenced. The cloned fragment contained an open reading frame, nadA, capable of encoding a 359-amino-acid protein (39.0 kDa) with substantial identity to various bacterial quinolinate synthetases. The nadA gene complemented quinolinic acid synthesis deficiency and niacin auxotrophy of the P. fluorescens 106 P nadA mutant.
KeywordMeSH Terms
Genes, Bacterial
129. Heeb  S, Valverde  C, Gigot-Bonnefoy  C, Haas  D,     ( 2005 )

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

FEMS microbiology letters 243 (1)
PMID : 15668026  :   DOI  :   10.1016/j.femsle.2004.12.008    
Abstract >>
In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Oxidative Stress
130. Favre-Bonté  S, Ranjard  L, Colinon  C, Prigent-Combaret  C, Nazaret  S, Cournoyer  B,     ( 2005 )

Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.

Environmental microbiology 7 (2)
PMID : 15658983  :   DOI  :   10.1111/j.1462-2920.2004.00670.x    
Abstract >>
The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.
KeywordMeSH Terms
Evolution, Molecular
Genetic Variation
131. McCarthy  CN, Woods  RG, Beacham  IR,     ( 2004 )

Regulation of the aprX-lipA operon of Pseudomonas fluorescens B52: differential regulation of the proximal and distal genes, encoding protease and lipase, by ompR-envZ.

FEMS microbiology letters 241 (2)
PMID : 15598539  :   DOI  :   10.1016/j.femsle.2004.10.027    
Abstract >>
The production of lipase and protease from psychrotrophic strains of Pseudomonas fluorescens may result in spoilage of dairy products. The lipase (lipA) and alkaline metalloprotease (aprX) genes of P. fluorescens B52 are regulated by temperature and are located at opposite ends of an operon which contains eight genes and spans 14 kb. In this report, we show that lipase activity in the supernatant of cultures of P. fluorescens strain B52 is also regulated by the homologue of the Escherichia coli EnvZ-OmpR two-component regulatory system. Differences in the regulation of lipase and protease may be related to the proximal and distal locations of aprX and lipA within the operon.
KeywordMeSH Terms
Gene Expression Regulation, Enzymologic
Operon
132. Blankenfeldt  W, Kuzin  AP, Skarina  T, Korniyenko  Y, Tong  L, Bayer  P, Janning  P, Thomashow  LS, Mavrodi  DV,     ( 2004 )

Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens.

Proceedings of the National Academy of Sciences of the United States of America 101 (47)
PMID : 15545603  :   DOI  :   10.1073/pnas.0407371101     PMC  :   PMC534541    
Abstract >>
Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function.
KeywordMeSH Terms
133. Capdevila  S, Martínez-Granero  FM, Sánchez-Contreras  M, Rivilla  R, Martín  M,     ( 2004 )

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization.

Microbiology (Reading, England) 150 (Pt 11)
PMID : 15528673  :   DOI  :   10.1099/mic.0.27362-0    
Abstract >>
The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants. Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation. This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113. A 6.5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made. Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained. These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization. A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type. However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes.
KeywordMeSH Terms
134. Parsons  JF, Calabrese  K, Eisenstein  E, Ladner  JE,     ( 2004 )

Structure of the phenazine biosynthesis enzyme PhzG.

Acta crystallographica. Section D, Biological crystallography 60 (Pt 11)
PMID : 15502343  :   DOI  :   10.1107/S0907444904022474    
Abstract >>
PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.
KeywordMeSH Terms
135. Black  MT, Munn  JG, Allsop  AE,     ( 1992 )

On the catalytic mechanism of prokaryotic leader peptidase 1.

The Biochemical journal 282 (Pt 2) (N/A)
PMID : 1546969  :   DOI  :   10.1042/bj2820539     PMC  :   PMC1130814    
Abstract >>
The catalytic mechanism of leader peptidase 1 (LP1) of the bacterium Escherichia coli has been investigated by a combination of site-directed mutagenesis, assays of enzyme activity in vivo utilizing a strain of E. coli which has a conditional defect in LP1 activity, and gene cloning. The biological activity of mutant forms of E. coli LP1 demonstrates that this enzyme belongs to a novel class of proteinases. The possibility that LP1 may be an aspartyl proteinase has been excluded on the basis of primary sequence comparison and mutagenesis. Assignment of LP1 to one of the other three recognized classes of proteinases (metalloproteinases, thiol proteinases and the classical serine proteinases) can also be excluded, as it is clearly demonstrated that none of the histidine or cysteine residues within LP1 are required for catalytic activity. The Pseudomonas fluorescens lep gene has been cloned and sequenced and the corresponding amino acid sequence compared with that of E. coli LP1. The E. coli LP1 and P. fluorescens LP1 primary sequences are 50% identical after insertion of gaps. The P. fluorescens LP1 has 39 fewer amino acids, a calculated molecular mass of 31903 Da and functions effectively in vivo in E. coli. None of the cysteine residues and only one of the histidine residues which are present in E. coli LP1 are conserved in sequence position in the P. fluorescens LP1 enzyme. The possibility that LP1 is a novel type of serine proteinase is discussed.
KeywordMeSH Terms
Membrane Proteins
Serine Endopeptidases
136. Parsons  JF, Song  F, Parsons  L, Calabrese  K, Eisenstein  E, Ladner  JE,     ( 2004 )

Structure and function of the phenazine biosynthesis protein PhzF from Pseudomonas fluorescens 2-79.

Biochemistry 43 (39)
PMID : 15449932  :   DOI  :   10.1021/bi049059z    
Abstract >>
Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms. Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved. Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement. PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains. Unlike diaminopimelate epimerase, PhzF is a dimer in solution. The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure. In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors. PhzA, -B, and -G have no activity toward DHHA. However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF. Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA. These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously. A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction. Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins. PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.
KeywordMeSH Terms
137. De Mot  R, Proost  P, Van Damme  J, Vanderleyden  J,     ( 1992 )

Homology of the root adhesin of Pseudomonas fluorescens OE 28.3 with porin F of P. aeruginosa and P. syringae.

Molecular & general genetics : MGG 231 (3)
PMID : 1538702  :   DOI  :   10.1007/bf00292721    
Abstract >>
The gene encoding the root adhesin from the outer membrane of Pseudomonas fluorescens OE 28.3 was isolated from a genomic lambda EMBL3 library and sequenced. The deduced protein (32104 daltons) displayed strong homology with the amino- and carboxyterminal parts of porin F (OprF) from P. aeruginosa and P. syringae. Significant homology was also found within the C-terminal domain of the OmpA proteins from Enterobacteria and major outer membrane proteins from Neisseria species. However, a cysteine-rich domain present in the OprFs of P. aeruginosa and P. syringae is absent from the adhesin of P. fluorescens. Instead, it contains a shorter sequence with eight alternating proline residues.
KeywordMeSH Terms
Adhesins, Bacterial
Genes, Fungal
Lectins
Porins
138. 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
139. Göbel  M, Kranz  OH, Kaschabek  SR, Schmidt  E, Pieper  DH, Reineke  W,     ( 2004 )

Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters.

Archives of microbiology 182 (2��3��)
PMID : 15340793  :   DOI  :   10.1007/s00203-004-0681-5    
Abstract >>
Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.
KeywordMeSH Terms
140. 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
141. 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
142. Kamerbeek  NM, Fraaije  MW, Janssen  DB,     ( 2004 )

Identifying determinants of NADPH specificity in Baeyer-Villiger monooxygenases.

European journal of biochemistry 271 (11)
PMID : 15153101  :   DOI  :   10.1111/j.1432-1033.2004.04126.x    
Abstract >>
The Baeyer-Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O(2) to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH. Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased K(m). However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP(+), confirming a role of the lysine in recognizing the 2'-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues.
KeywordMeSH Terms
143. Nakashima  N, Tamura  T,     ( 2004 )

Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter.

Biochemical and biophysical research communications 319 (2)
PMID : 15178458  :   DOI  :   10.1016/j.bbrc.2004.05.034    
Abstract >>
We have modified the cell-free coupled transcription/translation system of bacteria. The cell-free extract of Pseudomonas fluorescens was used for translation instead of Escherichia coli. In addition, transcription of the target gene was regulated by CspA promoter with endogenous RNA polymerase instead of by T7 promoter with exogenous T7 RNA polymerase. We could increase the yields of soluble proteins using different combinations of the S30 extract and the promoter and different temperatures for protein synthesis. Increasing the variety of synthesis systems allows production of large quantities of soluble proteins. In order to carry out efficient cell-free protein synthesis, versatile pCop-plasmids carrying CspA promoter were constructed and these plasmids were applicable to expression of recombinant proteins in E. coli cells.
KeywordMeSH Terms
Promoter Regions, Genetic
144. Abbas  A, McGuire  JE, Crowley  D, Baysse  C, Dow  M, O'Gara  F,     ( 2004 )

The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance.

Microbiology (Reading, England) 150 (Pt 7)
PMID : 15256586  :   DOI  :   10.1099/mic.0.27033-0    
Abstract >>
2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Heat-Shock Response
145. Dong  C, Kotzsch  A, Dorward  M, van Pée  KH, Naismith  JH,     ( 2004 )

Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens.

Acta crystallographica. Section D, Biological crystallography 60 (Pt 8)
PMID : 15272170  :   DOI  :   10.1107/S0907444904012521    
Abstract >>
Chlorination of natural products is often required for their biological activity; notable examples include vancomycin, the last-ditch antibiotic. It is now known that many chlorinated natural products are made not by haloperoxidases, but by FADH2-dependent halogenases. The mechanism of the flavin-containing enzymes is obscure and there are no structural data. Here, crystals of PrnA (tryptophan 7-halogenase), an enzyme that regioselectively chlorinates tryptophan, cocrystallized with tryptophan and FAD are reported. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 67.8, c = 276.9 A. A data set to 1.8 A with 93% completeness and an Rmerge of 7.1% has been collected from a single flash-cooled crystal. A method for incorporating selenomethionine in a Pseudomonas fluorescens expression system also is reported.
KeywordMeSH Terms
146. de Weert  S, Dekkers  LC, Kuiper  I, Bloemberg  GV, Lugtenberg  BJ,     ( 2004 )

Generation of enhanced competitive root-tip-colonizing Pseudomonas bacteria through accelerated evolution.

Journal of bacteriology 186 (10)
PMID : 15126477  :   DOI  :   10.1128/jb.186.10.3153-3159.2004     PMC  :   PMC400599    
Abstract >>
A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog. This gene is involved in the repair of A. G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.
KeywordMeSH Terms
Pest Control, Biological
147. Ge  Y, Huang  X, Wang  S, Zhang  X, Xu  Y,     ( 2004 )

Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18.

FEMS microbiology letters 237 (1)
PMID : 15268936  :   DOI  :   10.1016/j.femsle.2004.06.028    
Abstract >>
The biosynthesis of antimicrobial metabolites is controlled by the GacS/GacA two-component regulatory system in Pseudomonas species. The production of phenazine-1-carboxylic acid and pyoluteorin is differentially regulated by GacA in Pseudomonas sp. M18. Pyoluteorin was reduced to nondetectable level in culture of the gacA insertional mutant strain M18G grown in King's medium B broth, whereas phenazine-1-carboxylic acid production was increased 30-fold over that of the wild-type strain. Production of both antibiotics was restored to wild-type levels after complementation in trans with the wild-type gacA gene. Expression of the translational fusions phzA'-'lacZ and pltA'-'lacZ confirmed the effect of GacA on both biosynthetic operons.
KeywordMeSH Terms
148. 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
149. Matthijs  S, Baysse  C, Koedam  N, Tehrani  KA, Verheyden  L, Budzikiewicz  H, Schäfer  M, Hoorelbeke  B, Meyer  JM, De Greve  H, Cornelis  P,     ( 2004 )

The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway.

Molecular microbiology 52 (2)
PMID : 15066027  :   DOI  :   10.1111/j.1365-2958.2004.03999.x    
Abstract >>
To cope with iron deficiency fluorescent pseudomonads produce pyoverdines which are complex peptidic siderophores that very efficiently scavenge iron. In addition to pyoverdine some species also produce other siderophores. Recently, it was shown that Pseudomonas fluorescens ATCC 17400 produces the siderophore quinolobactin, an 8-hydroxy-4-methoxy-2-quinoline carboxylic acid (Mossialos, D., Meyer, J.M., Budzikiewicz, H., Wolff, U., Koedam, N., Baysse, C., Anjaiah, V., and Cornelis, P. (2000) Appl Environ Microbiol 66: 487-492). The entire quinolobactin biosynthetic, transport and uptake gene cluster, consisting out of two operons comprising 12 open reading frames, was cloned and sequenced. Based on the genes present and physiological complementation assays a biosynthetic pathway for quinolobactin is proposed. Surprisingly, this pathway turned out to combine genes derived from the eukaryotic tryptophan-xanthurenic acid branch of the kynurenine pathway and from the pathway for the biosynthesis of pyridine-2,6-bis(thiocarboxylic acid) from P. stutzeri, PDTC. These results clearly show the involvement of the tryptophan-kynurenine-xanthurenic acid pathway in the synthesis of an authentic quinoline siderophore.
KeywordMeSH Terms
Quinolines
150. Cheeseman  JD, Tocilj  A, Park  S, Schrag  JD, Kazlauskas  RJ,     ( 2004 )

Structure of an aryl esterase from Pseudomonas fluorescens.

Acta crystallographica. Section D, Biological crystallography 60 (Pt 7)
PMID : 15213385  :   DOI  :   10.1107/S0907444904010522    
Abstract >>
The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 C(alpha) atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.
KeywordMeSH Terms
151. Momany  C, Levdikov  V, Blagova  L, Lima  S, Phillips  RS,     ( 2004 )

Three-dimensional structure of kynureninase from Pseudomonas fluorescens.

Biochemistry 43 (5)
PMID : 14756555  :   DOI  :   10.1021/bi035744e    
Abstract >>
Kynureninase [E.C. 3.7.1.3] is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to anthranilic acid and l-alanine. Sequence alignment with other PLP-dependent enzymes indicated that kynureninase is in subgroup IVa of the aminotransferases, along with nifS, CsdB, and serine-pyruvate aminotransferase, which suggests that kynureninase has an aminotransferase fold. Crystals of Pseudomonas fluorescens kynureninase were obtained, and the structure was solved by molecular replacement using the CsdB coordinates combined with multiple isomorphous heavy atom replacement. The coordinates were deposited in the PDB (ID code 1QZ9). The structure, refined to an R factor of 15.5% to 1.85 A resolution, is dimeric and has the aminotransferase fold. The structure also confirms the prediction from sequence alignment that Lys-227 is the PLP-binding residue in P. fluorescens kynureninase. The conserved Asp-201, expected for an aminotransferase fold, is located near the PLP nitrogen, but Asp-132 is also strictly conserved and at a similar distance from the pyridinium nitrogen. Mutagenesis of both conserved aspartic acids shows that both contribute equally to PLP binding, but Asp-201 has a greater role in catalysis. The structure shows that Tyr-226 donates a hydrogen bond to the phosphate of PLP. Unusual among PLP-dependent enzymes, Trp-256, which is also strictly conserved in kynureninases from bacteria to humans, donates a hydrogen bond to the phosphate through the indole N1-hydrogen.
KeywordMeSH Terms
152. 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
153. 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
154. Valverde  C, Heeb  S, Keel  C, Haas  D,     ( 2003 )

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

Molecular microbiology 50 (4)
PMID : 14622422  :   DOI  :   10.1046/j.1365-2958.2003.03774.x    
Abstract >>
In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Regulator
155. De Souza  JT, De Boer  M, De Waard  P, Van Beek  TA, Raaijmakers  JM,     ( 2003 )

Biochemical, genetic, and zoosporicidal properties of cyclic lipopeptide surfactants produced by Pseudomonas fluorescens.

Applied and environmental microbiology 69 (12)
PMID : 14660362  :   DOI  :   10.1128/aem.69.12.7161-7172.2003     PMC  :   PMC309978    
Abstract >>
Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m-1. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m-1 and reached the critical micelle concentration at 25 micrograms ml-1. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.
KeywordMeSH Terms
156. van Berkel  W, Westphal  A, Eschrich  K, Eppink  M, de Kok  A,     ( 1992 )

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

European journal of biochemistry 210 (2)
PMID : 1459126  :   DOI  :   10.1111/j.1432-1033.1992.tb17436.x    
Abstract >>
The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.
KeywordMeSH Terms
157. Brillet  K, Reimmann  C, Mislin  GL, Noël  S, Rognan  D, Schalk  IJ, Cobessi  D,     ( 2011 )

Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition.

Journal of the American Chemical Society 133 (41)
PMID : 21902256  :   DOI  :   10.1021/ja205504z    
Abstract >>
Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.
KeywordMeSH Terms
Fluorescence
158. Shintani  M, Horisaki  T, Yamane  H, Ohkuma  M, Nojiri  H,     ( 2011 )

Evolution of the IncP-7 carbazole-degradative plasmid pCAR1 improves survival of its host Pseudomonas fluorescens Pf0-1 in artificial water microcosms.

Microbiology (Reading, England) 157 (Pt 8)
PMID : 21565929  :   DOI  :   10.1099/mic.0.049064-0    
Abstract >>
In our previous study, Pseudomonas fluorescens Pf0-1L, harbouring the IncP-7 carbazole-degradative plasmid pCAR1 : : rfp, was shown to be undetectable within 5 days post-inoculation in carbazole-contaminated artificial freshwater microcosms containing several plasmid-free bacteria in addition to Pf0-1L(pCAR1 : : rfp). Fourteen days after the inoculation, carbazole degraders become detectable. Here, we revealed that these isolates were not pCAR1 transconjugants, but Pf0-1L(pCAR1 : : rfp) mutants, based on RFLP and BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) analysis. Notably, the mutants displayed more rapid initiation of carbazole degradation than the parent strain Pf0-1L(pCAR1 : : rfp). The mutants were unable to degrade anthranilate due to a 163 bp deletion in the antA gene, which was overcome by their transformation with a wild-type antABC-expressing plasmid. Quantitative RT-PCR analysis indicated that the transcriptional induction of carbazole-, anthranilate- and catechol-degradative genes was comparable in both parent and mutant strains. The deletion mutants became dominant in the artificial water microcosm. The mutation caused anthranilate to accumulate instead of catechol, a toxic compound for the parent strain, and may be beneficial to host survival in artificial microcosms.
KeywordMeSH Terms
Evolution, Molecular
Plasmids
159. Bodilis  J, Nsigue Meilo  S, Cornelis  P, De Vos  P, Barray  S,     ( 2011 )

A long-branch attraction artifact reveals an adaptive radiation in pseudomonas.

Molecular biology and evolution 28 (10)
PMID : 21504889  :   DOI  :   10.1093/molbev/msr099    
Abstract >>
A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms.
KeywordMeSH Terms
160. Li  QA, Mavrodi  DV, Thomashow  LS, Roessle  M, Blankenfeldt  W,     ( 2011 )

Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE.

The Journal of biological chemistry 286 (20)
PMID : 21454481  :   DOI  :   10.1074/jbc.M110.183418     PMC  :   PMC3093893    
Abstract >>
PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 ? resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH(3) to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 ? in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 ? resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn(2+), Mn(2+), and Ni(2+) reduce the activity of PhzE.
KeywordMeSH Terms
Protein Multimerization
161. Ferrara  S, Mapelli  E, Sello  G, Di Gennaro  P,     ( 2011 )

Characterization of the aldol condensation activity of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from Pseudomonas fluorescens N3.

Biochimica et biophysica acta 1814 (5)
PMID : 21443971  :   DOI  :   10.1016/j.bbapap.2011.03.013    
Abstract >>
The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene. This enzyme is an aldolase of class I that reversibly catalyzes the transformation of the trans-o-hydroxybenzylidenepyruvate (t-HBP), releasing pyruvate and salicylaldehyde. The enzyme was expressed in Escherichia coli as a recombinant protein of 38kDa with a His6-Tag at its N-terminus. The recombinant protein His-tHBP-HA was purified by affinity chromatography and we present here the biochemical characterization of its activity in the aldol condensation reaction. The aldol condensation reaction parameters were determined using as acceptors both salicylaldehyde, which is the natural substrate taking part to the naphthalene degradative pathway, and benzaldehyde. In both cases, His-tHBP-HA shows similar apparent K(m) and apparent V(max) values. Further analyses showed that the optimal pH and temperature of His-tHBP-HA activity are 7.0 and 30�XC, respectively. The tHBP-HA catalytic rates and the availability of an efficient system to produce large amounts of purified protein are relevant from a biotechnological point of view.
KeywordMeSH Terms
162. 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
163. Pathma  J, Ayyadurai  N, Sakthivel  N,     ( 2010 )

Assessment of genetic and functional relationship of antagonistic fluorescent pseudomonads of rice rhizosphere by repetitive sequence, protein coding sequence and functional gene analyses.

Journal of microbiology (Seoul, Korea) 48 (6)
PMID : 21221925  :   DOI  :   10.1007/s12275-010-0064-3    
Abstract >>
Antagonistic fluorescent pseudomonads isolated from rice rhizospheric soil were characterized using biochemical, taxonomical and molecular tools. Production of cyclopropane fatty acid (CFA) was correlated with their antagonistic potential. Strains were grouped into 18 different genotypes on the basis of amplified ribosomal DNA restriction analysis (ARDRA) and repetitive (rep)-PCR based genotypic fingerprinting analyses. High phylogenetic resolution among antagonistic fluorescent pseudomonad strains was obtained based on the DNA gyrase B subunit (gyrB) and RNA polymerase sigma factor 70 (rpoD) gene sequence analyses. Combined gyrB and rpoD sequence analysis resulted in the accurate estimation of molecular phylogeny and provided a significant correlation between the genetic distances among strains. Present study demonstrated the genetic and functional relationship of fluorescent pseudomonads. The knowledge on genetic and functional potential of fluorescent pseudomonads associated with rice rhizosphere is useful to understand their ecological role and for their utilization in sustainable agriculture.
KeywordMeSH Terms
Rhizosphere
Soil Microbiology
164. Michelsen  CF, Stougaard  P,     ( 2011 )

A novel antifungal Pseudomonas fluorescens isolated from potato soils in Greenland.

