| 1. |
Kobayashi M,
Komeda H,
Yanaka N,
Nagasawa T,
Yamada H,
( 1992 ) Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue. PMID : 1400390 : Abstract >>
The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.
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2. |
Kobayashi M,
Yanaka N,
Nagasawa T,
Yamada H,
( 1992 ) Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue. PMID : 1390687 : DOI : 10.1021/bi00152a042 Abstract >>
Peptides obtained by cleavage of a Rhodococcus rhodochrous K22 nitrilase, which acts on aliphatic nitriles such as acrylonitrile, crotonitrile, and glutaronitrile, have been sequenced. The data allowed the design of oligonucleotide probes which were used to clone a nitrilase encoding gene. Plasmid pNK21, in which 2.05-kb sequence covering the region encoding the nitrilase was was placed under the control of the lac promoter, directed overproduction of enzymatically active nitrilase in response to addition of isopropyl beta-D-thiogalactopyranoside in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of nitrilase was about 40% of the total soluble proteins, leading to the establishment of a simple purification of the nitrilase. The nucleotide sequence of the nitrilase gene predicts a protein composed of 383 amino acids (M(r) = 42,275), including only one cysteine. The amino acid sequence homology between the Rhodococcus nitrilase and the Klebsiella ozaenae bromoxynil nitrilase [Stalker et al. (1988) J. Biol. Chem. 263, 6310-6314] was 38.3%, and a unique cysteinyl residue (Cys-170) in the former nitrilase was conserved at the corresponding position in the latter nitrilase. Cys-170 of the Rhodococcus nitrilase was replaced by Ala or Ser by site-directed mutagenesis. Both mutations resulted in the complete loss of nitrilase activity, clearly indicating that this cysteinyl residue is essential for the catalytic activity.
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3. |
Van Beilen JB,
Mourlane F,
Seeger MA,
Kovac J,
Li Z,
Smits TH,
Fritsche U,
Witholt B,
( 2003 ) Cloning of Baeyer-Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. PMID : 12588297 : Abstract >>
To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.
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4. |
Sone N,
Fukuda M,
Katayama S,
Jyoudai A,
Syugyou M,
Noguchi S,
Sakamoto J,
( 2003 ) QcrCAB operon of a nocardia-form actinomycete Rhodococcus rhodochrous encodes cytochrome reductase complex with diheme cytochrome cc subunit. PMID : 12615356 : DOI : 10.1016/s0005-2728(02)00394-8 Abstract >>
Structural genes encoding quinol-cytochrome c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete Rhodococcus rhodochrous. QcrC and qcrA encode diheme cytochrome cc and the Rieske Fe-S protein, respectively, while the qcrB product is a diheme cytochrome b. These amino acid sequences are similar to those of Corynebacterium and Mycobacterium, the members of high G+C content firmicutes. The presence of diheme cytochrome cc subunit as a sole c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome c oxidase. The N-terminal half of the Rieske Fe-S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved. A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with Thermus, but not with low G+C firmicutes.
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5. |
Seth-Smith HM,
Rosser SJ,
Basran A,
Travis ER,
Dabbs ER,
Nicklin S,
Bruce NC,
( 2002 ) Cloning, sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous. PMID : 12324318 : DOI : 10.1128/aem.68.10.4764-4771.2002 PMC : PMC126434 Abstract >>
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.
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6. |
Hirasawa K,
Ishii Y,
Kobayashi M,
Koizumi K,
Maruhashi K,
( 2001 ) Improvement of desulfurization activity in Rhodococcus erythropolis KA2-5-1 by genetic engineering. PMID : 11302154 : Abstract >>
Rhodococcus erythropolis KA2-5-1 can desulfurize dibenzothiophene (DBT) into 2-hydroxybiphenyl. A cryptic plasmid, pRC4, which was derived from R. rhodochrous IFO3338, was combined with an Escherichia coli vector to construct an E. coli-Rhodococcus shuttle vector. The complete nucleotide sequence of 2582-bp pRC4 was analyzed. Based on the characteristics of its putative replication genes, pRC4 was assigned to the family of pAL5000-related replicons. The desulfurization gene cluster, dszABC, and the related reductase gene, dszD, cloned from KA2-5-1, were reintroduced into KA2-5-1 and efficiently expressed. The DBT desulfurization ability of the transformant carrying two dszABC clusters and one dszD on the vector was about 4-fold higher than that of the parent strain, and the transformant also showed improved desulfurization activity for light gas oil (LGO). Sulfur components in LGO before and after the reaction were analyzed with gas chromatography-atomic emission detection.
