Identification and Characterization of a Bile Acid 7α-Dehydroxylation Operon in Clostridium sp. Strain TO-931, a Highly Active 7α-Dehydroxylating Strain Isolated from Human Feces

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Abstract
Clostridium sp. strain TO-931 can rapidly convert the primary bile acid cholic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucleotide probes were used to isolate a gene fragment encoding a putative bile acid transporter fromClostridium sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene fromEubacterium sp. strain VPI 12708, another bile acid-7α-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned fromClostridium sp. strain TO-931 and sequenced. Within this larger DNA fragment was a 7.9-kb region, containing six successive open reading frames (ORFs), that was encoded by a single 8.1-kb transcript, as determined by Northern blot analysis. The gene arrangement and DNA sequence of the Clostridium sp. strain TO-931 operon are similar to those of a Eubacterium sp. strain VPI 12708 bile acid-inducible operon containing nine ORFs. Several genes in theEubacterium sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7α-dehydroxylation. InClostridium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3α-hydroxysteroid dehydrogenase, bile acid 7α-dehydratase, bile acid-CoA hydrolase, and a bile acid transporter were similar in size and exhibited amino acid homology to similar gene products from Eubacterium sp. strain VPI 12708 (encoded by baiB, baiA, baiE,baiF, and baiG, respectively). However, no genes similar to Eubacterium sp. strain VPI 12708biaH or baiI were found in theClostridium sp. strain TO-931 bai operon, and the two putative Eubacterium sp. strain VPI 12708 genes,baiC and baiD, were arranged in one continuous ORF in Clostridium sp. strain TO-931. Intergene regions showed no significant DNA sequence similarity, but primer extension analysis identified a region 115 bp upstream from the first ORF that exhibited 58% identity to a bai operator/promoter region identified in Eubacterium sp. strain VPI 12708. These results indicate that the gene organization, gene product amino acid sequences, and promoters of the bile acid-inducible operons ofClostridium sp. strain TO-931 and Eubacteriumsp. strain VPI 12708 are highly conserved.