Current microbiology 62 (4)
PMID : 21165740  :   DOI  :   10.1007/s00284-010-9846-4    
Abstract >>
A rhizobacterium with high antifungal activity was isolated from a potato field at Inneruulalik, South Greenland. Phylogenetic analysis based on multi locus sequence typing showed that the bacterium was affiliated with strains of Pseudomonas fluorescens. The bacterium, denoted as Pseudomonas fluorescens In5, inhibited in vitro a broad range of phytopathogenic fungi, and the antifungal activity increased with decreasing temperature. Microcosm experiments demonstrated that P. fluorescens In5 protected tomato seedlings from Rhizoctonia solani. Transposon mutagenesis showed that the major cause for the antifungal activity of P. fluorescens In5 was a novel non-ribosomal peptide synthase (NRPS) gene. In addition, transposon mutagenesis showed that P. fluorescens In5 also contained a putative quinoprotein glucose dehydrogenase gene, which was involved in growth inhibition of phytopathogenic fungi. Although P. fluorescens In5 contained the capacity to synthesize hydrogen cyanide, �]-1,3-glucanase, protease, and chitinase, these did not seem to play a role in the in vitro and microcosm antifungal assays.
KeywordMeSH Terms
Antibiosis
Soil Microbiology
165. Isaki  L, Beers  R, Wu  HC,     ( 1990 )

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

Journal of bacteriology 172 (11)
PMID : 2121716  :   DOI  :   10.1128/jb.172.11.6512-6517.1990     PMC  :   PMC526840    
Abstract >>
The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.
KeywordMeSH Terms
Genes, Bacterial
Membrane Proteins
Serine Endopeptidases
166. Mavrodi  DV, Joe  A, Mavrodi  OV, Hassan  KA, Weller  DM, Paulsen  IT, Loper  JE, Alfano  JR, Thomashow  LS,     ( 2011 )

Structural and functional analysis of the type III secretion system from Pseudomonas fluorescens Q8r1-96.

Journal of bacteriology 193 (1)
PMID : 20971913  :   DOI  :   10.1128/JB.00895-10     PMC  :   PMC3019950    
Abstract >>
Pseudomonas fluorescens Q8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogen Pseudomonas syringae. We also screened a collection of 30 closely related P. fluorescens strains and detected the T3SS genes in all but one of them. The Q8r1-96 genome contained ropAA and ropM type III effector genes, which are orthologs of the P. syringae effector genes hopAA1-1 and hopM1, as well as a novel type III effector gene designated ropB. These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by both P. syringae and Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses.
KeywordMeSH Terms
167. 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
168. Upadhyay  A, Srivastava  S,     ( 2010 )

Evaluation of multiple plant growth promoting traits of an isolate of Pseudomonas fluorescens strain Psd.

Indian journal of experimental biology 48 (6)
PMID : 20882763  :  
Abstract >>
P. fluorescens strain Psd was isolated from the rhizosphere of Vigna mungo and evaluated for its multiple plant growth promoting and biocontrol properties against F. oxyspornum. Interestingly, this strain not only produces a range of antimicrobial compounds but also solubilizes complexed phosphates and synthesizes phytohormone (IAA). These properties can be assessed to elucidate the agronomic significance and rhizospheric competence of this soil isolate. Biocontrol action has been demonstrated in vitro against some other rhizospheric bacteria, and a phytopathogenic fungus along with wild type E. coli K-12. Genetic evidence for the antimicrobial status of strain Psd has been derived in terms of elucidating a unique combination of phenazine and pyrrolnitrin biosynthesis genes, not reported for any other P. fluorescens strain. The conserved part of antibiotics biosynthesis operon has been PCR amplified, cloned, sequenced and phylogenetic relationship based on similar genes from a few known Pseudomonads has been derived. The properties possessed by strain Psd may enable the bacterium to establish itself successfully in the rhizosphere.
KeywordMeSH Terms
169. 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
170. Zhu  X, van Pée  KH, Naismith  JH,     ( 2010 )

The ternary complex of PrnB (the second enzyme in the pyrrolnitrin biosynthesis pathway), tryptophan, and cyanide yields new mechanistic insights into the indolamine dioxygenase superfamily.

The Journal of biological chemistry 285 (27)
PMID : 20421301  :   DOI  :   10.1074/jbc.M110.120485     PMC  :   PMC2898318    
Abstract >>
Pyrrolnitrin (3-chloro-4-(2'-nitro-3'-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-l-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both l-tryptophan or 7-Cl-l-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In l-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr(321) and Ser(332) play a key role in binding these groups. The structures suggest that catalysis requires an l-configured substrate. Isothermal titration calorimetry data suggest d-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications.
KeywordMeSH Terms
171. Sperandio  D, Rossignol  G, Guerillon  J, Connil  N, Orange  N, Feuilloley  MG, Merieau  A,     ( 2010 )

Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032.

BMC microbiology 10 (N/A)
PMID : 20416103  :   DOI  :   10.1186/1471-2180-10-124     PMC  :   PMC2871272    
Abstract >>
MFN1032 is a clinical Pseudomonas fluorescens strain able to grow at 37 degrees C. MFN1032 cells induce necrosis and apoptosis in rat glial cells at this temperature. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides. Under laboratory conditions, this activity is not expressed at 37 degrees C. This activity is tightly regulated and is subject to phase variation. We found that MFN1032 displays a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis was expressed at 37 degrees C and was only detected in vitro in mid log growth phase in the presence of erythrocytes. We studied the regulation of this activity in the wild-type strain and in a mutant defective in the Gac two-component pathway. GacS/GacA is a negative regulator of this activity. In contrast to the Pseudomonas fluorescens strains PfO-1 and Pf5, whose genomes have been sequenced, the MFN1032 strain has the type III secretion-like genes hrcRST belonging to the hrpU operon. We showed that disruption of this operon abolished cell-associated hemolytic activity. This activity was not detected in P.fluorescens strains carrying similar hrc genes, as for the P. fluorescens psychrotrophic strain MF37. To our knowledge this the first demonstration of cell-associated hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this activity seems to be related to a functional hrpU operon and is independent of biosurfactant production. Precise link between a functional hrpU operon and cell-associated hemolytic activity remains to be elucidated.
KeywordMeSH Terms
Hemolysis
172. Mattheus  W, Gao  LJ, Herdewijn  P, Landuyt  B, Verhaegen  J, Masschelein  J, Volckaert  G, Lavigne  R,     ( 2010 )

Isolation and purification of a new kalimantacin/batumin-related polyketide antibiotic and elucidation of its biosynthesis gene cluster.

Chemistry & biology 17 (2)
PMID : 20189105  :   DOI  :   10.1016/j.chembiol.2010.01.014    
Abstract >>
Kal/bat, a polyketide, isolated to high purity (>95%) is characterized by strong and selective antibacterial activity against Staphylococcus species (minimum inhibitory concentration, 0.05 microg/mL), and no resistance was observed in strains already resistant to commonly used antibiotics. The kal/bat biosynthesis gene cluster was determined to a 62 kb genomic region of Pseudomonas fluorescens BCCM_ID9359. The kal/bat gene cluster consists of 16 open reading frames (ORF), encoding a hybrid PKS-NRPS system, extended with trans-acting tailoring functions. A full model for kal/bat biosynthesis is postulated and experimentally tested by gene inactivation, structural confirmation (using NMR spectroscopy), and complementation. The structural and microbiological study of biosynthetic kal/bat analogs revealed the importance of the carbamoyl group and 17-keto group for antibacterial activity. The mechanism of self-resistance lies within the production of an inactive intermediate, which is activated in a one-step enzymatic oxidation upon export. The genetic basis and biochemical elucidation of the biosynthesis pathway of this antibiotic will facilitate rational engineering for the design of novel structures with improved activities. This makes it a promising new therapeutic option to cope with multidrug-resistant clinical infections.
KeywordMeSH Terms
173. Choi  MH, Xu  J, Rho  JK, Zhao  XP, Yoon  SC,     ( 2010 )

Enhanced production of longer side-chain polyhydroxyalkanoic acid with omega-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid beta-oxidation inhibition.

Bioresource technology 101 (12)
PMID : 20153638  :   DOI  :   10.1016/j.biortech.2010.01.082    
Abstract >>
The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07.
KeywordMeSH Terms
174. Farajzadeh  D, Aliasgharzad  N, Sokhandan Bashir  N, Yakhchali  B,     ( 2010 )

Cloning and characterization of a plasmid encoded ACC deaminase from an indigenous Pseudomonas fluorescens FY32.

Current microbiology 61 (1)
PMID : 20049599  :   DOI  :   10.1007/s00284-009-9573-x    
Abstract >>
In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into alpha-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5alpha proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process.
KeywordMeSH Terms
175. Lu  W, Zhang  W, Bai  Y, Fu  Y, Chen  J, Geng  X, Wang  Y, Xiao  M,     ( 2010 )

A genetically engineered Pseudomonas fluorescens strain possesses the dual activity against phytopathogenic fungi and insects.

Journal of microbiology and biotechnology 20 (2)
PMID : 20208430  :  
Abstract >>
A Pseudomonas fluorescens strain was isolated and showed antagonistic activity against phytopathogenic fungi and found to possess a gene responsible for production of antibiotic 2, 4-diacetylphloroglucinol. For the extension of biocontrol range, a gene for an Androctonus australis Hector insect toxin 1 (AaHIT1), one of the most toxic known insect-selective peptides, was designed and synthesized according to the preferred codon usage of Pseudomonas fluorescens, cloned and transformed into the strain by pSUP106 vector, a broad-host-range plasmid. Bioassays indicated that the engineered strain was able to produce AaHIT1 with insecticidal activity, in the same time retained the activity against plant pathogen. The experiments for nonplanted soil and rhizosphere colonization showed that, similar to the population of the wild-type strain, that of the engineered strain remained relatively constant in the first 10 d, and the subsequent 50 d, suggesting that AaHIT expression in the bacterial cell does not substantially impair its long-term colonization. It is first reported that a Pseudomonas fluorescens strain expressing an active scorpion neurotoxin has dual activity against phytopathogenic fungi and insects, attractive for agronomic applications.
KeywordMeSH Terms
Genetic Engineering
176. Brandt  GS, Kneen  MM, Petsko  GA, Ringe  D, McLeish  MJ,     ( 2010 )

Active-site engineering of benzaldehyde lyase shows that a point mutation can confer both new reactivity and susceptibility to mechanism-based inhibition.

Journal of the American Chemical Society 132 (2)
PMID : 20030408  :   DOI  :   10.1021/ja907064w    
Abstract >>
Benzaldehyde lyase (BAL) from Pseudomonas putida is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the breakdown of (R)-benzoin. Here we report that a point mutant, BAL A28S, not only catalyzes the decarboxylation of benzoylformate but, like benzoylformate decarboxylase (BFDC), is also inactivated by the benzoylformate analogues methyl benzoylphosphonate (MBP) and benzoylphosphonate (BP). The latter has no effect on wild-type BAL, and the inactivation of the A28S variant is shown to result from phosphorylation of the newly introduced serine residue. This lends support to the proposal that an appropriately placed nucleophile facilitates the expulsion of carbon dioxide from the active site in many ThDP-dependent decarboxylases.
KeywordMeSH Terms
Point Mutation
Protein Engineering
177. Mavrodi  DV, Peever  TL, Mavrodi  OV, Parejko  JA, Raaijmakers  JM, Lemanceau  P, Mazurier  S, Heide  L, Blankenfeldt  W, Weller  DM, Thomashow  LS,     ( 2010 )

Diversity and evolution of the phenazine biosynthesis pathway.

Applied and environmental microbiology 76 (3)
PMID : 20008172  :   DOI  :   10.1128/AEM.02009-09     PMC  :   PMC2813009    
Abstract >>
Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.
KeywordMeSH Terms
Genes, Bacterial
178. Girlich  D, Poirel  L, Nordmann  P,     ( 2010 )

Novel ambler class A carbapenem-hydrolyzing beta-lactamase from a Pseudomonas fluorescens isolate from the Seine River, Paris, France.

Antimicrobial agents and chemotherapy 54 (1)
PMID : 19901091  :   DOI  :   10.1128/AAC.00961-09     PMC  :   PMC2798510    
Abstract >>
A Pseudomonas fluorescens isolate (PF-1) resistant to carbapenems was recovered during an environmental survey performed with water from the Seine River (Paris). It expressed a novel Ambler class A carbapenemase, BIC-1, sharing 68 and 59% amino acid identities with beta-lactamases SFC-1 from Serratia fonticola and the plasmid-encoded KPC-2, respectively. beta-Lactamase BIC-1 hydrolyzed penicillins, carbapenems, and cephalosporins except ceftazidime and monobactams. The bla(BIC-1) gene was chromosomally located and was also identified in two other P. fluorescens strains isolated from the Seine River 3 months later.
KeywordMeSH Terms
179. Hu  YH, Wang  HL, Zhang  M, Sun  L,     ( 2009 )

Molecular analysis of the copper-responsive CopRSCD of a pathogenic Pseudomonas fluorescens strain.

Journal of microbiology (Seoul, Korea) 47 (3)
PMID : 19557345  :   DOI  :   10.1007/s12275-008-0278-9    
Abstract >>
CopRS/CopABCD is one of the known systems that control copper homeostasis in bacteria. Although CopRS/CopABCD homologues are found to exist in Pseudomonas fluorescens, the potential role of this system in P. fluorescens has not been investigated. In this study a genetic cluster, consisting of copR, S, C, and D but lacking copAB, was identified in a pathogenic P. fluorescens strain (TSS) isolated from diseased fish. The copRSCD cluster was demonstrated to be required for full copper resistance and regulated at the transcription level by Cu. Expression of copCD is regulated directly by the two-component response regulator CopR, which also regulates its own expression. Interruption of the regulated expression of copR affected bacterial growth, biofilm formation, and tissue dissemination and survival. A mutant CopR, which lacks the N-terminal signal receiver domain and is constitutively active, was found to have an attenuating effect on bacterial virulence when expressed in TSS. To our knowledge, this is the first report that suggests a link between CopR and bacterial pathogenicity in P. fluorescens.
KeywordMeSH Terms
180. Heinaru  E, Vedler  E, Jutkina  J, Aava  M, Heinaru  A,     ( 2009 )

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20.

FEMS microbiology ecology 70 (3)
PMID : 19744238  :   DOI  :   10.1111/j.1574-6941.2009.00763.x    
Abstract >>
The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 ISPre1-like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 ISPre2-like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer - we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of ISPre2-like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.
KeywordMeSH Terms
Conjugation, Genetic
181. Wang  HR, Hu  YH, Zhang  WW, Sun  L,     ( 2009 )

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine.

Vaccine 27 (30)
PMID : 19501788  :   DOI  :   10.1016/j.vaccine.2009.04.023    
Abstract >>
Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P. fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections.
KeywordMeSH Terms
182. Stockwell  VO, Hockett  K, Loper  JE,     ( 2009 )

Role of RpoS in stress tolerance and environmental fitness of the phyllosphere bacterium Pseudomonas fluorescens strain 122.

Phytopathology 99 (6)
PMID : 19453227  :   DOI  :   10.1094/PHYTO-99-6-0689    
Abstract >>
Bacteria living epiphytically on aerial plant surfaces encounter severe and rapidly fluctuating environmental conditions, and their capacity to withstand environmental stress contributes to epiphytic fitness. The stationary phase sigma factor RpoS is a key determinant in stress response of gram-negative bacteria, including Pseudomonas spp. This study focused on the role of RpoS in stress response and epiphytic fitness of Pseudomonas fluorescens strain 122 on aerial plant surfaces. RpoS had a significant role in the response of the phyllosphere bacterium P. fluorescens 122 to stresses imposed by desiccation, UV irradiation, starvation, and an oxidative environment. While significant, the difference in stress response between an rpoS mutant and the parental strain was less for strain 122 than for the rhizosphere bacterium P. fluorescens Pf-5. No consistent influence of RpoS on epiphytic population size of strain 122 on pear or apple flowers or leaves was observed in field trials. These data may indicate that P. fluorescens occupies protected microsites on aerial plant surfaces where the bacteria escape exposure to environmental stress, or that redundant stress-response mechanisms are operating in this bacterium, thereby obscuring the role of RpoS in epiphytic fitness of the bacterium.
KeywordMeSH Terms
183. Tian  T, Wu  XG, Duan  HM, Zhang  LQ,     ( 2010 )

The resistance-nodulation-division efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24.

Microbiology (Reading, England) 156 (Pt 1)
PMID : 19833777  :   DOI  :   10.1099/mic.0.031161-0    
Abstract >>
The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of soil-borne plant diseases by Pseudomonas fluorescens 2P24. Two mutants (PM810 and PM820) with increased extracellular accumulation of 2,4-DAPG were isolated using transposon mutagenesis. The disrupted genes in these two mutants shared >80 % identity with the genes of the EmhR-EmhABC resistance-nodulation-division (RND) efflux system of P. fluorescens cLP6a. The deletion of emhA (PM802), emhB (PM803) or emhC (PM804) genes in strain 2P24 increased the extracellular accumulation of 2,4-DAPG, whereas the deletion of the emhR (PM801) gene decreased the biosynthesis of 2,4-DAPG. The promoter assay confirmed the elevated transcription of emhABC in the EmhR disrupted strain (PM801) and an indirect negative regulation of 2,4-DAPG biosynthetic locus transcription by the EmhABC efflux pump. Induction by exogenous 2,4-DAPG led to remarkable differences in transcription of chromosome-borne phlA : : lacZ fusion in PM901 and PM811 (emhA(-)) strains. Additionally, the EmhABC system in strain 2P24 was involved in the resistance to a group of toxic compounds, including ampicillin, chloramphenicol, tetracycline, ethidium bromide and crystal violet. In conclusion, our results suggest that the EmhABC system is an important element that influences the production of antibiotic 2,4-DAPG and enhances resistance to toxic compounds in P. fluorescens 2P24.
KeywordMeSH Terms
184. Zhang  WW, Hu  YH, Wang  HL, Sun  L,     ( 2009 )

Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain.

Veterinary microbiology 139 (1��2��)
PMID : 19464828  :   DOI  :   10.1016/j.vetmic.2009.04.026    
Abstract >>
Pseudomonas fluorescens is an aquaculture pathogen that can infect a number of fish species. The virulence mechanisms of aquatic P. fluorescens remain largely unknown. Many P. fluorescens strains are able to secrete an extracellular protease called AprX, yet no AprX-like proteins have been identified in pathogenic P. fluorescens associated with aquaculture. In this study, a gene encoding an AprX homologue was cloned from TSS, a pathogenic P. fluorescens strain isolated from diseased fish. In TSS, AprX is secreted into the extracellular milieu, and the production of AprX is controlled by growth phase and calcium. Mutation of aprX has multiple effects, which include impaired abilities in interaction with cultured host cells, adherence to host mucus, modulation of host immune response, and dissemination and survival in host tissues and blood. Purified recombinant AprX exhibits apparent proteolytic activity, which is optimal at pH 8.0 and 50 degrees C. The protease activity of recombinant AprX is enhanced by Ca2+ and Zn2+ and reduced by Co2+. Cytotoxicity analyses showed that purified recombinant AprX has profound toxic effect on cultured fish cells. These results demonstrate that AprX is an extracellular metalloprotease that is involved in bacterial virulence.
KeywordMeSH Terms
185. Hu  YH, Liu  CS, Hou  JH, Sun  L,     ( 2009 )

Identification, characterization, and molecular application of a virulence-associated autotransporter from a pathogenic Pseudomonas fluorescens strain.

Applied and environmental microbiology 75 (13)
PMID : 19447960  :   DOI  :   10.1128/AEM.00159-09     PMC  :   PMC2704808    
Abstract >>
A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.
KeywordMeSH Terms
186. Hagen  MJ, Stockwell  VO, Whistler  CA, Johnson  KB, Loper  JE,     ( 2009 )

Stress tolerance and environmental fitness of Pseudomonas fluorescens A506, which has a mutation in RpoS.

Phytopathology 99 (6)
PMID : 19453226  :   DOI  :   10.1094/PHYTO-99-6-0679    
Abstract >>
Establishment of suppressive populations of bacterial biological control agents on aerial plant surfaces is a critical phase in biologically based management of floral diseases. Periodically, biocontrol agents encounter inhospitable conditions for growth on plants; consequently, tolerance of environmental stresses may contribute to their fitness. In many gram-negative bacteria, including strains of Pseudomonas spp., the capacity to survive environmental stresses is influenced by the stationary phase sigma factor RpoS. This study focused on the role of RpoS in stress response and epiphytic fitness of Pseudomonas fluorescens A506, a well-studied bacterial biological control agent. We detected a frameshift mutation in the rpoS of A506 and demonstrated that the mutation resulted in a truncated, nonfunctional RpoS. Using site-directed mutagenesis, we deleted a nucleotide from rpoS, which then encoded a full-length, functional RpoS. We compared the stress response and epiphytic fitness of A506 with derivative strains having the functional full-length RpoS or a disrupted, nonfunctional RpoS. RpoS had little effect on stress response of A506 and no consistent influence on epiphytic population size of A506 on pear or apple leaves or flowers. Although the capacity of strain A506 to withstand exposure to environmental stresses was similar to that of other fluorescent pseudomonads, this capacity was largely independent of rpoS.
KeywordMeSH Terms
Mutation
187. de Werra  P, Péchy-Tarr  M, Keel  C, Maurhofer  M,     ( 2009 )

Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

Applied and environmental microbiology 75 (12)
PMID : 19376896  :   DOI  :   10.1128/AEM.00295-09     PMC  :   PMC2698339    
Abstract >>
The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.
KeywordMeSH Terms
188. Hoegy  F, Lee  X, Noel  S, Rognan  D, Mislin  GL, Reimmann  C, Schalk  IJ,     ( 2009 )

Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent pseudomonads.

The Journal of biological chemistry 284 (22)
PMID : 19297329  :   DOI  :   10.1074/jbc.M900606200     PMC  :   PMC2685677    
Abstract >>
Pyochelin (Pch) and enantio-pyochelin (EPch) are enantiomer siderophores that are produced by Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, under iron limitation. Pch promotes growth of P. aeruginosa when iron is scarce, and EPch carries out the same biological function in P. fluorescens. However, the two siderophores are unable to promote growth in the heterologous species, indicating that siderophore-mediated iron uptake is highly stereospecific. In the present work, using binding and iron uptake assays, we found that FptA, the Fe-Pch outer membrane transporter of P. aeruginosa, recognized (K(d) = 2.5 +/- 1.1 nm) and transported Fe-Pch but did not interact with Fe-EPch. Likewise, FetA, the Fe-EPch receptor of P. fluorescens, was specific for Fe-EPch (K(d) = 3.7 +/- 2.1 nm) but did not bind and transport Fe-Pch. Growth promotion experiments performed under iron-limiting conditions confirmed that FptA and FetA are highly specific for Pch and EPch, respectively. When fptA and fetA along with adjacent transport genes involved in siderophore uptake were swapped between the two bacterial species, P. aeruginosa became able to utilize Fe-EPch as an iron source, and P. fluorescens was able to grow with Fe-Pch. Docking experiments using the FptA structure and binding assays showed that the stereospecificity of Pch recognition by FptA was mostly due to the configuration of the siderophore chiral centers C4'' and C2'' and was only weakly dependent on the configuration of the C4' carbon atom. Together, these findings increase our understanding of the stereospecific interaction between Pch and its outer membrane receptor FptA.
KeywordMeSH Terms
189. Moynihan  JA, Morrissey  JP, Coppoolse  ER, Stiekema  WJ, O'Gara  F, Boyd  EF,     ( 2009 )

Evolutionary history of the phl gene cluster in the plant-associated bacterium Pseudomonas fluorescens.

