Identification and characterization of a bile acid 7 alpha-dehydroxylationoperon in Clostridium sp strain TO-931, a highly active 7 alpha-dehydroxylating strain isolated from human feces

Clostridium sp. strain TO-931 can rapidly convert the primary bile acid cho lic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucl eotide probes were used to isolate a gene fragment encoding a putative bile acid transporter from Clostridium sp, strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene fro m Eubacterium sp. strain VPI 12708, another bile acid-7 alpha-dehydroxylati ng intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from Clostridium sp, strain TO-931 and sequenced. Within this l arger DNA fragment was a 7.9-kb region, containing six successive open read ing frames (ORFs), that was encoded by a single 8.1-kb transcript, as deter mined by Northern blot analysis. The gene arrangement and DNA sequence of t he Clostridium sp, strain TO-931 operon are similar to those of a Eubacteri um sp, strain WI 12708 bile acid-inducible operon containing nine ORFs. Sev eral genes in the Eubacterium sp, strain WI 12708 operon have been shown to encode products required for bile acid 7 alpha-dehydroxylation. In Clostri dium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (Co A) ligase, 3 alpha-hydroxysteroid dehydrogenase, bile acid 7 alpha-dehydrat ase, bile acid-CoA hydrolase, and a bile acid transporter were similar in s ize and exhibited amino acid homology to similar gene products from Eubacte rium sp. strain VPI 12708 (encoded by baiB, baiA, baiE, baiF, and baiG, res pectively). However, no genes similar to Eubacterium sp, strain VPI 12708 b iaH or bail were found in the Clostridium sp, strain TO-931 bai operon, and the two putative Eubacterium sp, strain VPI 12708 genes, baiC and baiD, we re arranged in one continuous ORF in Clostridium sp, strain TO-931, Interge ne regions showed no significant DNA sequence similarity, but primer extens ion analysis identified a region 115 bp upstream from the first ORF that ex hibited 58% identity to a bai operator/promoter region identified in Eubact erium sp, strain VPI 12708, These results indicate that the gene organizati on, gene product amino acid sequences, and promoters of the bile acid-induc ible operons of Clostridium sp, strain TO-931 and Eubacterium sp, strain WI 12708 are highly conserved.