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7. |
Itagaki E,
Iwami M,
Sawamoto S,
Yamauchi Y,
( 1999 ) Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme. PMID : 10467180 : DOI : 10.1093/oxfordjournals.jbchem.a022494 Abstract >>
Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.
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8. |
Kulakov LA,
Larkin MJ,
Janssen DB,
( 1999 ) Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family. PMID : 10217489 : DOI : 10.1099/13500872-145-3-561 Abstract >>
A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116. Two copies of IS2112 were found in R. rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the 1-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp. HA1 or in several Rhodococcus strains which do not utilize haloalkanes. IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064, which harbours genes encoding utilization of 1-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA). Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.
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9. |
Degen O,
Eitinger T,
Shimizu S,
( 1999 ) Selective transport of divalent cations by transition metal permeases: the Alcaligenes eutrophus HoxN and the Rhodococcus rhodochrous NhlF. PMID : 10201093 : Abstract >>
nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli. 57CO2+ and 63Ni2+ transport of the recombinants was examined by means of a previously described physiological assay. Although the transporters are highly similar, different preferences for divalent transition metal cations were observed. HoxN was unable to transport 57CO2+, but mediated 63Ni2+ uptake. The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni2+. In contrast, NhlF transported both 57CO2+ and 63Ni2+ ion. NhlF-mediated 63Ni2+ uptake was markedly reduced in the presence of CO2+, while 57CO2+ uptake was only slightly lower in the presence of Ni2+. These results indicate different affinities of NhlF for CO2+ and Ni2+ and identified CO2+ ion as the preferred substrate.
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10. |
Taguchi K,
Motoyama M,
Iida T,
Kudo T,
( 2007 ) Polychlorinated biphenyl/biphenyl degrading gene clusters in Rhodococcus sp. K37, HA99, and TA431 are different from well-known bph gene clusters of Rhodococci. PMID : 17485846 : DOI : 10.1271/bbb.60551 Abstract >>
Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8(K37) gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.
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11. |
Kim YH,
Engesser KH,
Kim SJ,
( 2007 ) Physiological, numerical and molecular characterization of alkyl ether-utilizing rhodococci. PMID : 17504487 : DOI : 10.1111/j.1462-2920.2007.01269.x Abstract >>
Twenty-seven Gram-positive strains were characterized physiologically and numerically and classified them into four groups according to their specific activities for utilization of linear alkyl ethers (AEs), cyclic AEs, monoalkoxybenzenes and 1,4-diethoxybenzene. The comparative analysis of the 16S ribosomal RNA gene and 16S-23S intergenic spacer region showed that they belonged to the genera Rhodococcus and Gordonia. Alkyl ether-utilizing rhodococci appeared to involve various and diverse cytochromes P450 of the families CYP116 (25 positive strains from 27), CYP153 (5/27), CYP249 (1/27) and a new family P450RR1 (27/27). The presence of P450RR1 was strongly related to the specific activity for utilization of 2-methoxyphenol and 2-ethoxyphenol. In addition, 26 of 27 strains contained multiple alkB genes coding for probable non-haem iron containing alkane monooxygenases and hydroxylases. Similar DNA fragments coding for a tetrahydrofuran monooxygenase A subunit (ThmA) were found in all cyclic AE-utilizing strains and nearly identical DNA fragments coding for likely orthologues of a propane monooxygenase A subunit (PrmA) in all linear AE-utilizing strains. The substrate availability in the degradation of aryl AEs, cyclic AEs and linear AEs agreed with the molecular probing of the respective genes encoding cytochrome P450RR1, ThmA and PrmA.
