Two bile acid-inducible polypeptides from Eubacterium sp. strain VPI 12708 with molecular weights of 27,000 and approximately 45,000 have previously been shown to be encoded by genes residing on a 2.9-kb EcoRI fragment. We now report the cloning and sequencing of three additional overlapping DNA fragments upstream from this EcoRI fragment. Together, these four fragments contain a large segment of a bile acid-inducible operon which encodes the 27,000- and 45,000-Mr (now shown to be 47,500-Mr) polypeptides and open reading frames potentially coding for four additional polypeptides with molecular weights of 59,500, 58,000, 19,500, and 9,000 to 11,500. A bile acid-inducible polypeptide with an apparent Mr of 23,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity, and the N-terminal amino acid sequence that was obtained matched the sequence deduced from the open reading frame coding for the 19,500-Mr polypeptide. A short DNA segment containing the 3' downstream end of the gene coding for the 47,500-Mr polypeptide was not successfully cloned but was directly sequenced from DNA fragments synthesized by polymerase chain reaction. The mRNA initiation site for the bile acid-inducible operon was shown by primer extension to be immediately upstream from the gene encoding the 58,000-Mr polypeptide. A potential promoter region upstream from the mRNA initiation site displayed significant homology with the promoter regions of previously identified bile acid-inducible genes from Eubacterium sp. strain VPI 12708. We hypothesize that this bile acid-inducible operon codes for most of the enzymes involved in the bile acid 7 alpha-dehydroxylation pathway in this bacterium.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium that has inducible bile acid 7-dehydroxylation activity. At least four new polypeptides were synthesized after addition of primary bile acids to the growth medium. One of these, of molecular weight 27,000 (P-27), was shown to be involved in the 7-dehydroxylation reaction sequence. The gene coding for P-27 was cloned, and the entire DNA sequence for the protein-coding region was determined. In addition, sequence information was obtained for 294 bases upstream from the translational start codon and 329 bases downstream from the stop codon. Induction studies with a synthetic oligonucleotide probe (16-mer) revealed the presence of a cholic acid-inducible mRNA species approximately 900 bases long. A 5' terminus of this mRNA was detected by primer extension analysis, and the location of the 3' terminus of the mRNA was estimated by using Si nuclease mapping. The 3' terminus of the mRNA contained a large element with dyad symmetry of unknown function. The open reading frame contained 249 codons, and the corresponding polypeptide had a calculated molecular weight of 26,745. The amino acid sequence of P-27 showed significant homology to several previously described alcohol-polyol dehydrogenases ("nonzinc" dehydrogenases), especially in the region believed to contain a pyridine nucleotide-binding domain. The implications of this homology and the possible function of P-27 in bile acid 7-dehydroxylation are discussed.

The overlappingfdhA andfdhB genes of Methanobacteriumformicicum, which encode the a and 13 subunits, respectively, of formate dehydrogenase, were cotranscribed as part of an approximately 4.5-kb transcript. An additional gene (fdhC) upstream offdhA was cotranscribed with fdhA and fdhB. The deduced amino acid sequence suggested thatfdhC has the potential to encode a hydrophobic polypeptide with a calculated molecular weight of 29,417. A hydropathy plot of the hypothetical polypeptide indicated several potential membranespanning regions. The putativefdhC gene product had 28% identity with the deduced amino acid sequence of the nirC gene from Salmonella typhimurium. Northern (RNA) blot analyses and primer extension assays located a transcription start site 268 bp upstream of the initiation codon offdhC. A sequence identical to the consensus promoter sequence for methanogenic organisms was situated between -35 and -25 bp from the proposed transcription start site. In addition to the 4.5-kb transcript, Northern blot analyses detected a 1.1-kb transcript with anfdhC-specific probe and a 3.4-kb transcript with either anfdhA-orfdhB-specific probe. The levels of all three transcripts were significantly greater in cells grown in media supplemented with molybdate.Methanobacterium formicicum is an archaeon (32) which utilizes either formate or carbon dioxide and hydrogen as the sole sources of carbon and energy. Formate dehydrogenase catalyzes the oxidation of formate, which supplies electrons for the reduction of carbon dioxide to methane. The formate dehydrogenase of M. fonnicicum is an iron-sulfur enzyme containing molybdopterin guanine dinucleotide and flavin adenine dinucleotide (13,18,25,26). The enzyme contains two subunits with molecular weights of approximately 85,000 and 35,000 (25). The level of formate dehydrogenase protein in the cell is regulated in response to the amount of molybdate in the growth medium (18).The fdhA and fdhB genes, which encode the two subunits of the formate dehydrogenase from M. formicicum, have been cloned and sequenced (27). The deduced amino acid sequence of fdhA, encoding the large subunit, has 40% identity with the deduced amino acid sequence of fdhF, which encodes the 80,000-Da subunit of formate dehydrogenase-H from Escherichia coli (36). The fdhAB genes of M. formicicum overlap by 1 bp, which suggests that they may be cotranscribed as part of a polycistronic operon. Approximately 3.6 kbp upstream of the initiation codon offdhA is a 60-bp sequence with 61% identity to a sequence upstream of the fdhF gene of E. coli (4, 20). It was previously suggested that the fdhA and fdhB genes are cotranscribed as part of an approximately 12-kb transcript initiating within 50 bp downstream of this 60-bp sequence (20). Here, we reexamine the transcription offdhAB and present evidence that these genes are cotranscribed as part of a 4.5-kb transcript. In addition, we report the nucleotide sequence of fdhC, which was cotranscribed with fdhA and fdhB. MATERIALS AND METHODSBacterial strains, bacterio...

(27,28). Deoxycholic and lithocholic acids differ markedly from their 7-hydroxylated precursors in physicochemical properties as well as in physiological effects (3,6,30,33,36,37,41). Deoxycholic acid makes up approximately 20 to 25% of the total bile acid pool in humans (45). Intestinal bactena capable of 7-dehydroxylation ofbile acids have been isolated by several laboratories (15, 28). Most intestinal bacteria possessing 7-dehydroxylation activity have been identified as members of the Clostridium (21,23,47) or Eubacteruim (20,23) genus. It has been demonstrated that the fecal population of 7-dehydroxylating intestinal bacteria in humans and in rats is in the range of 103 to iO cultivable organisms per g (wet weight) of feces.Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium that possesses bile acid 7a-dehydroxylation activity which is induced by culturing the bacterium in the presence of C-24 bile acids containing a 7a-hydroxyl group. At least four new polypeptides with estimated molecular weights of 77,000, 45,000, 27,000, and 23,000 have been * Corresponding author.

Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708.