Applied and environmental microbiology 75 (7)
PMID : 19181839  :   DOI  :   10.1128/AEM.02052-08     PMC  :   PMC2663185    
Abstract >>
Pseudomonas fluorescens is of agricultural and economic importance as a biological control agent largely because of its plant association and production of secondary metabolites, in particular 2,4-diacetylphloroglucinol (2,4-DAPG). This polyketide, which is encoded by the eight-gene phl cluster, has antimicrobial effects on phytopathogens, promotes amino acid exudation from plant roots, and induces systemic resistance in plants. Despite its importance, 2,4-DAPG production is limited to a subset of P. fluorescens strains. Determination of the evolution of the phl cluster and understanding the selective pressures promoting its retention or loss in lineages of P. fluorescens will help in the development of P. fluorescens as a viable and effective inoculant for application in agriculture. In this study, genomic and sequence-based approaches were integrated to reconstruct the phylogeny of P. fluorescens and the phl cluster. It was determined that 2,4-DAPG production is an ancestral trait in the species P. fluorescens but that most lineages have lost this capacity through evolution. Furthermore, intragenomic recombination has relocated the phl cluster within the P. fluorescens genome at least three times, but the integrity of the cluster has always been maintained. The possible evolutionary and functional implications for retention of the phl cluster and 2,4-DAPG production in some lineages of P. fluorescens are discussed.
KeywordMeSH Terms
Evolution, Molecular
Multigene Family
190. Barret  M, Frey-Klett  P, Boutin  M, Guillerm-Erckelboudt  AY, Martin  F, Guillot  L, Sarniguet  A,     ( 2009 )

The plant pathogenic fungus Gaeumannomyces graminis var. tritici improves bacterial growth and triggers early gene regulations in the biocontrol strain Pseudomonas fluorescens Pf29Arp.

The New phytologist 181 (2)
PMID : 19121038  :   DOI  :   10.1111/j.1469-8137.2008.02675.x    
Abstract >>
In soil, some antagonistic rhizobacteria contribute to reduce root diseases caused by phytopathogenic fungi. Direct modes of action of these bacteria have been largely explored; however, commensal interaction also takes place between these microorganisms and little is known about the influence of filamentous fungi on bacteria. An in vitro confrontation bioassay between the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) and the biocontrol bacterial strain Pseudomonas fluorescens Pf29Arp was set up to analyse bacterial transcriptional changes induced by the fungal mycelium at three time-points of the interaction before cell contact and up until contact. For this, a Pf29Arp shotgun DNA microarray was constructed. Specifity of Ggt effect was assessed in comparison with one of two other filamentous fungi, Laccaria bicolor and Magnaporthe grisea. During a commensal interaction, Ggt increased the growth rate of Pf29Arp. Before contact, Ggt induced bacterial genes involved in mycelium colonization. At contact, genes encoding protein of stress response and a patatin-like protein were up-regulated. Among all the bacterial genes identified, xseB was specifically up-regulated at contact by Ggt but down-regulated by the other fungi. Data showed that the bacterium sensed the presence of the fungus early, but the main gene alteration occurred during bacterial-fungal cell contact.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
191. de Bruijn  I, Raaijmakers  JM,     ( 2009 )

Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease.

Journal of bacteriology 191 (6)
PMID : 19114474  :   DOI  :   10.1128/JB.01558-08     PMC  :   PMC2648375    
Abstract >>
Cyclic lipopeptides produced by Pseudomonas species exhibit potent surfactant and broad-spectrum antibiotic properties. Their biosynthesis is governed by large multimodular nonribosomal peptide synthetases, but little is known about the genetic regulatory network. This study provides, for the first time, evidence that the serine protease ClpP regulates the biosynthesis of massetolides, cyclic lipopeptides involved in swarming motility, biofilm formation, and antimicrobial activity of Pseudomonas fluorescens SS101. The results show that ClpP affects the expression of luxR(mA), the transcriptional regulator of the massetolide biosynthesis genes massABC, thereby regulating biofilm formation and swarming motility of P. fluorescens SS101. Transcription of luxR(mA) was significantly repressed in the clpP mutant, and introduction of luxR(mA) restored, in part, massetolide biosynthesis and swarming motility of the clpP mutant. Site-directed mutagenesis and expression analyses indicated that the chaperone subunit ClpX and the Lon protease are not involved in regulation of massetolide biosynthesis and are transcribed independently of clpP. Addition of Casamino Acids enhanced the transcription of luxR(mA) and massABC in the clpP mutant, leading to a partial rescue of massetolide production and swarming motility. The results further suggested that, at the transcriptional level, ClpP-mediated regulation of massetolide biosynthesis operates independently of regulation by the GacA/GacS two-component system. The role of amino acid metabolism and the putative mechanisms underlying ClpP-mediated regulation of cyclic lipopeptide biosynthesis, swarming motility, and growth in P. fluorescens are discussed.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
192. Pellegrini  C, Mercuri  PS, Celenza  G, Galleni  M, Segatore  B, Sacchetti  E, Volpe  R, Amicosante  G, Perilli  M,     ( 2009 )

Identification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location.

The Journal of antimicrobial chemotherapy 63 (5)
PMID : 19270313  :   DOI  :   10.1093/jac/dkp061    
Abstract >>
The aim of the study was the biochemical characterization of a new variant of the metallo-beta-lactamase, IMP-22. Moreover, the genetic environment of the bla(IMP-22) gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L'Aquila, Italy. Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (K(m) and V(max)) were determined for a large pattern of substrates. A new IMP metallo-beta-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The bla(IMP-22) encodes for a pre-protein of 246 amino acids and the N-terminus of the mature beta-lactamase (NH(2)-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (K(m) and V(max)), IMP-22 was found to hydrolyse narrow- and extended-spectrum beta-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC(50) of 800, 750 and 300 microM, respectively. The finding of the bla(IMP-22) gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of bla(MBL) genes in several bacterial species and in different environments.
KeywordMeSH Terms
Water Microbiology
193. Yan  Q, Gao  W, Wu  XG, Zhang  LQ,     ( 2009 )

Regulation of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by the PhoP/PhoQ two-component system.

Microbiology (Reading, England) 155 (Pt 1)
PMID : 19118353  :   DOI  :   10.1099/mic.0.020750-0    
Abstract >>
A quorum-sensing locus, pcoI/pcoR, which is involved in the regulation of root colonization and plant disease-suppressive ability, was previously identified in Pseudomonas fluorescens 2P24. In this study, we performed random mutagenesis using mini-Tn5 in order to screen the upstream transcriptional regulators of pcoI, a biosynthase gene responsible for the synthesis of N-acylhomoserine lactone signal molecules. Two mutants, PM400 and PM410, with elevated pcoI gene promoter activity, were identified from approximately 10,000 insertion clones. The amino acid sequences of the interrupted genes in these two mutants were highly similar to PhoQ, a sensor protein of the two-component regulatory system PhoP/PhoQ, which responds to environmental Mg2+ starvation and regulates virulence in Salmonella typhimurium and antimicrobial peptide resistance in Pseudomonas aeruginosa. The promoter activity of pcoI was also induced under low-Mg2+ conditions in the 2P24 strain of P. fluorescens. Deletion mutagenesis and complementation experiments demonstrated that the transcription of pcoI was negatively regulated by the sensor PhoQ but positively regulated by the response regulator PhoP. Genetic evidence also indicated that transcription of the outer-membrane protein gene oprH was induced by Mg2+ starvation through regulation of the wild-type PhoP/PhoQ system. Additionally, PhoQ was involved in biofilm formation by 2P24 under low-Mg2+ conditions through a PhoP-independent pathway.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Quorum Sensing
194. Ahuja  EG, Janning  P, Mentel  M, Graebsch  A, Breinbauer  R, Hiller  W, Costisella  B, Thomashow  LS, Mavrodi  DV, Blankenfeldt  W,     ( 2008 )

PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

Journal of the American Chemical Society 130 (50)
PMID : 19053436  :   DOI  :   10.1021/ja806325k    
Abstract >>
Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.
KeywordMeSH Terms
Biocatalysis
195. Upadhyay  A, Srivastava  S,     ( 2008 )

Characterization of a new isolate of Pseudomonas fluorescens strain Psd as a potential biocontrol agent.

Letters in applied microbiology 47 (2)
PMID : 18565138  :   DOI  :   10.1111/j.1472-765X.2008.02390.x    
Abstract >>
Evaluation of a new isolate of Pseudomonas fluorescens for its biocontrol properties. Strain Psd identified as Ps. fluorescens, produces secondary metabolites that are toxic to some plant-pathogenic fungi. Inhibition of fungal growth of Fusarium oxysporum and Verticillium dahliae in the presence of bacterial culture filtrate provided the first clue to its biocontrol properties. In order to determine the basis for antifungal properties, antibiotics were extracted and analysed by TLC. Both pyrrolnitrin and phenazines could be detected in the culture of Psd. Presence of response regulator gene gacA of the two component regulatory system (GacS/GacA) was established by PCR amplification and sequencing. Sequence comparison of gacA justified the taxonomic position of this strain among the known members of Pseudomonadaceae. Synthesis of other compounds like toxic lipodepsipeptide, siderophores, and HCN was also confirmed by appropriate biochemical tests. Characterization of strain Psd by various biochemical/plate tests followed by chromatographic identification of antibiotics, demonstrates its multifunctional biocontrol property. Response regulator gene gacA provides an additional genetic marker for the phylogenetic studies. Ps. fluorescens strain Psd with its multifunctional biocontrol property can be used to bioprotect the crop plants from phytopathogens.
KeywordMeSH Terms
Antibiosis
196. Yang  J, Zhang  B, Yan  Y,     ( 2009 )

Cloning and expression of Pseudomonas fluorescens 26-2 lipase gene in Pichia pastoris and characterizing for transesterification.

Applied biochemistry and biotechnology 159 (2)
PMID : 19005622  :   DOI  :   10.1007/s12010-008-8419-5    
Abstract >>
Pseudomonas lipases are important biocatalysts widely used in a variety of industrial fields. An extracellular lipase gene lipA with 1,854-bp open reading frame was cloned from Pseudomonas fluorescens 26-2. The multialignment assay of the putative amino acid and the secondary structure prediction revealed this enzyme could be classified into the lipolytic subfamily I.3 and secreted via adenosine-triphosphate-binding cassette pathway. The lipA gene was integrated into Pichia pastoris GS115, and the methanol-inducible recombinants with Mut(S) and Mut(+) phenotypes were acquired. The characteristics and the transesterification capacity shown by this enzyme suggested it is a useful biocatalyst for biodiesel preparation.
KeywordMeSH Terms
197. Calisti  C, Ficca  AG, Barghini  P, Ruzzi  M,     ( 2008 )

Regulation of ferulic catabolic genes in Pseudomonas fluorescens BF13: involvement of a MarR family regulator.

Applied microbiology and biotechnology 80 (3)
PMID : 18575856  :   DOI  :   10.1007/s00253-008-1557-4    
Abstract >>
In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to vanillic acid (ech, vdh and fcs) is expressed in ferulic but not in succinic-grown cells. In the upstream region, we identified a gene, ferR, encoding a protein homologous to transcriptional regulators of the MarR family. A ferR knockout mutant (BF13-89) showed a 3.5-fold increase in expression of an ech-reporter gene fusion compared with the parent strain in succinic-grown cells, indicating that the ferR gene product negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13. Consistent with the increased expression of the catabolic genes in the ferR mutant, BF13-89 showed a shorter (relative to its FerR(+) parent) lag phase during carbon source shift from succinic to ferulic acid. However, expression of ech-lacZ fusion did not increase in BF13-89 grown in the presence of ferulic acid, indicating that FerR has a second function as transcriptional activator. Expression of ech-lacZ in a feruloyl-CoA synthetase-deficient strain revealed unambiguously that FerR-mediated activation of the ferulic catabolic operon is dependent on the thioester product of the feruloyl-CoA synthetase reaction.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Multigene Family
198. Rossignol  G, Merieau  A, Guerillon  J, Veron  W, Lesouhaitier  O, Feuilloley  MG, Orange  N,     ( 2008 )

Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens.

BMC microbiology 8 (N/A)
PMID : 18973676  :   DOI  :   10.1186/1471-2180-8-189     PMC  :   PMC2613904    
Abstract >>
Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32 degrees C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37 degrees C in laboratory conditions. We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of P. fluorescens or the mesophilic opportunistic pathogen, Pseudomonas aeruginosa PAO1. We demonstrated the direct, and indirect - through increases in biosurfactant release - involvement of a phospholipase C in the hemolytic activity of this bacterium. Sequence analysis assigned this phospholipase C to a new group of phospholipases C different from those produced by P. aeruginosa. We show that changes in PlcC production have pleiotropic effects and that plcC overexpression and plcC extinction increase MFN1032 toxicity and colonization, respectively. This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production.
KeywordMeSH Terms
Hemolysis
199. Crespo  MC, Valverde  C,     ( 2009 )

A single mutation in the oprF mRNA leader confers strict translational control by the Gac/Rsm system in Pseudomonas fluorescens CHA0.

Current microbiology 58 (2)
PMID : 18979131  :   DOI  :   10.1007/s00284-008-9306-6    
Abstract >>
The rhizobacterium Pseudomonas fluorescens CHA0 is able to antagonize fungal phytopathogens on a variety of crop plants, mainly due to the production of secondary metabolites that are coordinately upregulated by the global regulatory Gac/Rsm cascade. The two-component system GacS/GacA activates transcription of the three small regulatory RNAs RsmX, RsmY, and RsmZ, which counteract translational repression of target mRNAs by RsmA and RsmE proteins. In a search for novel Gac/Rsm targets based on the minimal sequence on mRNA leaders required for RsmA/RsmE control, the leader region of the major porin OprF emerged as a candidate. Although an isogenic CHA0 oprF mutant showed a reduced ability to attach to cucumber and tomato roots, suggesting a role for OprF in root colonization as a requisite for pathogen antagonism, a translational oprF'-'lacZ fusion was weakly regulated by Gac/Rsm despite its high sequence similarity to the hcnA leader. A single base substitution put the modified oprF 5'-UTR under strict control by Gac/Rsm. The results highlight the subtle sequence requirements of Gac/Rsm targets.
KeywordMeSH Terms
5' Untranslated Regions
Gene Expression Regulation, Bacterial
Point Mutation
Protein Biosynthesis
200. Brandt  GS, Nemeria  N, Chakraborty  S, McLeish  MJ, Yep  A, Kenyon  GL, Petsko  GA, Jordan  F, Ringe  D,     ( 2008 )

Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester.

Biochemistry 47 (29)
PMID : 18570438  :   DOI  :   10.1021/bi8004413     PMC  :   PMC2729719     DOI  :   10.1021/bi8004413     PMC  :   PMC2729719    
Abstract >>
Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.
KeywordMeSH Terms
201. Moonen  MJ, Kamerbeek  NM, Westphal  AH, Boeren  SA, Janssen  DB, Fraaije  MW, van Berkel  WJ,     ( 2008 )

Elucidation of the 4-hydroxyacetophenone catabolic pathway in Pseudomonas fluorescens ACB.

Journal of bacteriology 190 (15)
PMID : 18502868  :   DOI  :   10.1128/JB.01944-07     PMC  :   PMC2493259    
Abstract >>
The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to beta-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.
KeywordMeSH Terms
202. Dufour  D, Nicodème  M, Perrin  C, Driou  A, Brusseaux  E, Humbert  G, Gaillard  JL, Dary  A,     ( 2008 )

Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them.

International journal of food microbiology 125 (2)
PMID : 18511140  :   DOI  :   10.1016/j.ijfoodmicro.2008.04.004    
Abstract >>
P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins. Several P. fluorescens synthesize an extracellular caseinolytic metalloprotease, called AprX. It is important to rapidly detect the presence of a contamination of raw milk by a strain, especially a P. fluorescens strain, having a high potential of depredation. If standard plate count procedures are often employed, they are time consuming and do not permit to rapidly evaluate the potential of depredation. An alternative method consists to search the aprX gene, but such a method remains of low sensitivity and does not allow evaluating the real potential of depredation of the contaminant. After a milk depredation event, three strains of Pseudomonas spp. (F, 2312 and 2313) have been isolated from a dairy plant. Using molecular and phenotypic approaches, these strains were identified as P. fluorescens strains. Their respective extracellular caseinolytic potential was characterized as well as that of several collection strains of P. fluorescens. It appeared that these strains secreted one protease of about 45 kDa, that their extracellular caseinolytic potential was highly variable for one strain to another and that the one of strain F was the highest. The protease secreted by the strain F was purified and its N-terminal sequence established. It shared 100% identity with the domain 14-34 of extracellular alkaline endoprotease sequences which are called AprX for some of them. Its gene was sequenced as well as that of two collection strains of P. fluorescens having a significant lower extracellular caseinolytic potential. The genomic environment of the aprX gene as well as its expression during the strain growth was investigated. It appears that the difference of extracellular caseinolytic potential which has been observed between the three strains does not mainly result from the AprX sequence/structure but it might rather result from the aprX level of expression.
KeywordMeSH Terms
203. Flecks  S, Patallo  EP, Zhu  X, Ernyei  AJ, Seifert  G, Schneider  A, Dong  C, Naismith  JH, van Pée  KH,     ( 2008 )

New insights into the mechanism of enzymatic chlorination of tryptophan.

Angewandte Chemie (International ed. in English) 47 (49)
PMID : 18979475  :   DOI  :   10.1002/anie.200802466     PMC  :   PMC3326536    
Abstract >>
N/A
KeywordMeSH Terms
Halogenation
204. Anderson  LM, Stockwell  VO, Loper  JE,     ( 2004 )

An Extracellular Protease of Pseudomonas fluorescens Inactivates Antibiotics of Pantoea agglomerans.

Phytopathology 94 (11)
PMID : 18944458  :   DOI  :   10.1094/PHYTO.2004.94.11.1228    
Abstract >>
ABSTRACT Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens strain A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas Pantoea agglomerans strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pantoea agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zinc- or calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, partially purified extracellular protease inactivated the antibiotics mccEh252 and herbicolin O, which are produced by Pantoea agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. Strain A506 inactivated mccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously unknown role of the extracellular protease produced by Pseudomonas fluorescens A506 in interactions among plant-associated microbes.
KeywordMeSH Terms
205. Ramazzina  I, Cendron  L, Folli  C, Berni  R, Monteverdi  D, Zanotti  G, Percudani  R,     ( 2008 )

Logical identification of an allantoinase analog (puuE) recruited from polysaccharide deacetylases.

The Journal of biological chemistry 283 (34)
PMID : 18550550  :   DOI  :   10.1074/jbc.M801195200    
Abstract >>
The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase beta/alpha barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution.
KeywordMeSH Terms
206. Bennett  JP, Bertin  L, Moulton  B, Fairlamb  IJ, Brzozowski  AM, Walton  NJ, Grogan  G,     ( 2008 )

A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism.

The Biochemical journal 414 (2)
PMID : 18479250  :   DOI  :   10.1042/BJ20080714    
Abstract >>
HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.
KeywordMeSH Terms
207. Warmink  JA, van Elsas  JD,     ( 2008 )

Selection of bacterial populations in the mycosphere of Laccaria proxima: is type III secretion involved?

The ISME journal 2 (8)
PMID : 18421258  :   DOI  :   10.1038/ismej.2008.41    
Abstract >>
The bacterial communities in the Laccaria proxima mycosphere (soil from beneath fruiting bodies) and the corresponding bulk soil were compared by cultivation-dependent and cultivation-independent methods. To assess the distribution of type III secretion systems (TTSS), a PCR-based system for the broad detection of a highly conserved gene involved in TTSS, that is hrcR, was developed and used to assay the cultured bacteria from the L. proxima mycosphere and surrounding bulk soil. PCR-DGGE based on the 16S ribosomal RNA gene showed the selection of presumably mycosphere-specific bacterial groups in the mycosphere of L. proxima compared to the bulk soil in 3 sampling years. Moreover, plate counts revealed that the numbers of culturable heterotrophic bacteria were increased in the mycosphere as compared to the bulk soil. Strikingly, the percentage of randomly picked isolates that carried the hrcR gene showed a significant increase, from 2.8 in the bulk to 13.4 in the mycosphere soil. The increase could be mainly attributed to the emergence of a hrcR positive Pseudomonas fluorescens, denoted BS053, which constituted the most dominant species in the culturable mycosphere communities. This organism was, together with a hrcR-positive Burkholderia terrae BS110, exclusively found in mycosphere soil. Direct detection of hrcR genes using a cultivation-independent approach showed the selection of several hrcR gene types uniquely in the mycosphere, indicating the selection of several TTSS-harboring bacterial species. Thus, different bacteria were found to be enriched in the L. proxima mycosphere and TTSS can be involved in some of the interactions with the fungal host.
KeywordMeSH Terms
Antibiosis
Biodiversity
Selection, Genetic
Soil Microbiology
208. Michaux  C, Massant  J, Kerff  F, Frère  JM, Docquier  JD, Vandenberghe  I, Samyn  B, Pierrard  A, Feller  G, Charlier  P, Van Beeumen  J, Wouters  J,     ( 2008 )

Crystal structure of a cold-adapted class C beta-lactamase.

The FEBS journal 275 (8)
PMID : 18312599  :   DOI  :   10.1111/j.1742-4658.2008.06324.x    
Abstract >>
In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.
KeywordMeSH Terms
Cold Temperature
209. Choi  O, Kim  J, Kim  JG, Jeong  Y, Moon  JS, Park  CS, Hwang  I,     ( 2008 )

Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16.

Plant physiology 146 (2)
PMID : 18055583  :   DOI  :   10.1104/pp.107.112748     PMC  :   PMC2245851    
Abstract >>
Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growth promotion by this organism, we mutagenized wild-type strain B16 using OmegaKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato (Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 1,000 nm of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings, confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.
KeywordMeSH Terms
Plant Development
210. Maraite  A, Schmidt  T, Ansörge-Schumacher  MB, Brzozowski  AM, Grogan  G,     ( 2007 )

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 A resolution.

Acta crystallographica. Section F, Structural biology and crystallization communications 63 (Pt 7)
PMID : 17620706  :   DOI  :   10.1107/S1744309107028576     PMC  :   PMC2335142     DOI  :   10.1107/S1744309107028576     PMC  :   PMC2335142    
Abstract >>
Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6 A using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005), FEBS J. 272, 6067-6076]. In this communication of parallel studies, BAL was crystallized in an alternative space group (P2(1)2(1)2(1)) and its structure refined to a resolution of 1.65 A, allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface.
KeywordMeSH Terms
211. Dun  BQ, Wang  XJ, Lu  W, Zhao  ZL, Hou  SN, Zhang  BM, Li  GY, Evans  TC, Xu  MQ, Lin  M,     ( 2007 )

Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco.

Applied and environmental microbiology 73 (24)
PMID : 17951442  :   DOI  :   10.1128/AEM.00956-07     PMC  :   PMC2168149    
Abstract >>
A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.
KeywordMeSH Terms
212. de Bruijn  I, de Kock  MJ, de Waard  P, van Beek  TA, Raaijmakers  JM,     ( 2008 )

Massetolide A biosynthesis in Pseudomonas fluorescens.

Journal of bacteriology 190 (8)
PMID : 17993540  :   DOI  :   10.1128/JB.01563-07     PMC  :   PMC2293227    
Abstract >>
Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.
KeywordMeSH Terms
213. Youard  ZA, Mislin  GL, Majcherczyk  PA, Schalk  IJ, Reimmann  C,     ( 2007 )

Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin.