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12. |
Matsui T,
Saeki H,
Shinzato N,
Matsuda H,
( 2007 ) Analysis of the 7.6-kb cryptic plasmid pNC500 from Rhodococcus rhodochrous B-276 and construction of Rhodococcus-E. coli shuttle vector. PMID : 17043815 : DOI : 10.1007/s00253-006-0660-7 Abstract >>
Four circular cryptic plasmids were detected from propene-degrading Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 and the smallest 7.6-kb plasmid, named pNC500 was used to construct Rhodococcus-E. coli shuttle vector, pNC5403. Sequence analysis of pNC500 revealed that the plasmid contains eight potential ORFs, namely 1 through 8. The deduced amino acid sequences for ORFs 3, 4, 6, and 7 show homology with those of Rep A, Rep B, DNA methyl-transferase (M.XamI), and restriction nuclease (R.XamI), respectively. The region responsible for replication in the potent oil-desulfurization bacterium, Rhodococcus opacus T09 was determined as 3.7 kb-XbaI/BalI fragment which contains ORFs 3 and 4, while no transformants were obtained when ORF 4 was partially deleted, suggesting that both are required for its replication. Alignment of the predicted amino acid sequences revealed that ORFs 3 and 4 were DNA binding protein and DNA primase, respectively. A compatibility test with pAL5000-related plasmid vector, pRHK1, which contains pRC4, revealed that pNC5403 was compatible with pRHK1 suggesting that each replication origin would be different. ORFs 3 and 4 containing a pNC5403 derivative, pN5DXB, was stably maintained for over 80 generations in the absence of antibiotic selective conditions.
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13. |
Gao B,
Gupta RS,
( 2005 ) Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria. PMID : 16280504 : DOI : 10.1099/ijs.0.63785-0 Abstract >>
Gram-positive bacteria with a high G+C content are currently recognized as a distinct phylum, Actinobacteria, on the basis of their branching in 16S rRNA trees. Except for an insert in the 23S rRNA, there are no unique biochemical or molecular characteristics known at present that can distinguish this group from all other bacteria. In this work, three conserved indels (i.e. inserts or deletions) are described in three widely distributed proteins that are distinctive characteristics of the Actinobacteria and are not found in any other groups of bacteria. The identified signatures are a 2 aa deletion in cytochrome-c oxidase subunit 1 (Cox1), a 4 aa insert in CTP synthetase and a 5 aa insert in glutamyl-tRNA synthetase (GluRS). Additionally, the actinobacterial specificity of the large insert in the 23S rRNA was also tested. Using primers designed for conserved regions flanking these signatures, fragments of most of these genes were amplified from 23 actinobacterial species, covering many different families and orders, for which no sequence information was previously available. All the 61 sequenced fragments, except two in GluRS, were found to contain the indicated signatures. The presence of these signatures in various species from 20 families within this phylum provides evidence that they are likely distinctive characteristics of the entire phylum, which were introduced in a common ancestor of this group. The absence of all four of these signatures in Symbiobacterium thermophilum suggests that this species, which is distantly related to other actinobacteria in 16S rRNA and CTP synthetase trees, may not be a part of the phylum Actinobacteria. The identified signatures provide novel molecular means for defining and circumscribing the phylum Actinobacteria. Functional studies on them should prove helpful in understanding novel biochemical and physiological characteristics of this group of bacteria.
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14. |
Taguchi K,
Motoyama M,
Kudo T,
( 2004 ) Multiplicity of 2,3-dihydroxybiphenyl dioxygenase genes in the Gram-positive polychlorinated biphenyl degrading bacterium Rhodococcus rhodochrous K37. PMID : 15118304 : DOI : 10.1271/bbb.68.787 Abstract >>
Rhodococcus rhodochrous K37, a Gram-positive bacterium grown under alkaline conditions, was isolated for its ability to metabolize PCBs. Analysis revealed that it has eight genes encoding extradiol dioxygenase, which has 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and these genes were designated bphC1 to bphC8. According to the classification of extradiol dioxygenases [Eltis, L. D., and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)], BphC3 and BphC6 belong to the type II enzyme group. The other six BphCs were classified as members of the type I extradiol dioxygenase group. BphC4 and BphC8 were classified into a new subfamily of type I, family 3. Two linear plasmids, 200 kb and 270 kb in size, were found in K37, and the bphC6 and bphC8 genes were located in the 200 kb linear plasmid. Northern hybridization analysis revealed that the bphC1, bphC2, and bphC7 genes were induced in the presence of testosterone, the bphC6 gene was induced by fluorene, and the bphC8 gene was induced by biphenyl. All eight BphC products exhibited much higher substrate activity for 2,3-dihydroxybiphenyl than for catechol, 3-methylcatechol, or 4-methylcatechol.
