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

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 from Clostridium sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene from Eubacterium sp. strain VPI 12708, another bile acid-7alpha-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from Clostridium 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 the Eubacterium sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7alpha-dehydroxylation. In Clostridium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3alpha-hydroxysteroid dehydrogenase, bile acid 7alpha-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 12708 biaH or baiI were found in the Clostridium 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 of Clostridium sp. strain TO-931 and Eubacterium sp. strain VPI 12708 are highly conserved.

FIG. 1

Overview of gene identity and organization for bile acid-inducible (bai) operons of Eubacterium sp. strain VPI 12078 and Clostridium sp. strain TO-931. No baiH or baiI gene was found downstream of Clostridium baiG. O/P, operator/promoter; FOR, flavin oxidoreductase; HSDH, hydroxysteroid dehydrogenase.

FIG. 2

Northern blot analysis of the bai operon of Clostridium TO-931. Total RNA was isolated at time zero and at 30 min following addition (+) of cholic acid (50 μM) to the culture medium. In a control culture, bile acid was not added (−). Approximately 10 μg of RNA was loaded onto each lane and probed with the baiCD gene. MW Markers, molecular size marker positions.

FIG. 3

Clustal alignment of peptide sequences for Eubacterium (Eub) sp. strain VPI 12708 bile acid-inducible proteins BaiC, BaiD, and BaiH and Clostridium (Clos.) sp. strain TO-931 bile acid-inducible protein BaiCD. Darkened residues (white letters on black background) denote identity and shaded residues (black letters on gray background) denote similarity between aligned sequences. The putative BaiCD peptide exhibits 46% identity and 51% similarity to BaiH, an enzyme with NADH:flavin oxidoreductase activity. Neither BaiC nor BaiD has been associated with an enzyme function.

FIG. 4

Autoradiograph of primer extension analysis products. Lanes A, C, G, and T represent dideoxy nucleotide termination of Clostridium sp. strain TO-931 DNA sequence reactions. Lanes 60", 30", and 0" denote primer extension of Clostridium sp. strain TO-931 mRNA isolated from cultures at 60, 30, and 0 min, respectively, after induction with 100 μM cholic acid.

FIG. 5

Alignment of bai operon promoter regions from Clostridium sp. strain TO-931 and Eubacterium sp. strain VPI 12708. Arrows denote transcription start sites as determined by primer extension analysis. The conserved regions are shaded with gray. The underlined sequences are the putative promoter binding (−10) sites. Clostridium sp. strain TO-931 DNA also had an inverted repeat (GATA/A/TATC) between the −10 site and the mRNA initiation site.

FIG. 6

Cholic acid 7α-dehydroxylation pathway in intestinal anaerobic bacteria. Bile acid-inducible (bai) gene products that participate in this pathway are indicated (also see Fig. 1). CoASH, coenzyme A-SH.