The Journal of biological chemistry 282 (49)
PMID : 17938167  :   DOI  :   10.1074/jbc.M707039200    
Abstract >>
The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.
KeywordMeSH Terms
214. Izumi  S, Yamamoto  M, Suzuki  K, Shimizu  A, Aranishi  F,     ( 2007 )

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region.

Journal of fish diseases 30 (7)
PMID : 17584436  :   DOI  :   10.1111/j.1365-2761.2007.00820.x    
Abstract >>
Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.
KeywordMeSH Terms
Polymerase Chain Reaction
215. De Laurentis  W, Khim  L, Anderson  JL, Adam  A, Johnson  KA, Phillips  RS, Chapman  SK, van Pee  KH, Naismith  JH,     ( 2007 )

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily.

Biochemistry 46 (43)
PMID : 17924666  :   DOI  :   10.1021/bi7012189     PMC  :   PMC3326534    
Abstract >>
Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.
KeywordMeSH Terms
216. Liddle  J, Beaufils  B, Binnie  M, Bouillot  A, Denis  AA, Hann  MM, Haslam  CP, Holmes  DS, Hutchinson  JP, Kranz  M, McBride  A, Mirguet  O, Mole  DJ, Mowat  CG, Pal  S, Rowland  P, Trottet  L, Uings  IJ, Walker  AL, Webster  SP,     ( 2017 )

The discovery of potent and selective kynurenine 3-monooxygenase inhibitors for the treatment of acute pancreatitis.

Bioorganic & medicinal chemistry letters 27 (9)
PMID : 28336141  :   DOI  :   10.1016/j.bmcl.2017.02.078    
Abstract >>
A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.
KeywordMeSH Terms
Kynurenine
Monooxygenase
Pancreatitis
Substrate
Tryptophan
217. van der Laan  JM, Schreuder  HA, Swarte  MB, Wierenga  RK, Kalk  KH, Hol  WG, Drenth  J,     ( 1989 )

The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation.

Biochemistry 28 (18)
PMID : 2819062  :   DOI  :   10.1021/bi00444a011    
Abstract >>
p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.
KeywordMeSH Terms
218. Walker  AL, Ancellin  N, Beaufils  B, Bergeal  M, Binnie  M, Bouillot  A, Clapham  D, Denis  A, Haslam  CP, Holmes  DS, Hutchinson  JP, Liddle  J, McBride  A, Mirguet  O, Mowat  CG, Rowland  P, Tiberghien  N, Trottet  L, Uings  I, Webster  SP, Zheng  X, Mole  DJ,     ( 2017 )

Development of a Series of Kynurenine 3-Monooxygenase Inhibitors Leading to a Clinical Candidate for the Treatment of Acute Pancreatitis.

Journal of medicinal chemistry 60 (8)
PMID : 28398044  :   DOI  :   10.1021/acs.jmedchem.7b00055    
Abstract >>
Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.
KeywordMeSH Terms
219. Gallique  M, Decoin  V, Barbey  C, Rosay  T, Feuilloley  MG, Orange  N, Merieau  A,     ( 2017 )

Contribution of the Pseudomonas fluorescens MFE01 Type VI Secretion System to Biofilm Formation.

PloS one 12 (1)
PMID : 28114423  :   DOI  :   10.1371/journal.pone.0170770     PMC  :   PMC5256989    
Abstract >>
Type VI secretion systems (T6SSs) are widespread in Gram-negative bacteria, including Pseudomonas. These macromolecular machineries inject toxins directly into prokaryotic or eukaryotic prey cells. Hcp proteins are structural components of the extracellular part of this machinery. We recently reported that MFE01, an avirulent strain of Pseudomonas fluorescens, possesses at least two hcp genes, hcp1 and hcp2, encoding proteins playing important roles in interbacterial interactions. Indeed, P. fluorescens MFE01 can immobilise and kill diverse bacteria of various origins through the action of the Hcp1 or Hcp2 proteins of the T6SS. We show here that another Hcp protein, Hcp3, is involved in killing prey cells during co-culture on solid medium. Even after the mutation of hcp1, hcp2, or hcp3, MFE01 impaired biofilm formation by MFP05, a P. fluorescens strain isolated from human skin. These mutations did not reduce P. fluorescens MFE01 biofilm formation, but the three Hcp proteins were required for the completion of biofilm maturation. Moreover, a mutant with a disruption of one of the unique core component genes, MFE01�GtssC, was unable to produce its own biofilm or inhibit MFP05 biofilm formation. Finally, MFE01 did not produce detectable N-acyl-homoserine lactones for quorum sensing, a phenomenon reported for many other P. fluorescens strains. Our results suggest a role for the T6SS in communication between bacterial cells, in this strain, under biofilm conditions.
KeywordMeSH Terms
Biofilms
220. 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
221. Cendron  L, Ramazzina  I, Puggioni  V, Maccacaro  E, Liuzzi  A, Secchi  A, Zanotti  G, Percudani  R,     ( 2016 )

The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism.

Biochemistry 55 (46)
PMID : 27797489  :   DOI  :   10.1021/acs.biochem.6b00881    
Abstract >>
The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (kcat/KM ? 5 �� 104 M-1 s-1) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13C and 1H nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution and conserved in spite of the substitution of catalytic residues.
KeywordMeSH Terms
Protein Domains
222. Ge  Y, Zhu  J, Ye  X, Yang  Y,     ( 2017 )

Spoilage potential characterization of Shewanella and Pseudomonas isolated from spoiled large yellow croaker (Pseudosciaena crocea).

Letters in applied microbiology 64 (1)
PMID : 27747903  :   DOI  :   10.1111/lam.12687    
Abstract >>
Ten strains were isolated from a spoiled large yellow croaker (Pseudosciaena crocea). All of them were able to grow aerobically from 4 to 30�XC, and reduce trimethylamine-N-oxide to trimethylamine (TMA) and produce H2 S except SB01, PF05 and PF07. Biochemical characterization and phylogenetic analysis of 16S rRNA gene showed that eight H2 S-producing isolates were closely related to Shewanella baltica, and two isolates PF05 and PF07 were identified as Pseudomonas fluorescens and Pseudomonas fragi respectively. However, of the eight Shewanella, seven isolates cluster with S. baltica and one with Shewanella glacialipiscicola based on the analysis of the gyrB gene. Shewanella baltica also had the ability to produce biogenic amines, while two Pseudomonas had high activities of proteinase and lipase, and failed to produce TMA and biogenic amines. In spoilage potential evaluation, the TVB-N value of S. baltica was significantly higher than that of Pseudomonas in sterile fish juice, although its growth was slower than Pseudomonas. Therefore, this work demonstrated that S. baltica was able to cause rapid and strong spoilage and was therefore identified as a specific spoilage organism in refrigerated P. crocea. Members of the bacterial genera Shewanella and Pseudomonas are widely known to be responsible for the specific spoilers in iced fish. Ten strains isolated from spoiled large yellow croaker (Pseudosciaena crocea) were identified as Shewanella baltica and Pseudomonas spp. S. baltica was demonstrated as the predominant spoiler in the refrigerated P. crocea due to its high metabolic activities. This work has generated baseline information for a better understanding of the role of various spoilage bacteria in chilled marine fish and for the control of contamination and growth of main spoilage bacteria to extend the shelf life of marine fish.
KeywordMeSH Terms
16S rRNA
Pseudomonas
Shewanella baltica
biogenic amine
enzyme
gyrB gene
16S rRNA
Pseudomonas
Shewanella baltica
biogenic amine
enzyme
gyrB gene
Food Contamination
Food Microbiology
223. Kondakova  T, Catovic  C, Barreau  M, Nusser  M, Brenner-Weiss  G, Chevalier  S, Dionnet  F, Orange  N, Poc  CD,     ( 2016 )

Response to Gaseous NO2 Air Pollutant of P. fluorescens Airborne Strain MFAF76a and Clinical Strain MFN1032.

Frontiers in microbiology 7 (N/A)
PMID : 27065229  :   DOI  :   10.3389/fmicb.2016.00379     PMC  :   PMC4814523    
Abstract >>
Human exposure to nitrogen dioxide (NO2), an air pollutant of increasing interest in biology, results in several toxic effects to human health and also to the air microbiota. The aim of this study was to investigate the bacterial response to gaseous NO2. Two Pseudomonas fluorescens strains, namely the airborne strain MFAF76a and the clinical strain MFN1032 were exposed to 0.1, 5, or 45 ppm concentrations of NO2, and their effects on bacteria were evaluated in terms of motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes. While 0.1 and 5 ppm of NO2did not lead to any detectable modification in the studied phenotypes of the two bacteria, several alterations were observed when the bacteria were exposed to 45 ppm of gaseous NO2. We thus chose to focus on this high concentration. NO2-exposed P. fluorescens strains showed reduced swimming motility, and decreased swarming in case of the strain MFN1032. Biofilm formed by NO2-treated airborne strain MFAF76a showed increased maximum thickness compared to non-treated cells, while NO2 had no apparent effect on the clinical MFN1032 biofilm structure. It is well known that biofilm and motility are inversely regulated by intracellular c-di-GMP level. The c-di-GMP level was however not affected in response to NO2 treatment. Finally, NO2-exposed P. fluorescens strains were found to be more resistant to ciprofloxacin and chloramphenicol. Accordingly, the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in the two P. fluorescens strains. Noticeably, similar phenotypes had been previously observed following a NO treatment. Interestingly, an hmp-homolog gene in P. fluorescens strains MFAF76a and MFN1032 encodes a NO dioxygenase that is involved in NO detoxification into nitrites. Its expression was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification. Taken together, our study provides evidences for the bacterial response to NO2 toxicity.
KeywordMeSH Terms
Pseudomonas fluorescens
air pollution
airborne
antibiotic sensitivity
biofilm
motility
nitrogen dioxide
224. Gro?kinsky  DK, Tafner  R, Moreno  MV, Stenglein  SA, García de Salamone  IE, Nelson  LM, Novák  O, Strnad  M, van der Graaff  E, Roitsch  T,     ( 2016 )

Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis.

Scientific reports 6 (N/A)
PMID : 26984671  :   DOI  :   10.1038/srep23310     PMC  :   PMC4794740    
Abstract >>
Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience.
KeywordMeSH Terms
225. Mole  DJ, Webster  SP, Uings  I, Zheng  X, Binnie  M, Wilson  K, Hutchinson  JP, Mirguet  O, Walker  A, Beaufils  B, Ancellin  N, Trottet  L, Bénéton  V, Mowat  CG, Wilkinson  M, Rowland  P, Haslam  C, McBride  A, Homer  NZ, Baily  JE, Sharp  MG, Garden  OJ, Hughes  J, Howie  SE, Holmes  DS, Liddle  J, Iredale  JP,     ( 2016 )

Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis.

Nature medicine 22 (2)
PMID : 26752518  :   DOI  :   10.1038/nm.4020     PMC  :   PMC4871268    
Abstract >>
Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 ? resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.
KeywordMeSH Terms
226. Xu  GC, Li  L, Han  RZ, Dong  JJ, Ni  Y,     ( 2016 )

Characterization and Soluble Expression of D-Hydantoinase from Pseudomonas fluorescens for the Synthesis of D-Amino Acids.

Applied biochemistry and biotechnology 179 (1)
PMID : 26821258  :   DOI  :   10.1007/s12010-015-1975-6    
Abstract >>
An active D-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as D-PfHYD. Sequence and consensus analysis suggests that D-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. D-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. D-PfHYD is a homotetramer with molecular weight of 215 kDa and specific activity of 20.9 U mg(-1). D-PfHYD showed the highest activity at pH 9.0 and 60 �XC. Metal ions such as Mn(2+), Fe(2+), and Fe(3+) could activate D-PfHYD with 20 % improvement. Substrate specificity analysis revealed that purified D-PfHYD preferred aliphatic to aromatic 5'-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of D-PfHYD. Employing 1.0 g L(-1) recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100 mM isobutyl hydantoin was converted into D-isoleucine with 98.7 % enantiomeric excess (ee), isolation yield of 78.3 %, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant D-PfHYD could be potentially applied in the synthesis of D-amino acids.
KeywordMeSH Terms
Chaperone
D-amino acids
D-hydantoinase
Pseudomonas fluorescens
Soluble expression
227. Siegel  JB, Smith  AL, Poust  S, Wargacki  AJ, Bar-Even  A, Louw  C, Shen  BW, Eiben  CB, Tran  HM, Noor  E, Gallaher  JL, Bale  J, Yoshikuni  Y, Gelb  MH, Keasling  JD, Stoddard  BL, Lidstrom  ME, Baker  D,     ( 2015 )

Computational protein design enables a novel one-carbon assimilation pathway.

Proceedings of the National Academy of Sciences of the United States of America 112 (12)
PMID : 25775555  :   DOI  :   10.1073/pnas.1500545112     PMC  :   PMC4378393    
Abstract >>
We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.
KeywordMeSH Terms
carbon fixation
computational protein design
pathway engineering
228. Schreuder  HA, Prick  PA, Wierenga  RK, Vriend  G, Wilson  KS, Hol  WG, Drenth  J,     ( 1989 )

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes.

Journal of molecular biology 208 (4)
PMID : 2553983  :   DOI  :   10.1016/0022-2836(89)90158-7    
Abstract >>
Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)
KeywordMeSH Terms
4-Hydroxybenzoate-3-Monooxygenase
Mixed Function Oxygenases
229. Johnston  CW, Skinnider  MA, Wyatt  MA, Li  X, Ranieri  MR, Yang  L, Zechel  DL, Ma  B, Magarvey  NA,     ( 2015 )

An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products.

Nature communications 6 (N/A)
PMID : 26412281  :   DOI  :   10.1038/ncomms9421     PMC  :   PMC4598715    
Abstract >>
Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC-MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products.
KeywordMeSH Terms
Genome, Bacterial
230. Michelsen  CF, Watrous  J, Glaring  MA, Kersten  R, Koyama  N, Dorrestein  PC, Stougaard  P,     ( 2015 )

Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a Greenlandic suppressive soil.

mBio 6 (2)
PMID : 25784695  :   DOI  :   10.1128/mBio.00079-15     PMC  :   PMC4453515    
Abstract >>
Potatoes are cultivated in southwest Greenland without the use of pesticides and with limited crop rotation. Despite the fact that plant-pathogenic fungi are present, no severe-disease outbreaks have yet been observed. In this report, we document that a potato soil at Inneruulalik in southern Greenland is suppressive against Rhizoctonia solani Ag3 and uncover the suppressive antifungal mechanism of a highly potent biocontrol bacterium, Pseudomonas fluorescens In5, isolated from the suppressive potato soil. A combination of molecular genetics, genomics, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) revealed an antifungal genomic island in P. fluorescens In5 encoding two nonribosomal peptides, nunamycin and nunapeptin, which are key components for the biocontrol activity by strain In5 in vitro and in soil microcosm experiments. Furthermore, complex microbial behaviors were highlighted. Whereas nunamycin was demonstrated to inhibit the mycelial growth of R. solani Ag3, but not that of Pythium aphanidermatum, nunapeptin instead inhibited P. aphanidermatum but not R. solani Ag3. Moreover, the synthesis of nunamycin by P. fluorescens In5 was inhibited in the presence of P. aphanidermatum. Further characterization of the two peptides revealed nunamycin to be a monochlorinated 9-amino-acid cyclic lipopeptide with similarity to members of the syringomycin group, whereas nunapeptin was a 22-amino-acid cyclic lipopeptide with similarity to corpeptin and syringopeptin. Crop rotation and systematic pest management are used to only a limited extent in Greenlandic potato farming. Nonetheless, although plant-pathogenic fungi are present in the soil, the farmers do not experience major plant disease outbreaks. Here, we show that a Greenlandic potato soil is suppressive against Rhizoctonia solani, and we unravel the key biocontrol components for Pseudomonas fluorescens In5, one of the potent biocontrol bacteria isolated from this Greenlandic suppressive soil. Using a combination of molecular genetics, genomics, and microbial imaging mass spectrometry, we show that two cyclic lipopeptides, nunamycin and nunapeptin, are important for the biocontrol activity of P. fluorescens In5 both in vitro and in microcosm assays. Furthermore, we demonstrate that the synthesis of nunamycin is repressed by the oomycete Pythium aphanidermatum. Overall, our report provides important insight into interkingdom interference between bacteria and fungi/oomycetes.
KeywordMeSH Terms
Peptide Biosynthesis, Nucleic Acid-Independent
Soil Microbiology
231. Liu  G, Feng  K, Guo  D, Li  R,     ( 2015 )

Overexpression and activities of 1-Cys peroxiredoxin from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode.

Folia microbiologica 60 (5)
PMID : 25720803  :   DOI  :   10.1007/s12223-015-0380-4    
Abstract >>
Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame (ORF) of 639 bp, which encoded a protein of 213 amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared 99, 97, and 97 % identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17 and 1-Cys peroxiredoxin of P. fluorescens Pf 0-1, respectively. The ORF was cloned into expressing vector pET-15b and introduced into Escherichia coli BL21 (DE3). Overexpression of a 27-kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni(2+) matrix column. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. Furthermore, E. coli expressing the ORF showed tolerance to hydrogen peroxide stress, which indicated that the gene might help P. fluorescens GcM5-1A resist hydrogen peroxide generated by host pines after pine wood nematode associated with this bacterium infected pine trees.
KeywordMeSH Terms
232. Huo  L, Davis  I, Liu  F, Andi  B, Esaki  S, Iwaki  H, Hasegawa  Y, Orville  AM, Liu  A,     ( 2015 )

Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action.

Nature communications 6 (N/A)
PMID : 25565451  :   DOI  :   10.1038/ncomms6935     PMC  :   PMC4286809    
Abstract >>
Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.
KeywordMeSH Terms
Models, Molecular
233. Mazurier  S, Merieau  A, Bergeau  D, Decoin  V, Sperandio  D, Crépin  A, Barbey  C, Jeannot  K, Vicré-Gibouin  M, Plésiat  P, Lemanceau  P, Latour  X,     ( 2015 )

Type III secretion system and virulence markers highlight similarities and differences between human- and plant-associated pseudomonads related to Pseudomonas fluorescens and P. putida.

Applied and environmental microbiology 81 (7)
PMID : 25636837  :   DOI  :   10.1128/AEM.04160-14     PMC  :   PMC4357948    
Abstract >>
Pseudomonas fluorescens is commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the related Pseudomonas aeruginosa nosocomial species and the occurrence of this secretion system in plant-associated P. fluorescens raise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging to P. fluorescens were compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41�XC) isolates from patients suffering from blood infections; these isolates were finally found to not belong to P. fluorescens but were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs from P. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged to P. fluorescens and harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associated P. fluorescens.
KeywordMeSH Terms
234. Shahbaz Mohammadi  H, Mostafavi  SS, Soleimani  S, Bozorgian  S, Pooraskari  M, Kianmehr  A,     ( 2015 )

Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

Protein expression and purification 108 (N/A)
PMID : 25591389  :   DOI  :   10.1016/j.pep.2015.01.002    
Abstract >>
Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25�XC, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.
KeywordMeSH Terms
Aqueous two-phase systems (ATPS)
Glucose dehydrogenase (GDH)
Partition
Purification
Response surface methodology (RSM)
d-Galactose dehydrogenase (GalDH)
Bacterial Proteins
Galactose Dehydrogenases
Glucose 1-Dehydrogenase
235. Oteino  N, Lally  RD, Kiwanuka  S, Lloyd  A, Ryan  D, Germaine  KJ, Dowling  DN,     ( 2015 )

Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates.

Frontiers in microbiology 6 (N/A)
PMID : 26257721  :   DOI  :   10.3389/fmicb.2015.00745     PMC  :   PMC4510416    
Abstract >>
The use of plant growth promoting bacterial inoculants as live microbial biofertilizers provides a promising alternative to chemical fertilizers and pesticides. Inorganic phosphate solubilization is one of the major mechanisms of plant growth promotion by plant associated bacteria. This involves bacteria releasing organic acids into the soil which solubilize the phosphate complexes converting them into ortho-phosphate which is available for plant up-take and utilization. The study presented here describes the ability of endophytic bacteria to produce gluconic acid (GA), solubilize insoluble phosphate, and stimulate the growth of Pisum sativum L. plants. This study also describes the genetic systems within three of these endophyte strains thought to be responsible for their effective phosphate solubilizing abilities. The results showed that many of the endophytic strains produced GA (14-169 mM) and have moderate to high phosphate solubilization capacities (~400-1300 mg L(-1)). When inoculated into P. sativum L. plants grown in soil under soluble phosphate limiting conditions, the endophytes that produced medium-high levels of GA displayed beneficial plant growth promotion effects.
KeywordMeSH Terms
PQQ
Pisum sativum L
Pseudomonas fluorescens
endophytes
plant growth promotion
236. Kim  YH, Song  W, Kim  JS, Jiao  L, Lee  K, Ha  NC,     ( 2015 )

Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA.

Applied and environmental microbiology 81 (15)
PMID : 26025888  :   DOI  :   10.1128/AEM.01289-15     PMC  :   PMC4495210    
Abstract >>
The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-? resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site.
KeywordMeSH Terms
237. Zhang  W, Zhao  Z, Zhang  B, Wu  XG, Ren  ZG, Zhang  LQ,     ( 2014 )

Posttranscriptional regulation of 2,4-diacetylphloroglucinol production by GidA and TrmE in Pseudomonas fluorescens 2P24.

Applied and environmental microbiology 80 (13)
PMID : 24747907  :   DOI  :   10.1128/AEM.00455-14     PMC  :   PMC4054201    
Abstract >>
Pseudomonas fluorescens 2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by the phlACBD locus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity against Rhizoctonia solani. Deletion of the gidA (PM701) or trmE (PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of the phlA gene was not affected, but the translation of the phlA and phlD genes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated by gidA and trmE, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production by gidA and trmE, however, was independent of the Gac/Rsm pathway. Both the gidA and trmE mutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in the gidA and trmE mutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Protein Biosynthesis
238. Cornelis  P, Bouia  A, Belarbi  A, Guyonvarch  A, Kammerer  B, Hannaert  V, Hubert  JC,     ( 1989 )

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa.

Molecular microbiology 3 (3)
PMID : 2473376  :   DOI  :   10.1111/j.1365-2958.1989.tb00187.x    
Abstract >>
The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.
KeywordMeSH Terms
DNA-Binding Proteins
Genes, Bacterial
239. Benen  JA, Van Berkel  WJ, Van Dongen  WM, Müller  F, De Kok  A,     ( 1989 )

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens.

Journal of general microbiology 135 (7)
PMID : 2515251  :   DOI  :   10.1099/00221287-135-7-1787    
Abstract >>
The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.
KeywordMeSH Terms
Genes, Bacterial
240. Matthijs  S, Vander Wauven  C, Cornu  B, Ye  L, Cornelis  P, Thomas  CM, Ongena  M,     ( 2014 )

Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin.