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15. |
Portevin D,
De Sousa-D'Auria C,
Houssin C,
Grimaldi C,
Chami M,
Daffé M,
Guilhot C,
( 2004 ) A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. PMID : 14695899 : DOI : 10.1073/pnas.0305439101 PMC : PMC314182 Abstract >>
Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.
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16. |
Wilbrink MH,
Petrusma M,
Dijkhuizen L,
van der Geize R,
( 2011 ) FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains. PMID : 21602385 : DOI : 10.1128/AEM.00380-11 PMC : PMC3127730 Abstract >>
The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode �]-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9�\-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32�GfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32�GfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.
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17. |
Kong F,
Wang H,
Zhang E,
Sintchenko V,
Xiao M,
Sorrell TC,
Chen X,
Chen SC,
( 2010 ) secA1 gene sequence polymorphisms for species identification of Nocardia species and recognition of intraspecies genetic diversity. PMID : 20810768 : DOI : 10.1128/JCM.01113-10 PMC : PMC3020853 Abstract >>
Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5'-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.
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18. |
Nagasawa T,
Takeuchi K,
Yamada H,
( 1991 ) Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1. PMID : 2013281 : DOI : 10.1111/j.1432-1033.1991.tb15853.x Abstract >>
A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.
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19. |
Petrusma M,
Dijkhuizen L,
van der Geize R,
( 2009 ) Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9alpha-hydroxylase, a two-component iron-sulfur-containing monooxygenase with subtle steroid substrate specificity. PMID : 19561185 : DOI : 10.1128/AEM.00066-09 PMC : PMC2725467 Abstract >>
This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9alpha-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9alpha-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5alpha-Eta, 5beta-Eta, Delta1, and Delta4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid Delta1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.
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20. |
Ros?oniec KZ,
Wilbrink MH,
Capyk JK,
Mohn WW,
Ostendorf M,
van der Geize R,
Dijkhuizen L,
Eltis LD,
( 2009 ) Cytochrome P450 125 (CYP125) catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcus jostii RHA1. PMID : 19843222 : DOI : 10.1111/j.1365-2958.2009.06915.x PMC : PMC5218833 Abstract >>
The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3-hydroxysterols such as cholesterol, but not on 3-oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side-chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side-chain. The RG32 Omega cyp125 mutant failed to transform the side-chain of cholesterol, but degraded that of 5-cholestene-26-oic acid-3beta-ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125(RHA1) was < 10% in the low-spin state, cholesterol (K(D)(app) = 0.20 +/- 0.08 microM), 5 alpha-cholestanol (K(D)(app) = 0.15 +/- 0.03 microM) and 4-cholestene-3-one (K(D)(app) = 0.20 +/- 0.03 microM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26-carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.
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21. |
Táncsics A,
Szoboszlay S,
Kriszt B,
Kukolya J,
Baka E,
Márialigeti K,
Révész S,
( 2008 ) Applicability of the functional gene catechol 1,2-dioxygenase as a biomarker in the detection of BTEX-degrading Rhodococcus species. PMID : 18479346 : DOI : 10.1111/j.1365-2672.2008.03832.x Abstract >>
Catechol 1,2-dioxygenase is a key enzyme in the degradation of monoaromatic pollutants. The detection of this gene is in focus today but recently designed degenerate primers are not always suitable. Rhodococcus species are important members of the bacterial community involved in the degradation of aromatic contaminants and their specific detection could help assess functions and activities in the contaminated environments. To reach this aim, specific PCR primer sets were designed for the detection of Rhodococcus related catechol 1,2-dioxygenase genes. Primers were tested with genetically well-characterized strains isolated in this study and community DNA samples were used as template for Rhodococcus specific PCR as well. The sequences of the catabolic gene in question were subjected to multiple alignment and a phylogenetic tree was created and compared to a 16S rRNA gene based Rhodococcus tree. A strong coherence was observed between the phylogenetic trees. The results strongly support the opinion that there was no recent lateral gene transfer among Rhodococcus species in the case of catechol 1,2-dioxygenase. In gasoline contaminated environments, aromatic hydrocarbon degrading Rhodococcus populations can be identified based upon the detection and sequence analysis of catechol 1,2-dioxygenase gene.
