7-Ketocholesterol Catabolism by
            <i>Rhodococcus jostii</i>
            RHA1

Mathieu, Jacques; Mohn, William W.; Eltis, Lindsay D.; LeBlanc, Justin C.; Stewart, Gord R.; Dresen, Carola; Okamoto, Kenji; Alvarez, Pedro J. J.

doi:10.1128/aem.02538-09

Cited by 29 publications

(31 citation statements)

References 20 publications

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“…It is unclear why in RHA1 cluster 3 is more tightly downregulated in the absence of cholate than cluster 1 in the absence of cholesterol. It is interesting that cluster 3 was also upregulated during growth on 7-keto-cholesterol (7KC) (18). However, only genes in cluster 1 were shown to be essential for 7KC catabolism, and it was concluded that induction of cluster 3 by 7KC is likely gratuitous.…”

Section: Discussionmentioning

confidence: 99%

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

et al. 2012

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bBile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9␣-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. Bile salts are surface-active steroids with an important role in the uptake and metabolism of lipophilic substrates in vertebrates. These steroids, which include cholate and chenodeoxycholate, are synthesized in the liver from cholesterol, and their eventual fate is excretion in feces or urine. Bile salts may be modified, either by microbiological activity in the duodenum or by host cell bioactivity, leading to their conjugation to glycine, taurine, or sulfate. As such, biodegradation of the various bile salts is a significant process in carbon cycling in soil and aquatic environments. The processes involved in microbial transformation of steroids are also relevant for biotechnological applications in the synthesis and/or selective modification of steroid-based drugs (29).Despite the cytotoxicity of cholate toward various prokaryotic and eukaryotic cells, several bacterial species, especially members of the Proteobacteria (27, 28) and Actinomycetales (9, 32), are able to efficiently metabolize this substrate to sustain growth. Recent studies on microbial bile salts degradation have focused on the Proteobacteria. Genes encoding several steps in cholate degradation were identified, mainly in Comamonas testosteroni TA441 and Pseudomonas sp. strain Chol1. In the former strain, genes responsible for oxidation of the steroid nucleus were found (10-13), while in the latter, genes responsible for degradation of the cholate side chain were identified, in...

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Section: Discussionmentioning

confidence: 99%

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

et al. 2012

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“…To test this prediction, we investigated the phenotype of RHA017, an hsaA deletion mutant of RHA1 (30).…”

Section: Resultsmentioning

confidence: 99%

“…Plasmids were electroporated into RHA017 (30) and E. coli using a MicroPulser from Bio-Rad (Hercules, CA) with Bio-Rad 0.1-cm GenePulser Cuvettes. The hsaA (Rv3570c) and hsaB (Rv3567c) genes were amplified from Mtb H37Rv A, cholesterol is transformed to 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) via previous enzymatic steps.…”

Section: Methodsmentioning

confidence: 99%

“…Expression of hsaB was induced when the culture attained an A 600 of 0.6 by adding NaCl to a final concentration of 0.3 M. Cells were further incubated for 16 h, harvested by centrifugation, washed twice with 20 mM sodium phosphate (pH 8.0) containing 10% glycerol, then frozen at Ϫ80°C until use. For metabolite analyses, R. jostii RHA1 ⌬hsaA mutant, RHA017 (30), was grown in W minimal medium (31) supplemented with 0.5 mM cholesterol as described previously (6). To produce HsaA, RHA017 containing pTipHA was grown at 30°C in LB supplemented with 25 g/ml chloramphenicol.…”

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A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Dresen

Lin

D’Angelo

et al. 2010

Journal of Biological Chemistry

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102

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Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-). However, 3,9-DHSA was a poorer substrate than 3-hy-). In the presence of 3-HSA the K mapp for O 2 was 100 ؎ 10 M. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val 367 -Val 394 ) could adopt two conformations differing by a rigid body rotation of 25°around Arg 366 . This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids. Mycobacterium tuberculosis (Mtb),6 the most devastating infectious agent of mortality worldwide (1), remains a primary public health threat due to synergy with the human immunodeficiency virus and the emergence of extensively drug-resistant strains. Mtb has the unusual ability to survive and replicate in the hostile environment of the macrophage (2, 3) utilizing largely unknown mechanisms. Genes essential to intracellular survival have been identified using genome-wide mutagenesis (4). A cluster of such genes was subsequently predicted to specify cholesterol catabolism (5). Targeted gene deletion studies subsequently indicated that cholesterol catabolism occurs throughout infection, contributing to the virulence of Mtb (6 -8). Although the exact role of cholesterol in infection remains unclear, the gene deletion studies are consistent with the high concentrations of cholesterol found within caseating granulomas and the observed congregation of bacteria around cholesterol foci (9).The cholesterol catabolic pathway of Mtb is very similar to that of other pathogenic and environmental actinomycetes (10), as initially predicted in Rhodococcus jostii RHA1 (5). Sidechain degradation is initiate...

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“…5␣-Cholestane (0.25 mM) was used as an internal standard. The extracts were derivatized at room temperature with 200 l bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane and analyzed with an Agilent 6890 series gas chromatograph equipped with an HP-5ms 30-m by 0.25-mm capillary column (Hewlett-Packard) and an HP 5973 mass-selective detector (22). The gas chromatography conditions were as follows: the injector temperature was set at 280°C, the transfer line temperature was 290°C, and the flow rate was 1 ml min Ϫ1 with helium.…”

Section: Transporters For Reassimilation Of Cholate Metabolitesmentioning

confidence: 99%

Two Transporters Essential for Reassimilation of Novel Cholate Metabolites by Rhodococcus jostii RHA1

et al. 2012

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The bacterial uptake of steroids and their metabolites remains poorly understood. We investigated two transporters associated with cholate catabolism in Rhodococcus jostii RHA1. Reverse transcriptase quantitative-PCR indicated that an ATP-binding cassette (ABC) transporter and a major facilitator superfamily (MFS) transporter were upregulated 16.7-and 174-fold, respectively, during the exponential phase of growth on cholate compared to growth on pyruvate. Gene knockout analysis established that these transporters are required for the reassimilation of distinct metabolites that accumulate during growth on cholate. The ABC transporter, encoded by camABCD, was essential for uptake of 1␤(2=-propanoate)-3a␣-H-4␣(3؆(R)-hydroxy-3؆-propanoate)-7a␤-methylhexahydro-5-indanone and a desaturated analog. The MFS transporter, encoded by camM, was essential for uptake of 3,7(R),12(S)-trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oate. These metabolites differ from cholate metabolites reported to be excreted by proteobacteria in that they retain an isopropanoyl side chain at C-17. The uptake of these metabolites was necessary for maximal growth on cholate: a ⌬camB mutant lacking the permease component of the ABC transporter and a ⌬camM mutant lacking the MFS transporter grew to 74% and 77%, respectively, of the yield of the wild type. This study demonstrates for the first time the requirement for specific transporters for uptake of cholate metabolites and highlights the importance and complexity of transport processes associated with bacterial steroid catabolism.

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7-Ketocholesterol Catabolism by Rhodococcus jostii RHA1

Cited by 29 publications

References 20 publications

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Two Transporters Essential for Reassimilation of Novel Cholate Metabolites by Rhodococcus jostii RHA1

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