Research in microbiology 165 (8)
PMID : 25303834  :   DOI  :   10.1016/j.resmic.2014.09.009    
Abstract >>
Mupirocin is a polyketide antibiotic with broad antibacterial activity. It was isolated and characterized about 40 years ago from Pseudomonas fluorescens NCIMB 10586. To study the phylogenetic distribution of mupirocin producing strains in the genus Pseudomonas a large collection of Pseudomonas strains of worldwide origin, consisting of 117 Pseudomonas type strains and 461 strains isolated from different biological origins, was screened by PCR for the mmpD gene of the mupirocin gene cluster. Five mmpD(+) strains from different geographic and biological origin were identified. They all produced mupirocin and were strongly antagonistic against Staphylococcus aureus. Phylogenetic analysis showed that mupirocin production is limited to a single species. Inactivation of mupirocin production leads to complete loss of in vitro antagonism against S. aureus, except on certain iron-reduced media where the siderophore pyoverdine is responsible for the in vitro antagonism of a mupirocin-negative mutant. In addition to mupirocin some of the strains produced lipopeptides of the massetolide group. These lipopeptides do not play a role in the observed in vitro antagonism of the mupirocin producing strains against S. aureus.
KeywordMeSH Terms
Massetolide
Mupirocin
Pseudomonas fluorescens NCIMB 10586
Pseudomonas sp. W2Aug9
Pyoverdine
241. Decoin  V, Barbey  C, Bergeau  D, Latour  X, Feuilloley  MG, Orange  N, Merieau  A,     ( 2014 )

A type VI secretion system is involved in Pseudomonas fluorescens bacterial competition.

PloS one 9 (2)
PMID : 24551247  :   DOI  :   10.1371/journal.pone.0089411     PMC  :   PMC3925238    
Abstract >>
Protein secretion systems are crucial mediators of bacterial interactions with other organisms. Among them, the type VI secretion system (T6SS) is widespread in Gram-negative bacteria and appears to inject toxins into competitor bacteria and/or eukaryotic cells. Major human pathogens, such as Vibrio cholerae, Burkholderia and Pseudomonas aeruginosa, express T6SSs. Bacteria prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the cognate toxins. We describe here an environmental P. fluorescens strain, MFE01, displaying an uncommon oversecretion of Hcp (hemolysin-coregulated protein) and VgrG (valine-glycine repeat protein G) into the culture medium. These proteins are characteristic components of a functional T6SS. The aim of this study was to attribute a role to this energy-consuming overexpression of the T6SS. The genome of MFE01 contains at least two hcp genes (hcp1 and hcp2), suggesting that there may be two putative T6SS clusters. Phenotypic studies have shown that MFE01 is avirulent against various eukaryotic cell models (amebas, plant or animal cell models), but has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinical bacteria. Depending on the prey cell, mutagenesis of the hcp2 gene in MFE01 abolishes or reduces this antibacterial killing activity. Moreover, the introduction of T6SS immunity proteins from S. marcescens, which is not killed by MFE01, protects E. coli against MFE01 killing. These findings suggest that the protein encoded by hcp2 is involved in the killing activity of MFE01 mediated by effectors of the T6SS targeting the peptidoglycan of Gram-negative bacteria. Our results indicate that MFE01 can protect potato tubers against Pectobacterium atrosepticum, which causes tuber soft rot. Pseudomonas fluorescens is often described as a major PGPR (plant growth-promoting rhizobacterium), and our results suggest that there may be a connection between the T6SS and the PGPR properties of this bacterium.
KeywordMeSH Terms
Bacterial Secretion Systems
Microbial Interactions
242. Lavilla Lerma  L, Benomar  N, Casado Muñoz  Mdel C, Gálvez  A, Abriouel  H,     ( 2014 )

Antibiotic multiresistance analysis of mesophilic and psychrotrophic Pseudomonas spp. isolated from goat and lamb slaughterhouse surfaces throughout the meat production process.

Applied and environmental microbiology 80 (21)
PMID : 25172860  :   DOI  :   10.1128/AEM.01998-14     PMC  :   PMC4249046    
Abstract >>
The aim of this study was to investigate the phenotypic and genotypic antibiotic resistance profiles of pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which were representative of areas that are possible sources of meat contamination. Mesophilic (85 isolates) and psychrotrophic (37 isolates) pseudomonads identified at the species level generally were resistant to sulfamethoxazole, erythromycin, amoxicillin, ampicillin, chloramphenicol, trimethoprim, rifampin, and ceftazidime (especially mesophiles), as well as colistin and tetracycline (especially psychrotrophes). However, they generally were sensitive to ciprofloxacin, gentamicin, imipenem, and kanamycin regardless of species identity. Worryingly, in the present study, we found multidrug resistance (MDR) to up to 13 antibiotics, which was related to intrinsic and acquired resistance mechanisms. Furthermore, a link between various antimicrobial resistance genes was shown for beta-lactams and tetracycline, trimethoprim, and sulfonamides. The distribution and resistome-based analysis of MDR pseudomonads in different slaughterhouse zones indicated that the main sources of the identical or related pseudomonad strains were the animals (feet and wool) and the slaughterhouse environment, being disseminated from the beginning, or entrance environment, to the environment of the finished meat products. Those facts must be taken into consideration to avoid cross-contamination with the subsequent flow of mobile resistance determinants throughout all slaughterhouse zones and then to humans and the environment by the application of adequate practices of hygiene and disinfection measures, including those for animal wool and feet and also the entrance environment.
KeywordMeSH Terms
Abattoirs
Drug Resistance, Multiple, Bacterial
Environmental Microbiology
Food Handling
243. Allison  SD, Lu  L, Kent  AG, Martiny  AC,     ( 2014 )

Extracellular enzyme production and cheating in Pseudomonas fluorescens depend on diffusion rates.

Frontiers in microbiology 5 (N/A)
PMID : 24782855  :   DOI  :   10.3389/fmicb.2014.00169     PMC  :   PMC3990056    
Abstract >>
Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of Pseudomonas fluorescens producer and cheater genotypes over time in mixed liquid cultures and on agar plates. In mixed culture inoculated with equal frequencies of cheaters and producers, enzyme concentration declined to zero after 20 days, consistent with our hypothesis. We observed a similar decline in cultures inoculated with producers only, suggesting that cheater mutants arose de novo and swept the population. DNA sequencing showed that genetic changes most likely occurred outside the protease operon. In one experimental replicate, the population regained the ability to produce protease, likely due to further genetic changes or population dynamics. Under spatially structured conditions on agar plates, cheaters did not sweep the population. Instead, we observed a significant increase in the variation of enzyme activity levels expressed by clones isolated from the population. Together these results suggest that restricted diffusion favors a diversity of enzyme production strategies. In contrast, well-mixed conditions favor population sweeps by cheater strains, consistent with theoretical predictions. Cheater and producer strategies likely coexist in natural environments with the frequency of cheating increasing with diffusion rate.
KeywordMeSH Terms
Pseudomonas fluorescens
cheating
diffusion
extracellular enzyme
protease
protein
social evolution
spatial structure
244. Sperka  S, Zehelein  E, Fiedler  S, Fischer  S, Sommer  R, Buckel  P,     ( 1989 )

Complete nucleotide sequence of Pseudomonas fluorescens D-galactose dehydrogenase gene.

Nucleic acids research 17 (13)
PMID : 2503815  :   DOI  :   10.1093/nar/17.13.5402     PMC  :   PMC318141    
Abstract >>
N/A
KeywordMeSH Terms
Genes
Genes, Bacterial
245. Andreani  NA, Martino  ME, Fasolato  L, Carraro  L, Montemurro  F, Mioni  R, Bordin  P, Cardazzo  B,     ( 2014 )

Tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group.

Food microbiology 39 (N/A)
PMID : 24387861  :   DOI  :   10.1016/j.fm.2013.11.012    
Abstract >>
The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of "blue mozzarella" that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) will facilitate the application of the scheme and the sharing of the data.
KeywordMeSH Terms
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
Blue mozzarella
Food spoilage
Multilocus Sequence Typing
Pseudomonas fluorescens group
246. Shearer  AG, Altman  T, Rhee  CD,     ( 2014 )

Finding sequences for over 270 orphan enzymes.

PloS one 9 (5)
PMID : 24826896  :   DOI  :   10.1371/journal.pone.0097250     PMC  :   PMC4020792    
Abstract >>
Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These 'orphan enzymes' represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes.
KeywordMeSH Terms
247. Kushwaha  N, Srivastava  S,     ( 2014 )

Gamma-glutamyl transpeptidase from two plant growth promoting rhizosphere fluorescent pseudomonads.

Antonie van Leeuwenhoek 105 (1)
PMID : 24232936  :   DOI  :   10.1007/s10482-013-0051-x    
Abstract >>
Glutathione is the most abundant non-protein thiol compound present in many cells. Because this molecule is involved in many physiological processes, each cell maintains a critical level of glutathione. Gamma-glutamyl transpeptidase (GGT, E.C.2.3.2.2) is the key enzyme involved in the glutathione cycle. In the present study, GGT was isolated from two plant growth promoting rhizosphere isolates, Pseudomonas protegens strain Pf-5 and Pseudomonas fluorescens strain PfT-1. GGT in these strains is located in the periplasm and possessed good hydrolytic activity at pH 8.0. Strains Pf-5 and PfT-1 showed maximum enzyme activity when grown at 30�V35 �XC. The ggt gene from both the strains was cloned in pGEM-T cloning vector and sequenced. Subsequently, GGT expressed in Escherichia coli BL21(DE3) using the pET-28a(+) expression vector was purified and characterized. The enzymes are active in a wide range of pH and some divalent cations significantly enhanced the hydrolytic activity. These enzymes showed higher thermal stability as compared to those of other mesophilic strains, as they retained ~50 % of activity at 50 �XC even after 12 h of incubation. The enzymes could also tolerate up to 3.0 M NaCl.
KeywordMeSH Terms
Rhizosphere
248. Crump  MP, Haines  AS, Dong  X, Song  Z, Farmer  R, Williams  C, Hothersall  J, P?osko?  E, Wattana-Amorn  P, Stephens  ER, Yamada  E, Gurney  R, Takebayashi  Y, Masschelein  J, Cox  RJ, Lavigne  R, Willis  CL, Simpson  TJ, Crosby  J, Winn  PJ, Thomas  CM,     ( 2013 )

A conserved motif flags acyl carrier proteins for �]-branching in polyketide synthesis.

Nature chemical biology 9 (11)
PMID : 24056399  :   DOI  :   10.1038/nchembio.1342     PMC  :   PMC4658705    
Abstract >>
Type I polyketide synthases often use programmed �]-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where �]-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with �]-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem �]-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting �]-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.
KeywordMeSH Terms
Conserved Sequence
249. Reimmann  C,     ( 2012 )

Inner-membrane transporters for the siderophores pyochelin in Pseudomonas aeruginosa and enantio-pyochelin in Pseudomonas fluorescens display different enantioselectivities.

Microbiology (Reading, England) 158 (Pt 5)
PMID : 22343350  :   DOI  :   10.1099/mic.0.057430-0    
Abstract >>
Iron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch (ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two AraC-type regulators PchR, which are activated by their cognate siderophore. Here, stereospecific siderophore recognition is shown to occur at the inner membrane also. In P. aeruginosa, translocation of ferriPch across the inner membrane is carried out by the single-subunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational fetA-gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination with either of the two siderophore receptors FetA and FptA in a siderophore-negative P. aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific siderophore recognition occurs at the inner membrane by the FetCDE transporter.
KeywordMeSH Terms
250. Michelsen  CF, Stougaard  P,     ( 2012 )

Hydrogen cyanide synthesis and antifungal activity of the biocontrol strain Pseudomonas fluorescens In5 from Greenland is highly dependent on growth medium.

Canadian journal of microbiology 58 (4)
PMID : 22417387  :   DOI  :   10.1139/w2012-004    
Abstract >>
Hydrogen cyanide (HCN) is a secondary metabolite produced by many antagonistic Pseudomonas species. In the present study, the gene cluster encoding HCN synthesis in a newly isolated Pseudomonas fluorescens strain, In5, from South Greenland was investigated. Sequence analysis showed that the Greenlandic hcn gene cluster comprises a novel hcn cluster. Transposon mutagenesis of strain In5 resulted in mutants In5-2E1 and In5-1H7 with no production of HCN, and mutant In5-6B9 with reduced HCN synthesis. In mutant In5-2E1, the transposon was inserted into the hcnC gene; in mutant In5-1H7, the Tn5 insertion was found in a region upstream of a putative malate:quinone oxidoreductase gene (mqo); and in mutant In5-6B9, the transposon disrupted a probable enoyl-CoA hydratase/isomerase gene. In vitro inhibition experiments with In5 (wild type) and In5-2E1 (mutant) showed that in nitrogen-rich Luria-Bertani medium, strain In5 but not the hcn mutant In5-2E1 produced HCN and inhibited the growth of hyphae of Rhizoctonia solani and Pythium aphanidermatum . In contrast, when cultivating the strains in the carbohydrate-rich potato dextrose medium, neither of the strains produced any HCN, and thus, they were unable to inhibit hyphal growth of fungi. These experiments strongly indicate that the synthesis of HCN is highly dependent on the growth medium used.
KeywordMeSH Terms
251. Que  L, Hasegawa  Y, Chen  L, Iwaki  H, Hosler  JP, Li  T,     ( 2012 )

Evidence for a dual role of an active site histidine in �\-amino-�]-carboxymuconate-�`-semialdehyde decarboxylase.

Biochemistry 51 (29)
PMID : 22746257  :   DOI  :   10.1021/bi300635b     PMC  :   PMC3419591    
Abstract >>
The previously reported crystal structures of �\-amino-�]-carboxymuconate-�`-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)(3)(Asp)(OH(2)) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved (59)Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 ?) and Co-substituted (2.4 ?) and Zn-substituted H228Y (2.0 ? resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 ?) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.
KeywordMeSH Terms
252. Martínez-Granero  F, Navazo  A, Barahona  E, Redondo-Nieto  M, Rivilla  R, Martín  M,     ( 2012 )

The Gac-Rsm and SadB signal transduction pathways converge on AlgU to downregulate motility in Pseudomonas fluorescens.

PloS one 7 (2)
PMID : 22363726  :   DOI  :   10.1371/journal.pone.0031765     PMC  :   PMC3282751    
Abstract >>
Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracellular and intracellular signalling with the levels of AlgU and a new physiological role for the Gac system in the downregulation of flagella biosynthesis during exponential growth.
KeywordMeSH Terms
Down-Regulation
Signal Transduction
253. Fischer  S, Godino  A, Quesada  JM, Cordero  P, Jofré  E, Mori  G, Espinosa-Urgel  M,     ( 2012 )

Characterization of a phage-like pyocin from the plant growth-promoting rhizobacterium Pseudomonas fluorescens SF4c.

Microbiology (Reading, England) 158 (Pt 6)
PMID : 22442306  :   DOI  :   10.1099/mic.0.056002-0    
Abstract >>
R-type and F-type pyocins are high-molecular-mass bacteriocins produced by Pseudomonas aeruginosa that resemble bacteriophage tails. They contain no head structures and no DNA, and are used as defence systems. In this report, we show that Pseudomonas fluorescens SF4c, a strain isolated from the wheat rhizosphere, produces a high-molecular-mass bacteriocin which inhibits the growth of closely related bacteria. A mutant deficient in production of this antimicrobial compound was obtained by transposon mutagenesis. Sequence analysis revealed that the transposon had disrupted a gene that we have named ptm, since it is homologous to that encoding phage tape-measure protein in P. fluorescens Pf0-1, a gene belonging to a prophage similar to phage-like pyocin from P. aeruginosa PAO1. In addition, we have identified genes from the SF4c pyocin cluster that encode a lytic system and regulatory genes. We constructed a non-polar ptm mutant of P. fluorescens SF4c. Heterologous complementation of this mutation restored the production of bacteriocin. Real-time PCR was used to analyse the expression of pyocin under different stress conditions. Bacteriocin was upregulated by mitomycin C, UV light and hydrogen peroxide, and was downregulated by saline stress. This report constitutes, to our knowledge, the first genetic characterization of a phage tail-like bacteriocin in a rhizosphere Pseudomonas strain.
KeywordMeSH Terms
Rhizosphere
254. Yang  MM, Mavrodi  DV, Mavrodi  OV, Bonsall  RF, Parejko  JA, Paulitz  TC, Thomashow  LS, Yang  HT, Weller  DM, Guo  JH,     ( 2011 )

Biological control of take-all by fluorescent Pseudomonas spp. from Chinese wheat fields.

Phytopathology 101 (12)
PMID : 22070279  :   DOI  :   10.1094/PHYTO-04-11-0096    
Abstract >>
Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces graminis var. tritici is one of the most important root diseases of wheat worldwide. Bacteria were isolated from winter wheat from irrigated and rainfed fields in Hebei and Jiangsu provinces in China, respectively. Samples from rhizosphere soil, roots, stems, and leaves were plated onto King's medium B agar and 553 isolates were selected. On the basis of in vitro tests, 105 isolates (19% of the total) inhibited G. graminis var. tritici and all were identified as Pseudomonas spp. by amplified ribosomal DNA restriction analysis. Based on biocontrol assays, 13 strains were selected for further analysis. All of them aggressively colonized the rhizosphere of wheat and suppressed take-all. Of the 13 strains, 3 (HC9-07, HC13-07, and JC14-07, all stem endophytes) had genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) but none had genes for the production of 2,4-diacetylphloroglucinol, pyoluteorin, or pyrrolnitrin. High-pressure liquid chromatography (HPLC) analysis of 2-day-old cultures confirmed that HC9-07, HC13-07, and JC14-07 produced PCA but no other phenazines were detected. HPLC quantitative time-of-flight 2 mass-spectrometry analysis of extracts from roots of spring wheat colonized by HC9-07, HC13-07, or Pseudomonas fluorescens 2-79 demonstrated that all three strains produced PCA in the rhizosphere. Loss of PCA production by strain HC9-07 resulted in a loss of biocontrol activity. Analysis of DNA sequences within the key phenazine biosynthesis gene phzF and of 16S rDNA indicated that strains HC9-07, HC13-07, and JC14-07 were similar to the well-described PCA producer P. fluorescens 2-79. This is the first report of 2-79-like bacteria being isolated from Asia.
KeywordMeSH Terms
Pest Control, Biological
255. Jiang  F, Huang  S, Imadad  K, Li  C,     ( 2012 )

Cloning and expression of a gene with phospholipase B activity from Pseudomonas fluorescens in Escherichia coli.

Bioresource technology 104 (N/A)
PMID : 22078969  :   DOI  :   10.1016/j.biortech.2011.09.112    
Abstract >>
A gene from Pseudomonasfluorescens BIT-18 encoding a protein with phospholipase B activity (Pf-PLB) was cloned in E. coli BL21 (DE3). The open reading frame consists of 1272 bp and potentially encodes a protein of 423 amino acid residues with a calculated molecular mass of 45.8 kDa. The nucleotide sequence of Pf-PLB is 45%, 42%, 41%, 40%, 33%, and 31% identical to that of Bifidobacterium animals, Mycobacterium parascrofulaceum, Acidobacterium capsulatum, Lactobacillus johnsonii, Moraxella bovis, and Moraxella catarrhalis, respectively. The His-tagged protein was purified by affinity chromatography and the eluted protein hydrolyzed both the 1- and 2-ester bond of phosphatidylcholine. The recombinant Pf-PLB had optimal activity at pH 6.0 and 30 �XC, and it showed 20.1% higher efficiency in the conversion rate of the phosphorus content than the wild-type.
KeywordMeSH Terms
256.     ( 1997 )

Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon-phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida.

Gene 195 (1)
PMID : 9300819  :   DOI  :   10.1016/s0378-1119(97)00151-0    
Abstract >>
The phnA gene encoding a novel carbon-phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.
KeywordMeSH Terms
257.     ( 1997 )

Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin.

Applied and environmental microbiology 63 (6)
PMID : 9172332  :   PMC  :   PMC168505    
Abstract >>
Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis.
KeywordMeSH Terms
Genes, Bacterial
258.     ( 1997 )

Cloning, nucleotide sequence and expression of a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106 in Escherichia coli.

Biochimica et biophysica acta 1351 (1��2��)
PMID : 9116029  :   DOI  :   10.1016/s0167-4781(96)00189-3    
Abstract >>
A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.
KeywordMeSH Terms
Genes, Bacterial
Histidine
259.     ( 1997 )

A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1 as an immunological phosphate-starvation marker.

Microbiology (Reading, England) 143 (Pt 3) (N/A)
PMID : 9084184  :   DOI  :   10.1099/00221287-143-3-1019    
Abstract >>
A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1, expressed at phosphate concentrations below 0.08-0.13 mM, was purified and characterized. The purification method involved separation of outer-membrane proteins by SDS-PAGE and extraction of the protein from nitrocellulose or PVDF membranes after electrotransfer of proteins to the membranes. The N-terminal amino acid sequence of the purified protein, called Psi1, did not show homology to any known proteins, and in contrast to the phosphate-specific porin OprP of P. aeruginosa its mobility in SDS-PAGE was not affected by solubilization temperature. An antiserum against Psi1 recognized a protein of M, 55,000 in four other P. fluorescens strains among 24 tested strains representing Pseudomonas rRNA homology group I, showing antigenic heterogeneity within this group. A method for immunofluorescence microscopy involving cell permeabilization was adapted to visualize cell-specific expression of Psi1 in P. fluorescens exposed to limiting amounts of phosphate. This approach should be useful for further exploration of Psi1 as a marker to study the availability of phosphate to P. fluorescens in natural environments.
KeywordMeSH Terms
260.     ( 1997 )

Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens.

Journal of bacteriology 179 (8)
PMID : 9098078  :   DOI  :   10.1128/jb.179.8.2761-2765.1997     PMC  :   PMC179029    
Abstract >>
The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.
KeywordMeSH Terms
261.     ( 1997 )

Single-step conjugative cloning of bacterial gene fusions involved in microbe-host interactions.

Molecular & general genetics : MGG 256 (1)
PMID : 9341682  :   DOI  :   10.1007/s004380050548    
Abstract >>
In vivo expression technology (IVET) is a genetic strategy for isolating genes expressed in vivo. In order to full exploit this technology, it is necessary to analyse large numbers of IVET-generated gene fusions, which must be recovered from the chromosome of host bacteria. In bacteria for which transductional methods are not available, the recovery of integrated fusion plasmids is problematic and currently limits broad application of IVET. We describe a rapid, single-step, triparental conjugative approach for recovering chromosomally integrated fusion plasmids from both Pseudomonas fluorescens and Salmonella typhimurium. This simple and broadly applicable conjugative cloning system extends the utility of the IVET approach to clinically and agronomically relevant microbes and may be employed to recover non-replicating and integrated plasmids in other systems.
KeywordMeSH Terms
Conjugation, Genetic
262.     ( 1997 )

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Applied and environmental microbiology 63 (11)
PMID : 9361421  :   PMC  :   PMC168754    
Abstract >>
Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.
KeywordMeSH Terms
263.     ( 1997 )

Intercontinental spread of promiscuous mercury-resistance transposons in environmental bacteria.