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22. |
Kobayashi M,
Nishiyama M,
Nagasawa T,
Horinouchi S,
Beppu T,
Yamada H,
( 1991 ) Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1. PMID : 1840499 : DOI : 10.1016/0167-4781(91)90208-4 Abstract >>
Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.
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23. |
Luo H,
Fan L,
Chang Y,
Ma J,
Yu H,
Shen Z,
( 2010 ) Gene cloning, overexpression, and characterization of the nitrilase from Rhodococcus rhodochrous tg1-A6 in E. coli. PMID : 18677653 : DOI : 10.1007/s12010-008-8324-y Abstract >>
A DNA fragment containing the entire coding sequence of nitrilase gene was amplified from Rhodococcus rhodochrous tg1-A6 with high nitrilase activity using PCR and sequenced. The open reading frame of the nitrilase gene contains 1,101 base pairs, which encodes a putative polypeptide of 366 amino acid residues. The nitrilase gene was cloned into an expression vector pET-28a and expressed in an Escherichia coli strain BL21(DE3). The enzymatic activity of nitrilase, which converts various nitriles to the corresponding carboxylic acids, was detected to reach 24.5 U/ml at 9 h in the recombinant bacteria.
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24. |
Liu Y,
Shen Y,
Qiao Y,
Su L,
Li C,
Wang M,
( 2016 ) The effect of 3-ketosteroid-�G(1)-dehydrogenase isoenzymes on the transformation of AD to 9�\-OH-AD by Rhodococcus rhodochrous DSM43269. PMID : 27377798 : DOI : 10.1007/s10295-016-1804-0 Abstract >>
Rhodococcus rhodochrous DSM43269 is well known for its 3-ketosteroid-9�\-hydroxylases. However, the function of its 3-ketosteroid-�G(1)-dehydrogenases (KSDD) remains unknown. This study compared the involvement of ksdds in the strain's androst-4-ene-3,17-dione (AD) transformation via gene deletion. The conversion was performed using AD as substrate or directly with 9�\-hydroxyandrost-4-ene-3,17-dione (9�\-OH-AD). The single deletion of ksdd1 or ksdd3 did not appear to result in the accumulation of 9�\-OH-AD, whereas the single mutant ��ksdd2 could preserve this compound to some extent. To further compare the role of ksdds in this strain, double mutants were constructed. All ksdd2 mutants combined with ksdd1 and/or ksdd3 resulted in the accumulation of 9�\-OH-AD, among which the double mutant ��ksdd2,3 behaved similarly to the single mutant ��ksdd2 in this process. The mutant that lacked both ksdd1 and ksdd3 was still displayed, with no effect on the degradation of 9�\-OH-AD. The triple mutant ��ksdd1,2,3 was then constructed and exhibited the same capability as ��ksdd1,2, accumulating more 9�\-OH-AD than ��ksdd2,3 and ��ksdd2. The transcription of KSDD1 and KSDD2 increased, whereas that of KSDD3 seemed to exhibit no change, despite the use of the inducer AD or 9�\-OH-AD. Thus, only ksdd1 and ksdd2 were involved in the transformation of AD to 9�\-OH-AD. ksdd2 had the main role, ksdd1 had a minor effect on 9�\-OH-AD degradation, and ksdd3 did not exhibit any action in this course.
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25. |
Táncsics A,
Benedek T,
Szoboszlay S,
Veres PG,
Farkas M,
Máthé I,
Márialigeti K,
Kukolya J,
Lányi S,
Kriszt B,
( 2015 ) The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus. PMID : 25466921 : DOI : 10.1016/j.syapm.2014.10.010 Abstract >>
Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus.
