Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

Abstract

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...

Degradation of the Acyl Side Chain of the Steroid Compound Cholate in Pseudomonas sp. Strain Chol1 Proceeds via an Aldehyde Intermediate

“…Nevertheless, it is still unclear whether substrate-specific CoA ligases act on the free acids or whether only one or two ligases with broad substrate specificities are involved. Recently, two CoA ligases, CasG and CasI, specific for the CoA activation of cholate and of DHOPDC-like steroids, respectively, were identified in R. jostii (33). As CoA activation is an energy-consuming process, it is also reasonable that CoA transferases could be involved, reducing the demand for ATP during the side chain degradation of steroid compounds.…”

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

“…In culling, characterized enzymes were preferentially retained, as were enzymes encoded by genes linked to characterized steroid degradation pathways. In addition, sequences from Thermomonospora curvata were included, since this strain harbors a cholate gene cluster (25). Similarly, the chromosome of Salinispora tropica contains an hsa operon and a kshA ortholog in the vicinity of the fadD3 and fadD19 homologs.…”

“…Attempts to remove the affinity tag by using factor Xa (Boehringer) resulted in FadD19 degradation. CasG was purified as previously described (25).…”

“…In the cholate cluster, two genes encoding acyl-CoA synthetases were recently described: casG and casI. Purified CasG was demonstrated to be a cholyl-CoA synthetase capable of forming CoA thioesters with steroids harboring a C 5 side chain, while CasI transformed isopropanoate-bearing steroid metabolites (25). These enzymes were not further characterized.…”

“…In RHA1, separate gene clusters encode cholate catabolism (25) and cholesterol catabolism (26) (see Fig. S1 in the supplemental material).…”