Gene cluster encoding cholate catabolism in Rhodococcus spp

Abstract

Bile 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-9α-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.

Fig 1

Growth of RHA1 and ΔkshA3 on cholate. RHA1 (solid symbols) and ΔkshA3 (open symbols) were grown in mineral medium supplemented with 2.0 mM cholate at 30°C. The error bars indicate the range in duplicate experiments.

Fig 2

Gene clusters in RHA1 encoding steroid catabolism. (A) Red regions indicate locations of gene clusters within the 9.7-Mb genome, comprising one linear chromosome plus three linear plasmids. (B) Organization and expression of genes.

Fig 3

RT-QPCR analysis of the expression of 10 genes during growth of RHA1 on three substrates. The error bars show standard deviations for triplicate cultures.

Fig 4

Transformation of cholate to cholyl-CoA by CasG. Dotted chromatogram, CasG reaction after 2 h; solid chromatogram, no-enzyme control after 2 h. Substrate peaks: 1, ATP; 3, CoASH. Product peaks: 2, AMP; 4, cholyl-CoA. Cholate is not detected under these conditions.

Fig 5

Dendrogram showing the phylogeny of HsaA homologs. AMED, Amycolatopsis mediterranei U32; AS9A, Amycolicicoccus subflavus DQS3-9A1; Gpol, Gordonia polyisoprenivorans VH2; KTR9, Gordonia sp. KTR9; MAB, Mycobacterium abscessus ATCC 19977; MAV, Mycobacterium avium 104; MSMEG, M. smegmatis strain MC2 155; Mtb, M. tuberculosis H37Rv; Mvan, Mycobacterium vanbaalenii PYR-1; nfa, Nocardia farcinica IFM 10152; RHA1, R. jostii RHA1; REQ, R. equi 103S; RER, R. erythropolis PR4; Strop, Salinispora tropica CNB-440; Tbis, Thermobispora bispora DSM 43833; Tcur, T. curvata DSM 43183; Tpau, Tsukamurella paurometabola DSM 20162. The following sequences from R. opacus B4 were omitted from the analysis due to their high amino acid identities with the RHA1 orthologs: ROP_44540 (99% identity with HsaA_RHA1), ROP_22160 (98% identity with HsaA2_RHA1), ROP_58630 (97% identity with HsaA3_RHA1), ROP_58740 (96% identity with HsaA3b_RHA1), and ROP_51300 (96% identity with RHA1_ro05068). The RER_38340 sequence from R. erythropolis PR4 was omitted from the analysis due to its very high amino acid identity (98%) with HsaA3_Rer.