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26. |
Casabon I,
Swain K,
Crowe AM,
Eltis LD,
Mohn WW,
( 2014 ) Actinobacterial acyl coenzyme A synthetases involved in steroid side-chain catabolism. PMID : 24244004 : DOI : 10.1128/JB.01012-13 PMC : PMC3911158 Abstract >>
Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for �]-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 �� 10(5) �� 0.03 �� 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1�](2'-propanoate)-3a�\-H-4�\(3?-propanoate)-7a�]-methylhexahydro-5-indanone (kcat/Km = 2.4 �� 10(5) �� 0.1 �� 10(5) M(-1) s(-1) and 3.2 �� 10(5) �� 0.3 �� 10(5) M(-1) s(-1), respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.
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27. |
Penfield JS,
Worrall LJ,
Strynadka NC,
Eltis LD,
( 2014 ) Substrate specificities and conformational flexibility of 3-ketosteroid 9�\-hydroxylases. PMID : 25049233 : DOI : 10.1074/jbc.M114.575886 PMC : PMC4162158 Abstract >>
KshA is the oxygenase component of 3-ketosteroid 9�\-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >10(5) s(-1) M(-1) for 4-estrendione, 5�\-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (Ki S ?100 �gM). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb�PADD, KshA1�P1,4-BNC-CoA, KshA5�PADD, and KshA5�P1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue "mouth loop," which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and �\-helices harboring its ligands were displaced up to 4.4 ? in the KshA5�Psubstrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.
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28. |
Franceschini S,
van Beek HL,
Pennetta A,
Martinoli C,
Fraaije MW,
Mattevi A,
( 2012 ) Exploring the structural basis of substrate preferences in Baeyer-Villiger monooxygenases: insight from steroid monooxygenase. PMID : 22605340 : DOI : 10.1074/jbc.M112.372177 PMC : PMC3391149 Abstract >>
Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP(+)-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP(+) is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.
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29. |
( 1997 ) Cryptic plasmid pKA22 isolated from the naphthalene degrading derivative of Rhodococcus rhodochrous NCIMB13064. PMID : 9281496 : DOI : 10.1006/plas.1997.1293 Abstract >>
Cryptic plasmids were found in Rhodococcus rhodochrous NCIMB13064 derivatives which had lost the ability to utilize short-chain 1-chloroalkanes (chain length C3-C10) and had acquired the ability to degrade naphthalene. The reversions of these derivatives to the original phenotype were accompanied by the loss of the cryptic plasmids. The 4969-bp pKA22 plasmid was cloned in Escherichia coli and sequenced. This plasmid encodes a putative 33,200-Da protein which contains motifs typical of theta replicase proteins and shows a high degree of similarity to a putative theta replicase from Brevibacterium linens plasmid pRBL1 and to a putative protein encoded by ORF1 of the plasmid pAL5000 from Mycobacterium fortuitum. Two sets of long direct repeats were found in pKA22 which may be involved in the replication of the plasmid and recombination processes.
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30. |
Petrusma M,
Hessels G,
Dijkhuizen L,
van der Geize R,
( 2011 ) Multiplicity of 3-Ketosteroid-9�\-Hydroxylase enzymes in Rhodococcus rhodochrous DSM43269 for specific degradation of different classes of steroids. PMID : 21642460 : DOI : 10.1128/JB.00274-11 PMC : PMC3147496 Abstract >>
The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9�\-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshA(DSM43269) homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshA(DSM43269) homologues are C-9 �\-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that kshA5 supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple kshA genes in the R. rhodochrous DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 �\-hydroxylation occurring at different levels during microbial steroid degradation.
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31. |
( 1997 ) Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors. PMID : 9353918 : DOI : 10.1099/00221287-143-10-3137 Abstract >>
The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.
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32. |
( 1996 ) Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase. PMID : 8855219 : DOI : 10.1073/pnas.93.20.10572 PMC : PMC38194 Abstract >>
The 1.4-kb downstream region from a nitrilase gene (nitA) of an actinomycete Rhodococcus rhodochrous J1, which is industrially in use, was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis in experiments using a Rhodococcus-Escherichia coli shuttle vector pK4 in a Rhodococcus strain. Sequence analysis of the 1.4-kb region revealed the existence of an open reading frame (nitR) of 957 bp, which would encode a protein with a molecular mass of 35,100. Deletion of the central and 3'-terminal portion of nitR resulted in the complete loss of nitrilase activity, demonstrating that nitR codes for a transcriptional positive regulator in nitA expression. The deduced amino acid sequence of nitR showed similarity to a positive regulator family including XylS from Pseudomonas putida and AraC from E. coli. By Northern blot analysis, the 1.4-kb transcripts for nitA were detected in R. rhodochrous J1 cells cultured in the presence of isovaleronitrile, but not those cultured in the absence of isovaleronitrile. The transcriptional start site for nitA was mapped to a C residue located 26 bp upstream of its translational start site. Deletion analysis to define the nitA promoter region suggested the possible participation of an inverted repeat sequence, centered on base pair -52, in induction of nitA transcription.