Molecular microbiology 24 (2)
PMID : 9159519  :   DOI  :   10.1046/j.1365-2958.1997.3261688.x    
Abstract >>
We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic-resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site-specific recombination. Our data suggest that the mercury-resistance operons of Gram-negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.
KeywordMeSH Terms
Cation Transport Proteins
DNA Transposable Elements
264.     ( 1997 )

Purification of the dissimilative nitrate reductase of Pseudomonas fluorescens and the cloning and sequencing of its corresponding genes.

Biochimica et biophysica acta 1350 (3)
PMID : 9061022  :   DOI  :   10.1016/s0167-4781(97)00007-9    
Abstract >>
The dissimilative membrane-bound nitrate reductase from Pseudomonas fluorescens strain AK15 was purified and the alpha subunit of the enzyme partially sequenced. On the basis of this partial amino acid sequence and of conserved stretches of amino acids between Escherichia coli and Bacillus subtilis, degenerate primers were design to amplify the narG gene and part of the narH gene in a PCR approach. The deduced amino acid sequence of narG shows 72% and 52% and narH 78% and 62% identity to the homologous subunit of E. coli and B. subtilis, respectively.
KeywordMeSH Terms
265.     ( 1997 )

Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST.

Applied and environmental microbiology 63 (6)
PMID : 9172343  :   PMC  :   PMC168516    
Abstract >>
The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.
KeywordMeSH Terms
Genes, Bacterial
266.     ( 1996 )

A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400.

Molecular microbiology 21 (4)
PMID : 8878040  :   DOI  :   10.1046/j.1365-2958.1996.391399.x    
Abstract >>
Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens.
KeywordMeSH Terms
Oligopeptides
267.     ( 1996 )

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome.

Molecular microbiology 19 (3)
PMID : 8830243  :   DOI  :   10.1046/j.1365-2958.1996.391926.x    
Abstract >>
Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 PacI, 53 SpeI, 71 XbaI) were placed on the physical map and complete maps of the circular chromosome were obtained for both PacI and SpeI; only XbaI fragments linking SpeI fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by southern hybridization and 31 genes were positioned including oriC, rDNA operons (rnnA-E), recA, gacA, and pyvD.
KeywordMeSH Terms
Chromosome Mapping
268.     ( 1996 )

1-Aminocyclopropane-1-carboxylate deaminase genes from Pseudomonas strains.

FEMS microbiology letters 138 (2��3��)
PMID : 9026447  :   DOI  :   10.1111/j.1574-6968.1996.tb08158.x    
Abstract >>
Microbial ACC deaminase catalyses the conversion of 1-aminocyclopropane-1-carboxylate (ACC), the precursor to the phytohormone ethylene, to ammonia and alpha-ketobutyrate. We screened microorganisms for ACC degrading ability and cloned and sequenced the ACC deaminase genes from two Pseudomonas strains which displayed high enzyme activity. One of the genes was homologous with two previously sequenced ACC deaminase genes, but the other was different.
KeywordMeSH Terms
Carbon-Carbon Lyases
Genes, Bacterial
269.     ( 1996 )

The non-haem chloroperoxidase from Pseudomonas fluorescens and its relationship to pyrrolnitrin biosynthesis.

Microbiology (Reading, England) 142 (Pt 8) (N/A)
PMID : 8760926  :   DOI  :   10.1099/13500872-142-8-2129    
Abstract >>
The non-haem chloroperoxidase gene (cpoF) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe. Based on the overexpression of cpoF in Escherichia coli and the stability of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity. Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs. To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette. Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis.
KeywordMeSH Terms
270.     ( 1996 )

Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01.

Applied and environmental microbiology 62 (12)
PMID : 8953719  :   PMC  :   PMC168274    
Abstract >>
We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases. Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P. fluorescens IP01. After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 [cumH], ORF12 [cumD], and ORF13) were identified. The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons. Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity. The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y. Wang, M. Ralings, D. T. Gibson, D. Labb?, H. Bergeron, R. Brousseau, and P. C. K. Lau, Mol. Gen. Genet. 246:570-579, 1995). The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y. Yamada, P.-D. Lee, and T. Kosuge, Proc. Natl. Acad. Sci. USA 83:8263-8267, 1982). By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P. fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.
KeywordMeSH Terms
Genes, Bacterial
271.     ( 1995 )

The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F.

European journal of biochemistry 234 (1)
PMID : 8529644  :   DOI  :   10.1111/j.1432-1033.1995.225_c.x    
Abstract >>
A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown on phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively. The purified enzyme had an apparent Km of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate. The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.
KeywordMeSH Terms
272.     ( 1996 )

A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

Proceedings of the National Academy of Sciences of the United States of America 93 (14)
PMID : 8692990  :   DOI  :   10.1073/pnas.93.14.7315     PMC  :   PMC38981    
Abstract >>
A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.
KeywordMeSH Terms
Chromosomes, Bacterial
273.     ( 1995 )

The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.

Proceedings of the National Academy of Sciences of the United States of America 92 (26)
PMID : 8618880  :   DOI  :   10.1073/pnas.92.26.12255     PMC  :   PMC40335    
Abstract >>
Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces.
KeywordMeSH Terms
274.     ( 1994 )

In vitro characterization of a phosphate starvation-independent carbon-phosphorus bond cleavage activity in Pseudomonas fluorescens 23F.

Journal of bacteriology 176 (2)
PMID : 8288524  :   DOI  :   10.1128/jb.176.2.320-324.1994     PMC  :   PMC205052    
Abstract >>
A novel, metal-dependent, carbon-phosphorus bond cleavage activity, provisionally named phosphonoacetate hydrolase, was detected in crude extracts of Pseudomonas fluorescens 23F, an environmental isolate able to utilize phosphonoacetate as the sole carbon and phosphorus source. The activity showed unique specificity toward this substrate; its organic product, acetate, was apparently metabolized by the glyoxylate cycle enzymes of the host cell. Unlike phosphonatase, which was also detected in crude extracts of P. fluorescens 23F, phosphonoacetate hydrolase was inducible only in the presence of its sole substrate and did not require phosphate starvation.
KeywordMeSH Terms
275.     ( 1993 )

Cloning, nucleotide sequence, and expression of a p-hydroxybenzoate hydroxylase isozyme gene from Pseudomonas fluorescens.

The Journal of biological chemistry 268 (23)
PMID : 8349594  :  
Abstract >>
A gene encoding for a putative isozyme of p-hydroxy-benzoate hydroxylase (PHBH) has been isolated from Pseudomonas fluorescens (ATCC 13525). A comparison of the translated amino acid sequence with that of the known PHBH from P. fluorescens revealed that the new enzyme contains 3 additional amino acids and has 73% absolute homology to the previously known enzyme; conservation of secondary and active-site structures implied that the isozyme and known enzyme share the same general tertiary structure. Subsequent expression of the isozyme in Escherichia coli produced an enzyme with a specific activity about half that of the previously characterized PHBHs from P. fluorescens and Pseudomonas aeruginosa; in addition, somewhat weaker binding affinities for both NADPH and p-hydroxybenzoate were observed. Speculations are made on the reason for the existence of the isozyme, which does not appear to be expressed routinely in P. fluorescens.
KeywordMeSH Terms
Genes, Bacterial
276.     ( 1996 )

Cloning of a pectate lyase gene from Xanthomonas campestris pv. malvacearum and comparison of its sequence relationship with pel genes of soft-rot Erwinia and Pseudomonas.

Molecular plant-microbe interactions : MPMI 9 (1)
PMID : 8589419  :  
Abstract >>
The cotton blight pathogen, Xanthomonas campestris pv. malvacearum strain B414, produces an extracellular pectate lyase (Pel) with an estimated M(r) of 41,000 and pI of 9.7. The gene coding for this enzyme initially identified in a 1.8-kb PstI genomic DNA fragment was cloned. The nucleotide sequences of this 1.8-kb fragment and two pel genes previously cloned from Pseudomonas fluorescens and P. viridiflava were determined. These pel genes encoded pre-Pel proteins consisting of 377 to 380 amino acids (a.a.). A signal peptide consisting of 26 to 29 a.a. was present at the amino-terminus of each pre-Pel. Multiple sequence analysis revealed that Pel proteins of non-Erwinia phytopathogens including Xanthomonas, Pseudomonas, and Bacillus constituted a distinct cluster, which showed 20 to 43% a.a. identity to the four established Pel families of Erwinia. Homologous pel sequences were detected in various pathovars or strains of X. campestris. All of these xanthomonads produced an alkaline Pel and were capable of causing soft-rot in potato tuber slices and green pepper fruits.
KeywordMeSH Terms
Genes, Bacterial
277.     ( 1995 )

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Applied and environmental microbiology 61 (11)
PMID : 8526497  :   PMC  :   PMC167690    
Abstract >>
Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.
KeywordMeSH Terms
Genes, Bacterial
278.     ( 1994 )

Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host-range plasmids of the pQF series.

Plasmid 31 (3)
PMID : 8058819  :   DOI  :   10.1006/plas.1994.1028    
Abstract >>
The nucleotide sequence of the replicative origin (OriR) region of the small cryptic broad-host-range plasmid, pRO1600, which forms the basis of a number of useful cloning vectors has been determined. In addition it has been subjected to Tn5 mutagenesis, deletion analysis, and subcloning in order to define the regions essential for replication in Pseudomonas aeruginosa. The sequence (1894 bp) contains a fragment derived from transposon Tn1. The OriR region is structurally related to other replication (Rep) protein-dependent origins in that it has an A-T-rich region upstream of four 17-bp direct repeats (iterons) which presumably function in initiator protein binding. The sequence also contains a DNA-A-binding site and an open reading frame which could encode a basic (pI 10.6) 25,343-Da Rep protein with homology to RepA from the Neisseria gonorrhoeae beta-lactamase plasmid pFA3. The possible evolutionary origin of this plasmid in P. aeruginosa (RP1) is discussed.
KeywordMeSH Terms
DNA Helicases
DNA-Binding Proteins
Plasmids
Trans-Activators
279.     ( 1994 )

A putative regulatory gene downstream of recA is conserved in gram-negative and gram-positive bacteria.

Nucleic acids research 22 (7)
PMID : 8165147  :   DOI  :   10.1093/nar/22.7.1313     PMC  :   PMC523658    
Abstract >>
N/A
KeywordMeSH Terms
Conserved Sequence
Genes, Bacterial
Genes, Regulator
280.     ( 1994 )

A spontaneous point mutation in the aac(6')-Ib' gene results in altered substrate specificity of aminoglycoside 6'-N-acetyltransferase of a Pseudomonas fluorescens strain.

FEMS microbiology letters 115 (2��3��)
PMID : 8138142  :   DOI  :   10.1111/j.1574-6968.1994.tb06654.x    
Abstract >>
The aac(6')-Ib' gene from Pseudomonas fluorescens BM2687, encoding an aminoglycoside 6'-N-acetyltransferase type II which confers resistance to gentamicin but not to amikacin, was characterized. Nucleotide sequence determination indicated total identity between aac(6')-Ib' and the aac(6')-Ib gene from Pseudomonas aeruginosa BM2656 [1] with the exception of a C-to-T transition that results in a serine to leucine substitution at position 83 of the deduced polypeptide. The aac(6')-Ib gene specifies a type I enzyme which confers resistance to amikacin but not to gentamicin [2]. It thus appears that the point mutation detected is responsible for enzymic altered substrate specificity.
KeywordMeSH Terms
281.     ( 1993 )

Characterization of the recA gene from Pseudomonas fluorescens OE 28.3 and construction of a recA mutant.

Journal of general microbiology 139 (1)
PMID : 8450308  :   DOI  :   10.1099/00221287-139-1-49    
Abstract >>
The recA gene of Pseudomonas fluorescens OE 28.3 was isolated by complementation of the Fec- phenotype of recombinant lambda EMBL3 phages in a RecA- Escherichia coli strain. The subcloned recA restored resistance to UV and methyl methanesulphonate (MMS) exposure in recA mutants of E. coli. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acid residues, was preceded by an SOS box highly similar to those of Pseudomonas aeruginosa and Azotobacter vinelandii. The deduced amino acid sequence displayed highest homology to the RecA proteins from P. aeruginosa (87.8% identity) and A. vinelandii (84.3% identity). In both the regulatory region and the structural gene, a relatively high degree of sequence divergence from the Pseudomonas cepacia gene was observed. A mutant of P. fluorescens was constructed by inserting a kanamycin resistance cassette into its recA gene. This mutant exhibited an increased sensitivity to UV irradiation and MMS, and was strongly impaired in homologous recombinational activity.
KeywordMeSH Terms
282.     ( 1994 )

The sequence of the mer operon of pMER327/419 and transposon ends of pMER327/419, 330 and 05.

Gene 146 (1)
PMID : 8063107  :   DOI  :   10.1016/0378-1119(94)90835-4    
Abstract >>
Three different, independently isolated mercury-resistance-conferring plasmids, pMER327/419, pMER330 and pMER05, from cultures originating from the river Mersey (UK), contain identical regulatory merR genes and transposon ends. The mer determinant from pMER327/419 contains an additional potential ORF (ORF F) located between merP and merA when compared with the archetypal Tn501. Although these plasmids confer narrow-spectrum resistance (resistance to Hg2+, but not organomercurials) their merR genes encode a potential organomercurial-sensing protein. Transposition of the mer of pMER05 into plasmid RP4 was demonstrated and, as with Tn502 and Tn5053, insertion occurred at a specific region. The sequence of pMER05 is identical at the 'left' and 'right' termini and across merR to Tn5053, which was independently isolated from the chromosome of a Xanthomonas sp. bacteria from the Khaidarkan mercury mine in Kirgizia, former Soviet Union [Kholodii et al., J. Mol. Biol. 230 (1993a) 1103-1107]. The transpositional unit of pMER05 is, like that of Tn5053, bounded by DNA homologous to the imperfect 25-bp inverted repeats (IR) of the In2 integron, which brackets antibiotic-resistance cassettes in Tn21 subgroup transposons. At one end of the transposable element, and internal to the In2-like IR, is a 38-bp IR which closely resembles the IR that bounds Tn21.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
Operon
Plasmids
283.     ( 1994 )

The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes.

Gene 144 (1)
PMID : 8026764  :   DOI  :   10.1016/0378-1119(94)90196-1    
Abstract >>
The bph locus of Pseudomonas sp. LB400, encoding biphenyl/polychlorinated biphenyl (PCB) degradation, contains a region of about 3.5 kb of hitherto unknown function, between bphC and bphD. This DNA segment has now been characterized. Four structural genes have been located and identified by a combination of expression cloning, enzyme activity tests and DNA sequencing. The region contains four closely spaced cistrons (bphKHJI) encoding a glutathione S-transferase (GST), a 2-hydroxypenta-2,4-dienoate hydratase, an acetaldehyde dehydrogenase (acylating) and a 4-hydroxy-2-oxovalerate aldolase, respectively. The latter three are enzymes required for conversion of the aliphatic end product of bphABCD-encoded catabolism of biphenyls to Krebs cycle intermediates. The discovery of these genes provides a rationale for growth of the strain on chlorinated biphenyls which yield chlorinated benzoates as dead-end metabolites. The sequences of the enzymes involved are 54-71% identical to those of homologous enzymes encoded by the dmp and xyl operons. The role of the GST in the degradation of biphenyls is less clear, but since it was found to contain, in the putative xenobiotic substrate-binding domain, a region which shares about 29% of identical amino acids with a bacterial tetrachlorohydroquinone dehalogenase, it may be involved in dehalogenation of PCB-degradative intermediates.
KeywordMeSH Terms
284.     ( 1994 )

Sequence of the cobA gene encoding S-adenosyl-L-methionine: uroporhyrinogen III methyltransferase of Pseudomonas fluorescens.

Gene 150 (1)
PMID : 7959054  :   DOI  :   10.1016/0378-1119(94)90886-9    
Abstract >>
Sequence analysis of the region downstream of oprF, from Pseudomonas fluorescens OE 28.3, revealed the presence of cobA homologue encoding a putative S-adenosyl-L-methionine: uroporhyrinogen III methyltransferase. A similar gene organization exists in P. aeruginosa.
KeywordMeSH Terms
285.     ( 1994 )

Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product.

Journal of bacteriology 176 (22)
PMID : 7961472  :   DOI  :   10.1128/jb.176.22.7065-7073.1994     PMC  :   PMC197081    
Abstract >>
phaZPfi, the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized. It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma 70-like promoter 32 bp upstream of the ATG start codon. The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus. When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P. fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively. The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases. Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZPfl with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.
KeywordMeSH Terms
286.     ( 1994 )

Cloning and sequencing of the gene encoding benzaldehyde lyase from Pseudomonas fluorescens biovar I.

Gene 144 (1)
PMID : 8026749  :   DOI  :   10.1016/0378-1119(94)90218-6    
Abstract >>
The gene (bzl) encoding benzaldehyde lyase (BL) from Pseudomonas fluorescens biovar I has been cloned and characterized. The nucleotide sequence contains an open reading frame encoding a protein of 563 amino acids. The deduced BL protein shares little homology with proteins contained in the NBRF-PIR data bank. However, the higher homologies (up to about 28%) were obtained with enzymes that also utilize thiamine pyrophosphate as a cofactor.
KeywordMeSH Terms
Genes, Bacterial
287.     ( N/A )

Global regulation of expression of antifungal factors by a Pseudomonas fluorescens biological control strain.

Molecular plant-microbe interactions : MPMI 7 (4)
PMID : 8075420  :  
Abstract >>
The root-colonizing bacterium Pseudomonas fluorescens BL915 protects a variety of seedlings from damping-off disease caused by the fungal pathogen Rhizoctonia solani. Spontaneous pleiotropic mutants of P. fluorescens strain BL915 which fail to synthesize antifungal factors such as chitinase, cyanide, and pyrrolnitrin and exhibit altered colony morphology were isolated. Such mutants fail to inhibit the growth of R. solani in vitro, and their biological control capability is sharply reduced. We characterized a genomic DNA fragment from strain BL915 which, when introduced into these pleiotropic mutants, restored the lost functions, the wild-type colony morphology, and bio-control activity. DNA sequence analysis of the genomic fragment revealed the presence of genes homologous to those of numerous bacterial global regulatory systems and identified a cluster of genes identical in organization to the Escherichia coli gene cluster consisting of uvrY, uvrC, pgsA, and glyW. Coordinate biosynthesis of multiple antifungal products in some heterologous Pseudomonas strains in response to the introduction of the strain BL915 genomic fragment confirmed the regulatory nature of sequences contained on this fragment. Further genetic analysis indicated a gene homologous to response regulators of bacterial two-component systems was sufficient to complement the pleiotropic mutants and to activate antifungal genes in heterologous strains. Marker exchange of a truncated version of this gene into the P. fluorescens BL915 chromosome generated pleiotropic mutants indistinguishable from the original spontaneous mutants. Cloning and sequencing of the response regulator gene from several spontaneous mutants allowed identification of various nucleotide changes associated with the gene in such mutants.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
288.     ( 1993 )

Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties.

Journal of molecular biology 230 (4)
PMID : 8487301  :   DOI  :   10.1006/jmbi.1993.1236    
Abstract >>
The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.
KeywordMeSH Terms
Protein Conformation
289. Corbell  N, Loper  JE,     ( 1995 )

A global regulator of secondary metabolite production in Pseudomonas fluorescens Pf-5.

Journal of bacteriology 177 (21)
PMID : 7592389  :   DOI  :   10.1128/jb.177.21.6230-6236.1995     PMC  :   PMC177464    
Abstract >>
Mutations in the apdA (for antibiotic production) gene of the plant root-colonizing bacterium Pseudomonas fluorescens Pf-5 pleiotropically abolish the production of an array of antibiotics, including pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol, as well as the production of tryptophan side chain oxidase, hydrogen cyanide, and an extracellular protease. The lack of production of secondary metabolites by ApdA- mutants was correlated with the loss of inhibition of the phytopathogenic fungus Rhizoctonia solani in culture. Sequencing of the apdA region identified an open reading frame of 2,751 bp. The predicted amino acid sequence of the apdA gene contains conserved domains of the histidine kinases that serve as sensor components of prokaryotic two-component regulatory systems. The apdA nucleotide and predicted amino acid sequences are strikingly similar to the sequences of lemA and repA, genes encoding putative sensor kinases that are required for the pathogenicity of Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, respectively. Introduction of the cloned apdA+ gene restored the wild-type phenotype to both LemA- mutants of P. syringae and ApdA- mutants of Pf-5. The 101-kDa ApdA protein reacted with an anti-LemA antiserum, further demonstrating the similarity of ApdA to LemA. These results show that apdA encodes a putative sensor kinase component of a classical two-component regulatory system that is required for secondary-metabolite production by P. fluorescens Pf-5.
KeywordMeSH Terms
290. van Berkel  WJ, Eppink  MH, Schreuder  HA,     ( 1994 )

Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.

Protein science : a publication of the Protein Society 3 (12)
PMID : 7756982  :   DOI  :   10.1002/pro.5560031210     PMC  :   PMC2142777    
Abstract >>
The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.
KeywordMeSH Terms
291. Yokoigawa  K, Kawai  H, Endo  K, Lim  YH, Esaki  N, Soda  K,     ( 1993 )

Thermolabile alanine racemase from a psychotroph, Pseudomonas fluorescens: purification and properties.

Bioscience, biotechnology, and biochemistry 57 (1)
PMID : 7763424  :   DOI  :   10.1271/bbb.57.93    
Abstract >>
A psychotrophic bacterium that produces a thermolabile alanine racemase was isolated from raw milk, and identified as Pseudomonas fluorescens TM5-2. The enzyme was purified to homogeneity from the cell extract, and characterized to be compared with enzymes from mesophiles (Bacillus subtilis and Salmonella typhimurium) and a thermophile (Bacillus stearothermophilus). The enzyme has a molecular weight of about 76,000 and consists of two subunits identical in molecular weight (38,000). The enzyme contains two mol of pyridoxal 5'-phosphate per mol as a coenzyme. The amino acid composition was different from those of other alanine racemases in content of valine. The amino acid sequence of the amino terminal region (from 1Met to 25Gly) had 21-33% homology with those of other alanine racemases. Kinetic parameters of the enzyme were similar to those of other alanine racemases. The enzyme is extremely labile over 30 degrees C, and shows the high catalytic activity even at 0 degrees C; it is thermolabile and psychotrophic.
KeywordMeSH Terms
292. Sexton  R, Gill  PR, Callanan  MJ, O'Sullivan  DJ, Dowling  DN, O'Gara  F,     ( 1995 )

Iron-responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a transcription-activating factor, PbrA.

Molecular microbiology 15 (2)
PMID : 7746151  :   DOI  :   10.1111/j.1365-2958.1995.tb02244.x    
Abstract >>
In response to iron limitation. Pseudomonas fluorescens M114 induces a number of genes including an iron-scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ-induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron-regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron-regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseudobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the sigma 70 sigma factor family, including fecl required for expression of the ferric dicitrate outer-membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.
KeywordMeSH Terms
Bacterial Proteins
293.     ( 1994 )

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

Journal of bacteriology 176 (19)
PMID : 7928951  :   DOI  :   10.1128/jb.176.19.5912-5918.1994     PMC  :   PMC196807    
Abstract >>
A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.
KeywordMeSH Terms
294. Kojima  Y, Yokoe  M, Mase  T,     ( 1994 )

Purification and characterization of an alkaline lipase from Pseudomonas fluorescens AK102.