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33. |
( 1997 ) Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis. PMID : 9226278 : DOI : 10.1128/jb.179.14.4635-4638.1997 PMC : PMC179304 Abstract >>
Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.
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34. |
( 1997 ) A novel transporter involved in cobalt uptake. PMID : 8990157 : DOI : 10.1073/pnas.94.1.36 PMC : PMC19232 Abstract >>
Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57Co in their cells, but not of 63Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.
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35. |
( 1997 ) The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064. PMID : 9025284 : DOI : 10.1099/00221287-143-1-109 Abstract >>
The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhlA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13,064 dehalogenase. A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10. Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected. The analysis of NCIMB 13,064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid. It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R. rhodochrous NCIMB 13064. A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.
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36. |
( 1996 ) Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1. PMID : 8633053 : DOI : 10.1073/pnas.93.9.4267 PMC : PMC39524 Abstract >>
The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).
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37. |
( 1996 ) A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction product. PMID : 8662959 : DOI : 10.1074/jbc.271.26.15796 Abstract >>
The 3.5 kilobases (kb) of the 5'-upstream region from nhlBA encoding a cobalt-containing low molecular mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 was found to be required for the amide-dependent expression of nhlBA in experiments using a Rhodococcus transformation system. Sequence analysis of the 3.5-kb fragment revealed the presence of two open reading frames (nhlD and nhlC) in this fragment. NhlD has similarity to regulators MerR, CadC, and ArsR. NhlC has similarity to the regulators AmiC, for the expression of an aliphatic amidase from Pseudomonas aeruginosa, and NhhC, for the expression of a high molecular mass nitrile hydratase from R. rhodochrous J1. Assays of NHase activity of transformants carrying nhlD deletion or nhlC deletion mutations suggest a negative regulatory role for nhlD and a positive regulatory role for nhlC in the process of the L-NHase formation. Assays of NHase and amidase activities and Western blot analyses of each Rhodococcus transformant carrying various deletion plasmids, have shown that nhlBA and amdA encoding an amidase, which is located 1.9 kb downstream of nhlBA, were regulated in the same manner. These findings present the genetic evidence for a novel gene cluster controlling the expression of L-NHase, which is induced by the reaction product (amide) in the "practical microorganism" R. rhodochrous J1.
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38. |
( 1993 ) Purification and characterization of cytochrome P450RR1 from Rhodococcus rhodochrous. PMID : 8477696 : DOI : 10.1111/j.1432-1033.1993.tb17750.x Abstract >>
A soluble cytochrome P450 whose synthesis is induced by and that binds 2-ethoxyphenol was purified to apparent homogeneity from Rhodococcus rhodochrous strain 116. The enzyme had a subunit molecular mass of 44.5 kDa as determined by SDS/PAGE and a pI of 5.2. The electronic absorption spectrum indicates that the native cytochrome in the absence of substrate is predominantly in the low-spin state (13% high-spin state in 50 mM Mops, pH 7.0 25 degrees C). 2-Methoxyphenol binds to the cytochrome with a macroscopic dissociation constant of 0.53 +/- 0.03 microM (50 mM Mops, pH 7.0, 25 degrees C) and induces a 99.7% transition of the heme iron to the pentacoordinate high-spin form. Using a reconstituted in-vitro activity assay, it was demonstrated that P450RR1 catalyzed the O-dealkylation of 2-ethoxyphenol and 2-methoxyphenol to produce catechol. The cytochrome binds other ortho-substituted phenols, including 2-ethoxyphenol, 2-methylphenol (o-cresol) and 2-chlorophenol. The affinity of P450RR1 for these compounds is lower than that of 2-methoxyphenol and they are less effective than 2-methoxyphenol at inducing a transition in the heme iron to the high-spin state. Para-substituted and meta-substituted ether phenols did not induce a spin transition.