Bioscience, biotechnology, and biochemistry 58 (9)
PMID : 7765474  :   DOI  :   10.1271/bbb.58.1564    
Abstract >>
An extracellular, novel alkaline lipase produced by Pseudomonas fluorescens AK102 was purified by ultrafiltration, ammonium sulfate precipitation, and DEAE-Toyopearl 650M and Phenyl-Toyopearl 650M column chromatographies. The purified enzyme was homogeneous on SDS-PAGE. The molecular weight was estimated to be about 33,000 by SDS-PAGE. The isoelectric point was pH 4.0 by isoelectric focusing. The pH stability was 4 to 10 and the optimum pH was 8 to 10. The optimum temperature was 55 degrees C and the enzyme was stable below 50 degrees C. The enzyme unspecifically liberated short chain to long chain fatty acids from p-nitrophenyl esters, methyl esters, and triglycerides. In the presence of an anionic surfactant, the enzyme was characteristically stable. These results suggested that the enzyme can be used as a home laundry product ingredient.
KeywordMeSH Terms
295. Hosoya  H, Nakamura  K,     ( 1994 )

DNA sequence of proline permease gene from Pseudomonas fluorescens and predicted structure of proline permease.

Bioscience, biotechnology, and biochemistry 58 (11)
PMID : 7765603  :   DOI  :   10.1271/bbb.58.2099    
Abstract >>
The proline permease gene of Pseudomonas fluorescens has been isolated from the promoter region isolated by using a promoter probe (transposon Tn5-B21). By DNA sequencing of 2222 bp the primary structure of permease (494 aa) was deduced. The DNA sequence is 71.0% and 70.7% identical and the amino acid sequence is 75.8% and 76.0% similar to those of Escherichia coli and Salmonella typhimurium, respectively.
KeywordMeSH Terms
Amino Acid Transport Systems, Neutral
Genes, Bacterial
296. Hosoya  H, Nakamura  K, Furukawa  K,     ( 1994 )

Promoter screening from Pseudomonas species by a promoter probe transposon and its structure.

Bioscience, biotechnology, and biochemistry 58 (11)
PMID : 7765602  :   DOI  :   10.1271/bbb.58.2096    
Abstract >>
By use of the promoter probe transposon Tn5-B21, two promoter fragments were isolated. One promoter (822 bp) (GC = 63.5%) isolated from P. putida loses all of its promoter activity by exonuclease or restriction enzyme deletion. Another promoter (264 bp) (GC = 49.2%) isolated from P. fluorescens could be shortened to 154 bp by exonuclease deletion without any effect on its promoter activity in several Pseudomonas species.
KeywordMeSH Terms
DNA Transposable Elements
Promoter Regions, Genetic
297. Kim  YS, Lee  HB, Choi  KD, Park  S, Yoo  OJ,     ( 1994 )

Cloning of Pseudomonas fluorescens carboxylesterase gene and characterization of its product expressed in Escherichia coli.

Bioscience, biotechnology, and biochemistry 58 (1)
PMID : 7764506  :   DOI  :   10.1271/bbb.58.111    
Abstract >>
A gene (estC) coding for an esterase (esterase III) of Pseudomonas fluorescens was cloned into Escherichia coli JM83. DNA sequencing showed a single open reading frame with GTG as a translation initiation codon for esterase III. This was confirmed by N-terminal amino acid sequence analysis of the purified esterase III protein from an E. coli clone. The promoter sequence and a potential Shine-Dalgarno sequence were followed by the coding sequence of the estC gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterase. The esterase III expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. The native form of the enzyme was a monomer with a molecular weight of 41,000. The results of substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3.1.1.1) and a serine residue is present at the active site of the enzyme.
KeywordMeSH Terms
298. Solinas  F, Marconi  AM, Ruzzi  M, Zennaro  E,     ( 1995 )

Characterization and sequence of a novel insertion sequence, IS1162, from Pseudomonas fluorescens.

Gene 155 (1)
PMID : 7698671  :   DOI  :   10.1016/0378-1119(94)00922-f    
Abstract >>
We have determined the nucleotide sequence of IS1162, a new insertion sequence (IS) isolated from Pseudomonas fluorescens (Pf) strain ST. This IS element is present in two copies on the pEG plasmid harboured by Pf ST and in a single copy on the chromosome, adjacent to the styrene catabolic genes. IS1162 is 2634 bp in length with 12-bp terminal inverted repeats (IR), and could encode four proteins (ORFs), two for each strand. One strand, Pro1 (62,990 Da), showed a helix-turn-helix motif at the N-terminal region, and Pro2 (25,997 Da) was characterized by the presence of the A and B motives of the NTP (ATP/GTP)-binding site. Comparison of IS1162 of Pf with known IS showed a high homology with IS408 of Burkholderia cepacia [Byrne and Lessie, Plasmid 31 (1994) 138-147]. Pro1 and Pro2 were found to be homologous to the corresponding ORFs of IS408, IS21 [Reimmann et al., Mol. Gen. Genet. 215 (1989) 416-424], IS232 [Menou et al., J. Bacteriol. 172 (1990) 6689-6696] and IS5376 [Xu et al., Plasmid 29 (1993) 1-9]. IS1162 transposed at low frequency and no cointegrates were found among the transposition products. The target duplication sites, variable in length, showed the presence of homologous motives, suggesting a certain degree of specificity of the IS1162 insertion site.
KeywordMeSH Terms
299. Pelletier  I, Altenbuchner  J,     ( 1995 )

A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity.

Microbiology (Reading, England) 141 (Pt 2) (N/A)
PMID : 7704276  :   DOI  :   10.1099/13500872-141-2-459    
Abstract >>
Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides. However, this homology was limited to two distinct domains of the published esterase sequence. As errors in the published sequence were suspected, the esterase gene was sequenced again. The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence. In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology. The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon. The esterase is thus a bifunctional enzyme. The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family. It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad. By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad. The triad comprised the residues Ser95, Asp223 and His252. Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains. The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation.
KeywordMeSH Terms
300.     ( 1994 )

Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase.

The Journal of biological chemistry 269 (39)
PMID : 7929087  :  
Abstract >>
To elucidate the mode of action of pseudomonic acid, we have compared the deduced amino acid sequences of isoleucyl-tRNA synthetases (ILeRS) from wild-type Escherichia coli strain MC4100, a pseudomonic acid-resistant mutant (strain PS102) of MC4100, and a pseudomonic acid-producing strain, Pseudomonas fluorescens. Compared with the wild-type enzyme, the deduced amino acid sequence of E. coli mutant ileS gene in strain PS102 shows a single amino acid substitution of leucine for phenylalanine at residue 594 of the IleRS. This mutational alteration in IleRS of an E. coli pseudomonic acid-resistant mutant resides in a region of the enzyme in close proximity to one of the consensus sequences of class I aminoacyl-tRNA synthetases, the KMSKS sequence between residues 602 and 606 of the E. coli IleRS. DNA sequence of the cloned ileS gene predicts that the P. fluorescens IleRS consists of 943 amino acids with 54% identity with the E. coli IleRS. The P. fluorescens ileS gene and the wild type and PS102 alleles of E. coli ileS were cloned into an expression vector, pEXPCR, and the sensitivities of E. coli DH5 alpha cells harboring each of these plasmids were compared. The cells harboring the P. fluorescens ileS were found to be most resistant to pseudomonic acid, while the transformants expressing the PS102 IleRS were more resistant than those containing the wild-type E. coli IleRS. IleRS purified from the wild-type E. coli was specifically cleaved by trypsin between Lys605 and Ser606 in the region of K602MSKS606. The protection of the IleRS from the trypsin digestion was found with pseudomonic acid or ATP, but not with isoleucine or tRNA(1Ile). Based on these results, we propose that pseudomonic acid binds to IleRS in the vicinity of the KMSKS sequence that is an ATP-binding subsite, and that pseudomonic acid is a bifunctional inhibitor with characteristics of both isoleucine and ATP, for example, an analog of isoleucyladenylate.
KeywordMeSH Terms
301.     ( 1994 )

Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp).

Journal of bacteriology 176 (9)
PMID : 7909543  :   DOI  :   10.1128/jb.176.9.2569-2576.1994     PMC  :   PMC205394    
Abstract >>
The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonas fluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (M(r) 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacillus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.
KeywordMeSH Terms
302. De Mot  R, Schoofs  G, Roelandt  A, Declerck  P, Proost  P, Van Damme  J, Vanderleyden  J,     ( 1994 )

Molecular characterization of the major outer-membrane protein OprF from plant root-colonizing Pseudomonas fluorescens.

Microbiology (Reading, England) 140 (Pt 6) (N/A)
PMID : 7521711  :   DOI  :   10.1099/00221287-140-6-1377    
Abstract >>
N-terminal sequence analysis of peptides generated by proteolytic treatment of the Pseudomonas fluorescens OE 28.3 major outer-membrane protein OprF, embedded in outer membranes or present in whole cells, indicated a surface-exposed location for the proline-rich region of the protein. This region is absent from the P. aeruginosa and P. syringae OprFs. Evidence was obtained for the presence of additional exposed but less accessible regions in the carboxy half of OprF. Four OprF-specific monoclonal antibodies were all directed to the C-terminal part of the protein but did not recognize a surface-exposed epitope as shown by flow cytometry. Our data support the model previously proposed for P. aeruginosa OprF in which the entire protein is embedded in the outer membrane, unlike the topology proposed for the major outer-membrane protein from Escherichia coli, OmpA, whose carboxy half resides in the periplasmic space. For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes. The proline-rich domain represented the most conserved region of the different P. fluorescens OprFs. Based on the sequence of its oprF gene, it was shown that the mushroom pathogen P. tolaasii is quite closely related to P. fluorescens. Comparative sequence analysis further showed that the carboxy half of OprF contains a sequence motif that is well conserved in the enterobacterial OmpA proteins but is also present in a number of other outer-membrane proteins, including peptidoglycan-associated lipoproteins.
KeywordMeSH Terms
303. Vereijken  JM, Hofsteenge  J, Bak  HJ, Beintema  JJ,     ( 1980 )

The amino-acid sequence of the three smallest CNBr peptides from p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

European journal of biochemistry 113 (1)
PMID : 6780353  :   DOI  :   10.1111/j.1432-1033.1980.tb06149.x    
Abstract >>
After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: (Formula: See Text).
KeywordMeSH Terms
4-Hydroxybenzoate-3-Monooxygenase
Mixed Function Oxygenases
304. Hofsteenge  J, Vereijken  JM, Weijer  WJ, Beintema  JJ, Wierenga  RK, Drenth  J,     ( 1980 )

Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide.

European journal of biochemistry 113 (1)
PMID : 6780352  :  
Abstract >>
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.
KeywordMeSH Terms
4-Hydroxybenzoate-3-Monooxygenase
Mixed Function Oxygenases
305. Weijer  WJ, Hofsteenge  J, Beintema  JJ, Wierenga  RK, Drenth  J,     ( 1983 )

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 2. Fitting of the amino-acid sequence to the tertiary structure.

European journal of biochemistry 133 (1)
PMID : 6406227  :   DOI  :   10.1111/j.1432-1033.1983.tb07435.x    
Abstract >>
The complete primary and tertiary structure of p-hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron-density map at 0.25-nm resolution. The parts of the polypeptide chain contributing the residues to the FAD-binding site and the residues of the substrate-binding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p-hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD. Many side chains from the C-terminal part of the polypeptide chain are involved in subunit-subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.
KeywordMeSH Terms
306. Eppink  MH, Schreuder  HA, Van Berkel  WJ,     ( 1995 )

Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase implications for NADPH binding.

European journal of biochemistry 231 (1)
PMID : 7628466  :   DOI  :   10.1111/j.1432-1033.1995.0157f.x    
Abstract >>
Arg44, located at the si-face side of the flavin ring in 4-hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180 degrees in 50% of the molecules. The phi, psi angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.
KeywordMeSH Terms
307. Dé  E, De Mot  R, Orange  N, Saint  N, Molle  G,     ( 1995 )

Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3.

FEMS microbiology letters 127 (3)
PMID : 7538961  :   DOI  :   10.1111/j.1574-6968.1995.tb07484.x    
Abstract >>
The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media. Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3).
KeywordMeSH Terms
308. Schreuder  HA, Mattevi  A, Obmolova  G, Kalk  KH, Hol  WG, van der Bolt  FJ, van Berkel  WJ,     ( 1994 )

Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.

Biochemistry 33 (33)
PMID : 7520279  :   DOI  :   10.1021/bi00199a044    
Abstract >>
The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively. In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution. The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit. The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region. Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385. As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72. It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site. Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site. The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation). It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave.
KeywordMeSH Terms
Mutation
Protons
309. Weijer  WJ, Hofsteenge  J, Vereijken  JM, Jekel  PA, Beintema  JJ,     ( 1982 )

Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

Biochimica et biophysica acta 704 (2)
PMID : 6809053  :   DOI  :   10.1016/0167-4838(82)90170-4    
Abstract >>
The amino acid sequence of the p-hydroxybenzoate hydroxylase (4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) monomer from Pseudomonas fluorescens has been determined. The sequence was elucidated by a combination of the results from an X-ray crystallographic study at 0.25 nm resolution (Wierenga, R.K., de Jong, R.J., Kalk, K.H., Hol, W.G.J. and Drenth, J. (1979) J. Mol. Biol. 131, 55-73) and from protein sequence analysis. The polypeptide chain of the monomer contains 394 amino acids and has a molecular weight of 44 299.
KeywordMeSH Terms
4-Hydroxybenzoate-3-Monooxygenase
Mixed Function Oxygenases
310. Blachnitzky  EO, Wengenmayer  F, Kurz  G,     ( 1974 )

D-Galactose dehydrogenase from Pseudomonas fluorescens. Purification, properties and structure.

European journal of biochemistry 47 (2)
PMID : 4153311  :   DOI  :   10.1111/j.1432-1033.1974.tb03687.x    
Abstract >>
N/A
KeywordMeSH Terms
311. Wierenga  RK, de Jong  RJ, Kalk  KH, Hol  WG, Drenth  J,     ( 1979 )

Crystal structure of p-hydroxybenzoate hydroxylase.

Journal of molecular biology 131 (1)
PMID : 40036  :   DOI  :   10.1016/0022-2836(79)90301-2    
Abstract >>
N/A
KeywordMeSH Terms
4-Hydroxybenzoate-3-Monooxygenase
Mixed Function Oxygenases
312. Hofsteenge  J, Weijer  WJ, Jekel  PA, Beintema  JJ,     ( 1983 )

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 1. Completion of the elucidation of the primary structure.

European journal of biochemistry 133 (1)
PMID : 6406229  :   DOI  :   10.1111/j.1432-1033.1983.tb07433.x    
Abstract >>
As a final step in the elucidation of the primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111-276), that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346-347) in peptide CB2. The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map. The sequence of residues 343-346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map. An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys-C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.
KeywordMeSH Terms
313. Torres-Cortés  G, Garcia  BJ, Compant  S, Rezki  S, Jones  P, Préveaux  A, Briand  M, Roulet  A, Bouchez  O, Jacobson  D, Barret  M,     ( 2019 )

Differences in resource use lead to coexistence of seed-transmitted microbial populations.

Scientific reports 9 (1)
PMID : 31040301  :   DOI  :   10.1038/s41598-019-42865-9     PMC  :   PMC6491768    
Abstract >>
Seeds are involved in the vertical transmission of microorganisms in plants and act as reservoirs for the plant microbiome. They could serve as carriers of pathogens, making the study of microbial interactions on seeds important in the emergence of plant diseases. We studied the influence of biological disturbances caused by seed transmission of two phytopathogenic agents, Alternaria brassicicola Abra43 (Abra43) and Xanthomonas campestris pv. campestris 8004 (Xcc8004), on the structure and function of radish seed microbial assemblages, as well as the nutritional overlap between Xcc8004 and the seed microbiome, to find seed microbial residents capable of outcompeting this pathogen. According to taxonomic and functional inference performed on metagenomics reads, no shift in structure and function of the seed microbiome was observed following Abra43 and Xcc8004 transmission. This lack of impact derives from a limited overlap in nutritional resources between Xcc8004 and the major bacterial populations of radish seeds. However, two native seed-associated bacterial strains belonging to Stenotrophomonas rhizophila displayed a high overlap with Xcc8004 regarding the use of resources; they might therefore limit its transmission. The strategy we used may serve as a foundation for the selection of seed indigenous bacterial strains that could limit seed transmission of pathogens.
KeywordMeSH Terms
314. Schreuder  HA, van der Laan  JM, Hol  WG, Drenth  J,     ( 1988 )

Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate.

Journal of molecular biology 199 (4)
PMID : 3351945  :   DOI  :   10.1016/0022-2836(88)90307-5    
Abstract >>
Crystals of the flavin-containing enzyme p-hydroxybenzoate hydroxylase (PHBHase) complexed with its reaction product were investigated in order to obtain insight into the catalytic cycle of this enzyme involving two substrates and two cofactors. PHBHase was crystallized initially with its substrate, p-hydroxybenzoate and the substrate was then converted into the product 3,4-dihydroxybenzoate by allowing the catalytic reaction to proceed in the crystals. In addition, crystals were soaked in mother liquor containing a high concentration of this product. Data up to 2.3 A (1 A = 0.1 nm) were collected by the oscillation method and the structure of the enzyme product complex was refined by alternate restrained least-squares procedures and model building by computer graphics techniques. A total of 273 solvent molecules could be located, four of them being presumably sulfate ions. The R-factor for 14,339 reflections between 6.0 A and 2.3 A is 19.3%. The 3-hydroxyl group of the product introduced by the enzyme is clearly visible in the electron density, showing unambiguously which carbon atom of the substrate is hydroxylated. A clear picture of the hydroxylation site is obtained. The plane of the product is rotated 21 degrees with respect to the plane of the substrate in the current model of enzyme-substrate complex. The 4-hydroxyl group of the product is hydrogen bonded to the hydroxyl group of Tyr201, its carboxyl group is interacting with the side-chains of Tyr222, Arg214 and Ser212, while the newly introduced 3-hydroxyl group makes a hydrogen bond with the backbone carbonyl oxygen of Pro293.
KeywordMeSH Terms
315. Warren  G, Corotto  L, Wolber  P,     ( 1986 )

Conserved repeats in diverged ice nucleation structural genes from two species of Pseudomonas.

Nucleic acids research 14 (20)
PMID : 3774551  :   DOI  :   10.1093/nar/14.20.8047     PMC  :   PMC311833    
Abstract >>
Sequence analysis shows that an ice nucleation gene (inaW) from Pseudomonas fluorescens is related to the inaZ gene of Pseudomonas syringae. The two genes have diverged by many amino acid substitutions, and have effectively randomized the third bases of homologous codons. By reference to their potential for change, it is shown that certain conserved features must have been maintained by selection pressure. In particular, their conservation of internal sequence repetition, with three orders of repeat periodicity in each gene, suggests that the pattern of repetition is significant to the gene products' function. We propose models for the structure of the gene products in which each order of periodicity would be required for the nucleation function.
KeywordMeSH Terms
Ice
316. Takagi  JS, Tokushige  M, Shimura  Y,     ( 1986 )

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens.

Journal of biochemistry 100 (3)
PMID : 3096982  :   DOI  :   10.1093/oxfordjournals.jbchem.a121762    
Abstract >>
The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.
KeywordMeSH Terms
Genes, Bacterial
317. Tamulaitiene  G, Manakova  E, Jovaisaite  V, Tamulaitis  G, Grazulis  S, Bochtler  M, Siksnys  V,     ( 2019 )

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family.

Nucleic acids research 47 (2)
PMID : 30445642  :   DOI  :   10.1093/nar/gky1137     PMC  :   PMC6344858    
Abstract >>
Restriction endonucleases (REs) of the CCGG-family recognize a set of 4-8 bp target sequences that share a common CCGG or CCNGG core and possess PD�KD/ExK nuclease fold. REs that interact with 5 bp sequence 5'-CCNGG flip the central N nucleotides and 'compress' the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5'-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E�KExD�KK active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not 'compress' the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.
KeywordMeSH Terms
318. Gao  J, Yao  L, Xia  T, Liao  X, Zhu  D, Xiang  Y,     ( 2018 )

Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 32 (4)
PMID : 29208702  :   DOI  :   10.1096/fj.201700397RR    
Abstract >>
The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ?110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-�\-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.
KeywordMeSH Terms
neurodegeneration diseases
neurologic disorders
therapeutic target
Allosteric Site
319. Pavkov-Keller  T, Schmidt  NG, Ż?d?o-Dobrowolska  A, Kroutil  W, Gruber  K,     ( 2019 )

Structure and Catalytic Mechanism of a Bacterial Friedel-Crafts Acylase.

Chembiochem : a European journal of chemical biology 20 (1)
PMID : 30318713  :   DOI  :   10.1002/cbic.201800462     PMC  :   PMC6392133    
Abstract >>
C-C bond-forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel-Crafts C-acylation of phenolic substrates in aqueous solution, reaching up to >99 % conversion without the need for CoA-activated reagents. We determined X-ray crystal structures of the native and ligand-bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A2 C2)2 B4 composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1-(2,4,6-trihydroxyphenyl)ethanone together with site-directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.
KeywordMeSH Terms
Friedel-Crafts acylation
X-ray diffraction
acyltransferases
multicomponent enzymes
solid-state structures
transferases
320. Chung  J, Kim  S, Kwon  SK, Cha  H, Son  J, Cho  JM, Hwang  KY, Kim  HT, Na  BK,     ( 2018 )

Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase.

Cell chemical biology 25 (4)
PMID : 29429898  :   DOI  :   10.1016/j.chembiol.2018.01.008    
Abstract >>
Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via �k-�k interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal �\ helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors.
KeywordMeSH Terms
KMO inhibitor
drug design
flavin reduction
hydrogen peroxide
kynurenine 3-monooxygenase
321. Price  MN, Wetmore  KM, Waters  RJ, Callaghan  M, Ray  J, Liu  H, Kuehl  JV, Melnyk  RA, Lamson  JS, Suh  Y, Carlson  HK, Esquivel  Z, Sadeeshkumar  H, Chakraborty  R, Zane  GM, Rubin  BE, Wall  JD, Visel  A, Bristow  J, Blow  MJ, Arkin  AP, Deutschbauer  AM,     ( 2018 )

Mutant phenotypes for thousands of bacterial genes of unknown function.

Nature 557 (7706)
PMID : 29769716  :   DOI  :   10.1038/s41586-018-0124-0    
Abstract >>
One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.
KeywordMeSH Terms
Molecular Sequence Annotation
Mutation
Phenotype
Uncertainty
322.     ( 2013 )

The dar genes of Pseudomonas chlororaphis PCL1606 are crucial for biocontrol activity via production of the antifungal compound 2-hexyl, 5-propyl resorcinol.

Molecular plant-microbe interactions : MPMI 26 (5)
PMID : 23547906  :   DOI  :   10.1094/MPMI-01-13-0012-R    
Abstract >>
To determine the genetic basis by which 2-hexyl, 5-propyl resorcinol (HPR) is produced by the biocontrol rhizobacterium Pseudomonas chlororaphis (formerly known as P. fluorescens) PCL1606, the presence and role of dar genes were investigated. To accomplish this aim, the pCGNOV-1 plasmid was isolated from a PCL1606 genomic library and was shown to hybridize to various dar probes by Southern blot. An analysis of the pCGNOV-1 genomic DNA revealed the presence of five open reading frames that were homologous to dar genes and had an organization that resembled the arrangement of previously described P. chlororaphis strains. Phylogenetic studies resulted in the clustering of PCL1606 with the P. chlororaphis subgroup, which supported the renaming of this strain from P. fluorescens to P. chlororaphis PCL1606. The construction of insertional mutants for each homologous dar gene in P. chlororaphis PCL1606 along with their corresponding complemented derivative strains restored HPR production and confirmed the key role of the dar A and darB genes in HPR production and in the antagonistic phenotype. Finally, biocontrol assays were performed on avocado-Rosellinia and tomato-Fusarium test systems using the HPR-defective and -complemented derivative strains generated here and demonstrated the crucial role of the biosynthetic dar genes in the biocontrol phenotype of P. chlororaphis PCL1606. This biocontrol phenotype is dependent on the dar genes via their production of the HPR antibiotic. Some of the dar genes not directly involved in the biosynthesis of HPR, such as darS or darR, might contribute to regulatory features of HPR production.
KeywordMeSH Terms
323.     ( 2013 )

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil, shale oil or diesel fuel.

Microbiological research 168 (7)
PMID : 23510642  :   DOI  :   10.1016/j.micres.2013.02.006    
Abstract >>
The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.
KeywordMeSH Terms
Biodiversity
Ecosystem
324. Chevalier  S, Bodilis  J, Jaouen  T, Barray  S, Feuilloley  MG, Orange  N,     ( 2007 )

Sequence diversity of the OprD protein of environmental Pseudomonas strains.

Environmental microbiology 9 (3)
PMID : 17298381  :   DOI  :   10.1111/j.1462-2920.2006.01191.x    
Abstract >>
KeywordMeSH Terms
Environmental Microbiology
Genetic Variation
Sequence Analysis, DNA
325. Huo  L, Davis  I, Chen  L, Liu  A,     ( 2013 )

The power of two: arginine 51 and arginine 239* from a neighboring subunit are essential for catalysis in �\-amino-�]-carboxymuconate-epsilon-semialdehyde decarboxylase.

The Journal of biological chemistry 288 (43)
PMID : 24019523  :   DOI  :   10.1074/jbc.M113.496869     PMC  :   PMC3829401    
Abstract >>
KeywordMeSH Terms
Crystal Structure
Decarboxylase
Dimerization
Enzyme Mechanisms
Hybridization
Metabolic Regulation
Protein Assembly
Protein Folding
326.     ( 2012 )

Multiple-level regulation of 2,4-diacetylphloroglucinol production by the sigma regulator PsrA in Pseudomonas fluorescens 2P24.

PloS one 7 (11)
PMID : 23209661  :   DOI  :   10.1371/journal.pone.0050149     PMC  :   PMC3510223    
Abstract >>
Pseudomonas fluorescens 2P24 is a rhizospheric bacterium that aggressively colonizes the plant roots. It produces the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG), which contributes to the protection of various crop plants against soil borne diseases caused by bacterial and fungal pathogens. The biosynthesis of 2,4-DAPG is regulated at the transcriptional level in the expression of the phlACBD operon as well as at the posttranscriptional level by the Gac/Rsm signal transduction pathway. However, the detailed mechanism of such regulation is not clear. In this study, we identified a binding site for the sigma regulator PsrA in the promoter region of the phlA gene. Electrophoretic mobility shift experiments revealed direct and specific binding of PsrA to the phlA promoter region. Consistent with the fact that its binding site locates within the promoter region of phlA, PsrA negatively regulates phlA expression, and its inactivation led to significant increase in 2,4-DAPG production. Interestingly, PsrA also activates the expression of the sigma factor RpoS, which negatively regulates 2,4-DAPG production by inducing the expression of the RNA-binding protein RsmA. These results suggest that PsrA is an important regulator that modulates 2,4-DAPG biosynthesis at both transcriptional and posttranscriptional levels.
KeywordMeSH Terms
Gene Expression Regulation
Gene Expression Regulation, Bacterial
327.     ( 1998 )

Molecular cloning and analysis of a lipase gene from Pseudomonas fluorescens No. 33.

Bioscience, biotechnology, and biochemistry 62 (11)
PMID : 9972244  :  
Abstract >>
The gene encoding an extracellular lipase from Pseudomonas fluorescens No. 33 was cloned and sequenced. A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized. Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide. The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.
KeywordMeSH Terms
Genes, Bacterial
328.     ( 2012 )

Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy.

Molecular biology reports 39 (12)
PMID : 23090479  :   DOI  :   10.1007/s11033-012-1994-0    
Abstract >>
Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA(P.fluorescens) indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K (i) for glyphosate compared to those of the AroA(E.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA (P.fluorescens) gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.
KeywordMeSH Terms
Genomic Library
329.     ( 2013 )

Evaluation of oprI and oprL genes as molecular markers for the genus Pseudomonas and their use in studying the biodiversity of a small Belgian River.

Research in microbiology 164 (3)
PMID : 23246592  :   DOI  :   10.1016/j.resmic.2012.12.001    
Abstract >>
A multiplex PCR based on oprI and oprL, coding for the outer membrane lipoprotein I and the peptidoglycan-associated lipoprotein OprL, respectively, was developed for the detection of Pseudomonas strains from a bacterial collection isolated from a small river. To study the diversity of these Pseudomonas isolates, an oprI-oprL gene sequence database of 94 Pseudomonas type strains was constructed. Phylogenetic analysis of the concatenated oprI and oprL gene sequences of the Pseudomonas type strains showed that they were largely congruent with the classification based on the MLSA approach based on 16S rRNA, gyrB, rpoB and rpoD gene sequences of Mulet et al. in 2010. Identification of the isolates demonstrated a high diversity of Pseudomonas isolates at the source of the river located in a forest of which most isolates belonged to the Pseudomonas fluorescens lineage. On the other hand, the Pseudomonas population isolated at an anthropized site at the mouth of the river, receiving waste water from both households and industry, was very different and contained many Pseudomonas aeruginosa isolates.
KeywordMeSH Terms
330.     ( 1999 )

Pseudomonas aeruginosa fur overlaps with a gene encoding a novel outer membrane lipoprotein, OmlA.

Journal of bacteriology 181 (4)
PMID : 9973334  :   PMC  :   PMC93485    
Abstract >>
A novel outer membrane lipoprotein in Pseudomonas aeruginosa is encoded by the omlA gene, which was identified immediately upstream of the fur (ferric uptake regulator) gene. The omlA and fur genes were divergently transcribed and had overlapping promoter regions. The proximal fur P2 promoter and the omlA promoter shared a 5-bp DNA motif for their -10 promoter elements. The distal fur P1 promoter was located within the omlA coding sequence, and the omlA and fur T1 mRNAs overlapped by 154 nucleotides. Optimal expression of both fur and omlA required roughly 200 bp of DNA upstream of the promoter regions, suggesting the presence of cis-acting transcriptional activation elements located within the omlA and fur genes, respectively. The levels of Fur and OmlA proteins had no influence on omlA or fur expression, excluding any trans-acting cross-regulation between fur and omlA. Expression of omlA was constitutive regardless of growth phase, oxygen tension, iron concentration, pH, and temperature. OmlA contained a signal sequence typical of bacterial lipoproteins, with a cysteine as a putative cleavage and lipid attachment site. Inhibition of signal peptidase II by globomycin resulted in failure to process OmlA, thus giving strong evidence that OmlA is a lipoprotein. Cell fractionation followed by Western blot analysis indicated that all OmlA protein is localized in the outer membrane. Mature OmlA was an acidic (pI = 4. 5) protein of 17.3 kDa and had close to 40% amino acid sequence identity to SmpA (small protein A) of Escherichia coli, Vibrio cholerae, and Haemophilus influenzae, a protein of unknown function. All P. aeruginosa strains tested as well as Pseudomonas fluorescens were found to produce OmlA. A mutant strain with impaired production of OmlA but no change in the expression of the overlapping fur gene was constructed. The omlA mutant was hypersusceptible to anionic detergents such as sodium dodecyl sulfate and deoxycholate, and it showed increased susceptibility to various antibiotics, including nalidixic acid, rifampin, novobiocin, and chloramphenicol. A structural role of OmlA in maintaining the cell envelope integrity is proposed.
KeywordMeSH Terms
Genes, Bacterial
331.     ( 1998 )

A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365.

Proceedings of the National Academy of Sciences of the United States of America 95 (12)
PMID : 9618537  :   DOI  :   10.1073/pnas.95.12.7051     PMC  :   PMC22735    
Abstract >>
A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the lambda integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe-plant interactions.
KeywordMeSH Terms
Escherichia coli Proteins
Integrases
Mutation
332.     ( 1998 )

Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation.

Molecular microbiology 28 (3)
PMID : 9632260  :   DOI  :   10.1046/j.1365-2958.1998.00821.x    
Abstract >>
mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E. coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild-type E. coli channel, and a C-terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.
KeywordMeSH Terms
Escherichia coli Proteins
333.     ( 1998 )

Sequence diversity of the oprI gene, coding for major outer membrane lipoprotein I, among rRNA group I pseudomonads.

Journal of bacteriology 180 (24)
PMID : 9851998  :   PMC  :   PMC107757    
Abstract >>
The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.
KeywordMeSH Terms
334.     ( 1998 )

Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants.

The Journal of biological chemistry 273 (33)
PMID : 9694855  :   DOI  :   10.1074/jbc.273.33.21031    
Abstract >>
The conserved residues His-162 and Arg-269 of the flavoprotein p-hydroxybenzoate hydroxylase (EC 1.14.13.2) are located at the entrance of the interdomain cleft that leads toward the active site. To study their putative role in NADPH binding, His-162 and Arg-269 were selectively changed by site-specific mutagenesis. The catalytic properties of H162R, H162Y, and R269K were similar to the wild-type enzyme. However, less conservative His-162 and Arg-269 replacements strongly impaired NADPH binding without affecting the conformation of the flavin ring and the efficiency of substrate hydroxylation. The crystal structures of H162R and R269T in complex with 4-hydroxybenzoate were solved at 3.0 and 2.0 A resolution, respectively. Both structures are virtually indistinguishable from the wild-type enzyme-substrate complex except for the substituted side chains. In contrast to wild-type p-hydroxybenzoate hydroxylase, H162R is not inactivated by diethyl pyrocarbonate. NADPH protects wild-type p-hydroxybenzoate hydroxylase from diethylpyrocarbonate inactivation, suggesting that His-162 is involved in NADPH binding. Based on these results and GRID calculations we propose that the side chains of His-162 and Arg-269 interact with the pyrophosphate moiety of NADPH. An interdomain binding mode for NADPH is proposed which takes a novel sequence motif (Eppink, M. H. M., Schreuder, H. A., and van Berkel, W. J. H. (1997) Protein Sci. 6, 2454-2458) into account.
KeywordMeSH Terms
335.     ( 1999 )

Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones.

Applied and environmental microbiology 65 (2)
PMID : 9925571  :   PMC  :   PMC91050    
Abstract >>
A genomic library of Pseudomonas fluorescens DSM 50106 in a lambdaRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43 degreesC. The showed highest purified enzyme activities towards lactones. The activity increased from gamma-butyrolactone (18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.
KeywordMeSH Terms
336.     ( 1998 )

Structural investigation of the cofactor-free chloroperoxidases.

Journal of molecular biology 279 (4)
PMID : 9642069  :   DOI  :   10.1006/jmbi.1998.1802    
Abstract >>
The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A. The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity. Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates. Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites.
KeywordMeSH Terms
337.     ( 1998 )

The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigmaS and the stress response in Pseudomonas fluorescens Pf-5.

Journal of bacteriology 180 (24)
PMID : 9852008  :   PMC  :   PMC107767    
Abstract >>
Three global regulators are known to control antibiotic production by Pseudomonas fluorescens. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation in rpoS, which encodes the stationary-phase sigma factor sigmaS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. The gacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigmaS levels, and oxidative stress response of Pf-5 was determined. We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, sigmaS content in gacS and gacA mutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with an rpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations in gacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.
KeywordMeSH Terms
Genes, Regulator
Oxidative Stress
338.     ( 1998 )

Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and of NADH:ubiquinone oxidoreductase (nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365.

Molecular plant-microbe interactions : MPMI 11 (8)
PMID : 9675892  :   DOI  :   10.1094/MPMI.1998.11.8.763    
Abstract >>
Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.
KeywordMeSH Terms
339.     ( 1998 )

Reverse transcription-PCR analysis of the regulation of ethylbenzene dioxygenase gene expression in Pseudomonas fluorescens CA-4.

FEMS microbiology letters 166 (2)
PMID : 9770272  :   DOI  :   10.1111/j.1574-6968.1998.tb13886.x    
Abstract >>
Pseudomonas fluorescens strain CA-4 is a bioreactor isolate previously characterised by the presence of a side chain oxidation pathway for ethylbenzene breakdown. In this report a second pathway involving ethylbenzene ring dioxygenation has been identified in this strain. We examine here second substrate inhibition of the genes encoding the initial enzymes of this pathway, using reverse transcription (RT)-PCR. The genes of the ring-dioxygenation have been cloned and sequenced. They exhibit near identity to the gene clusters encoding the aromatic ring dioxygenase enzymes of two previously described isopropyl degrading strains, Pseudomonas sp. strain JR1 and P. fluorescens IP01. This dioxygenase pathway appears to be the major pathway for ethylbenzene degradation in this strain. The expression of these genes appears to be affected by the presence of second carbon substrates. Using RT-PCR we demonstrate that the negative effect of glutamate present in the growth medium together with ethylbenzene on the rate of ethylbenzene metabolism is mediated at the transcriptional level on the ethylbenzene dioxygenase genes.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
340.     ( 1998 )

Biochemical and genetic characterization of an extracellular protease from Pseudomonas fluorescens CY091.

Applied and environmental microbiology 64 (3)
PMID : 9501431  :   PMC  :   PMC106346    
Abstract >>
Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl2 or SrCl2 but not in media containing ZnCl2, MgCl2, or MnCl2. The requirement of Ca2+ (or Sr2+) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca2+ and Zn2+ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.
KeywordMeSH Terms
341.     ( 1997 )

Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species.

Applied and environmental microbiology 63 (12)
PMID : 9406411  :   PMC  :   PMC168818    
Abstract >>
Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.
KeywordMeSH Terms
Operon
342.     ( 1998 )

A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365.

Molecular plant-microbe interactions : MPMI 11 (1)
PMID : 9425686  :   DOI  :   10.1094/MPMI.1998.11.1.45    
Abstract >>
We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.
KeywordMeSH Terms
343.     ( 1998 )

The mannitol utilization genes of Pseudomonas fluorescens are regulated by an activator: cloning, nucleotide sequence and expression of the mtlR gene.

Gene 215 (1)
PMID : 9666063  :   DOI  :   10.1016/s0378-1119(98)00274-1    
Abstract >>
A plasmid with the galK gene under control of the promoter of the mannitol utilization genes (mtl) from Pseudomonas fluorescens DSM 50106 was constructed to isolate the mtl regulatory gene. An Escherichia coli galK- mtl- strain with this plasmid was used to screen a genomic library of P. fluorescens for the presence of the regulatory gene by plating on McConkey agar plates supplemented with galactose and mannitol. Clones carrying the regulatory gene were isolated and by complemention assays, deletion analysis and DNA sequencing an open reading frame (mtlR) of 906nt identified encoding the regulator. The deduced protein MtlR with a calculated molecular mass of 34.7kDa showed a low overall similarity to several other regulatory proteins of the XylS/AraC family. When mtlR was cloned and expressed in E. coli, the protein was produced as inclusion bodies. Complete denaturation followed by subsequent slow refolding led to low amounts of active protein. The activity was shown in gel mobility shift assays by binding of MtlR to a DNA fragment containing the promoter/operator region of the P. fluorescens mtl genes.
KeywordMeSH Terms
Escherichia coli Proteins
344.     ( 1998 )

Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding.

European journal of biochemistry 253 (1)
PMID : 9578477  :   DOI  :   10.1046/j.1432-1327.1998.2530194.x    
Abstract >>
The conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3-OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence-comparison studies suggest that Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH.
KeywordMeSH Terms
345.     ( 1997 )

Identification and sequence analysis of the genes encoding a polyketide synthase required for pyoluteorin biosynthesis in Pseudomonas fluorescens Pf-5.

Gene 204 (1��2��)
PMID : 9434161  :   DOI  :   10.1016/s0378-1119(97)00501-5    
Abstract >>
Pyoluteorin is a chlorinated antifungal metabolite of mixed polyketide/amino-acid origin produced by certain strains of Pseudomonas spp., including the soil bacterium Pseudomonas fluorescens Pf-5. Sequence analysis of a gene cluster required for pyoluteorin biosynthesis by Pf-5 (Kraus, J., Loper, J., 1995. Appl. Environ. Microbiol. 61, 849-854) has identified two genes whose deduced peptide sequences exhibit characteristics of both fungal and bacterial Type I polyketide synthases (PKSs). The pyoluteorin PKS does not contain a loading domain that is typically present in bacterial Type I PKSs. Furthermore, this PKS possesses an acyltransferase domain that does not contain the conserved residues surrounding the active-site motif typically found in domains of similar function. Based on the organization of the functional domains within the pyoluteorin PKS, we propose a biosynthetic pathway analogous to non-aromatic polyketide biosynthesis within the Actinomycete bacteria that is responsible for the formation of the resorcinol moiety of pyoluteorin.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
346.     ( 1997 )

Crystal structure of carboxylesterase from Pseudomonas fluorescens, an alpha/beta hydrolase with broad substrate specificity.

Structure (London, England : 1993) 5 (12)
PMID : 9438866  :  
Abstract >>
A group of esterases, classified as carboxylesterases, hydrolyze carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters. One such carboxylesterase from Pseudomonas fluorescens is a homodimeric enzyme, consisting of 218-residue subunits. It shows a limited sequence similarity to some members of the alpha/beta hydrolase superfamily. Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterases is very limited. This study was undertaken in order to provide such information and to understand a structural basis for the substrate specificity of this carboxylesterase. In this study, the crystal structure of carboxylesterase from P. fluorescens has been determined by the isomorphous replacement method and refined to 1.8 A resolution. Each subunit consists of a central seven-stranded beta sheet flanked by six alpha helices. The structure reveals the catalytic triad as Ser 114-His 199-Asp 168. The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 . The inhibitor is covalently attached to Ser 114 of both subunits, with the aromatic ring occupying a hydrophobic site defined by the aliphatic sidechains of Leu23, Ile58, Ile70, Met73 and Val170. No large structural changes are observed between the free and inhibitor-bound structures. Carboxylesterase from P. fluorescens has the alpha/beta hydrolase fold and the Ser-His-Asp catalytic triad. The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue. Three of the active-site loops in each subunit are involved in a head-to-head subunit interaction to form a dimer; it may be these extra structural elements, not seen in other esterases, that account for the inability of carboxylesterase to hydrolyze long chain fatty acids. As a result of dimerization, the active-site clefts from the two subunits merge to form holes in the dimer. The active-site clefts are relatively open and thus the catalytic residues are exposed to the solvent. An oxyanion hole, formed by nitrogen atoms of Leu23 and Gln115, is present in both the free and inhibitor-bound structures. An open active site, as well as a large binding pocket for the acid part of substrates, in P. fluorescens carboxylesterase may contribute to its relatively broad substrate specificity.
KeywordMeSH Terms
Crystallography, X-Ray
347.     ( 1998 )

A seven-gene locus for synthesis of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79.

Journal of bacteriology 180 (9)
PMID : 9573209  :   DOI  :   10113/34349     PMC  :   PMC107199    
Abstract >>
Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kb BglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all phz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the phzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5'-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the phzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes, phzX and phzY, that are homologous to phzA and phzB, respectively, were cloned and sequenced from P. aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.
KeywordMeSH Terms
Genes, Bacterial
348.     ( 1997 )

Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere.

Applied and environmental microbiology 63 (12)
PMID : 9406412  :   PMC  :   PMC168819    
Abstract >>
The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon. DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil. Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats. The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere. This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.
KeywordMeSH Terms
Genes, Bacterial
349.     ( 1998 )

Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens.

Journal of bacteriology 180 (7)
PMID : 9537395  :   PMC  :   PMC107110    
Abstract >>
Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of L-tryptophan to form 7-chloro-L-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-L-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.
KeywordMeSH Terms
Genes, Bacterial
350.     ( 1998 )

Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester.

The Journal of biological chemistry 273 (7)
PMID : 9461612  :   DOI  :   10.1074/jbc.273.7.4163    
Abstract >>
A gene encoding a novel enoyl-SCoA hydratase/lyase enzyme for the hydration and nonoxidative cleavage of feruloyl-SCoA to vanillin and acetyl-SCoA was isolated and characterized from a strain of Pseudomonas fluorescens. Feruloyl-SCoA is the CoASH thioester of ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid), an abundant constituent of plant cell walls and a degradation product of lignin. The gene was isolated by a combination of mutant complementation and biochemical approaches, and its function was demonstrated by heterologous expression in Escherichia coli under the control of a T7 RNA polymerase promoter. The gene product is a member of the enoyl-SCoA hydratase/isomerase superfamily.
KeywordMeSH Terms
351.     ( 1998 )

Structure and function of the genes involved in mannitol, arabitol and glucitol utilization from Pseudomonas fluorescens DSM50106.

Gene 206 (1)
PMID : 9461423  :   DOI  :   10.1016/s0378-1119(97)00574-x    
Abstract >>
A DNA fragment from Pseudomonas fluorescens DSM50106 containing the genes for the uptake and utilization of mannitol, arabitol and glucitol was cloned in Escherichia coli and sequenced. Seven open reading frames (mtlEFGKDYZ) were identified on the 10031 bp fragment. The deduced amino acid sequences of the first four open reading frames (mtlEFGK) revealed significant similarity to the components of the maltose transport system in E. coli and Salmonella typhimurium. The gene mtlD encoding a polyol dehydrogenase was located downstream of mtlK. The deduced proteins of the last two genes on the fragment showed a high similarity to a fructokinase from Vibrio alginolyticus (MtlZ) and a xylulose kinase from Streptomyces rubiginosus (MtlY), respectively. Both genes were expressed in E. coli. MtlZ phosphorylated fructose, glucose and glucitol whereas MtlY was highly specific for xylulose. Upstream of mtlE, a putative promoter/operator region was identified by promoter probe studies which was active in P. fluorescens but not in E. coli.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family

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