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39. |
Miyamoto M,
Matsumoto J,
Iwaya T,
Itagaki E,
( 1995 ) Bacterial steroid monooxygenase catalyzing the Baeyer-Villiger oxidation of C21-ketosteroids from Rhodococcus rhodochrous: the isolation and characterization. PMID : 7669800 : DOI : 10.1016/0167-4838(95)00090-h Abstract >>
Steroid monooxygenase from Rhodococcus rhodochrous, isolated in homogeneity with a high yield, catalyzes Baeyer-Villiger oxidation of progesterone to produce testosterone acetate with the stoichiometric consumptions of NADPH and molecular oxygen. It is a flavoenzyme with the molecular size of 60 kDa in the monomeric form and the isoelectric point of 4.9. The absorption spectrum has the maxima at 278, 376, and 439 nm and the shoulders at 360 and 465 nm, indicating a strong hypsochromic shift (blue-shift) of the absorption peak in the visible wavelength region. The prosthetic group of the enzyme was identified to be FAD, and the Kd value was estimated to be 0.95 microM. The enzyme catalyzed only the oxidative esterification of progesterone, 11 alpha- and 11 beta-hydroxyprogesterone and not the oxidative lactonization of androstenedione. Km for progesterone was 100 microM, for NADPH was 3.3 microM, and the turnover number was 185 min-1. Kd values for progesterone, 11 alpha-hydroxyprogesterone, deoxycorticosterone, and androstenedione were 110, 130, 2000, and 450 microM, respectively. The optimum pH of the reaction was about 8.5. The reaction was inhibited competitively by 17 alpha-hydroxyprogesterone and androstenedione. Amino terminal sequences of the enzymes from the bacterium and also from fungus, Cylindrocarpon radiocicola were considerably different, and the potential flavin-binding site could be detected on the amino-terminal region of the fungus enzyme but not on that of the bacterial enzyme. Western blotting analyses of the two steroid monooxygenases resulted that mouse antiserum raised for each enzyme reacted only with the antigenic enzyme protein but did not show the cross-reactions. It is clarified that bacterial steroid monooxygenase is distinctly different from the fungal enzyme in the molecular and enzymic properties.
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( 1993 ) Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product. PMID : 7916690 : DOI : 10.1111/j.1432-1033.1993.tb18250.x Abstract >>
The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-determined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi. The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.
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( 2012 ) Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM 43269. PMID : 23059973 : DOI : 10.1099/mic.0.059501-0 Abstract >>
A previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of Rhodococcus rhodochrous, which selectively degrades sterol side-chains. The ltp3 and ltp4 deletion mutants were each completely blocked in side-chain degradation of �]-sitosterol and campesterol, but not of cholesterol. These results indicated a role for ltp3 and ltp4 in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in �]-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side-chain carbons of �]-sitosterol was previously shown to proceed via aldolytic cleavage rather than by �]-oxidation. Our results therefore suggest that ltp3 and ltp4 probably encode aldol-lyases rather than thiolases. This is the first report, to our knowledge, on the molecular characterization of genes with specific and essential roles in carbon-carbon bond cleavage of C24-branched chain sterols in Rhodococcus strains, most likely acting as aldol-lyases. The results are a clear contribution to our understanding of sterol degradation in actinobacteria.
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( 2013 ) A comparative study of nitrilases identified by genome mining. PMID : 23475593 : DOI : 10.1007/s12033-013-9656-6 Abstract >>
Escherichia coli strains expressing different nitrilases transformed nitriles or KCN. Six nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetonitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising nitrilases for the transformation of industrially important substrates were found: the nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the nitrilases from N. crassa and A. niger for (R,S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine.
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( 1998 ) 3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme. PMID : 9792929 : DOI : 10.1093/oxfordjournals.jbchem.a022195 Abstract >>
The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.
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( 1998 ) Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene. PMID : 9677336 : DOI : 10.1042/bj3330741 PMC : PMC1219640 Abstract >>
A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria.
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( 1998 ) Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains. PMID : 9579069 : DOI : 10.1099/00221287-144-4-955 Abstract >>
Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. 11 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.
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