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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 expresses inducible bile acid 7␣-dehydroxylation activity via a multistep pathway. The genes encoding several of the inducible proteins involved in the pathway have been previously mapped to a bile acid-inducible (bai) operon in Eubacterium sp. strain VPI 12708. We now report the cloning, sequencing, and characterization of the baiG gene, which is part of the bai operon. The predicted amino acid sequence of the BaiG polypeptide shows significant homology to several membrane transport proteins, including sugar and antibiotic resistance transporters, which are members of the major facilitator superfamily. Hydrophilicity plots of BaiG show a high degree of similarity to class K and L TetA proteins from gram-positive bacteria, and, like these classes of TetA proteins, BaiG has 14 proposed transmembrane domains. The baiG gene was cloned into Escherichia coli and shown to confer an energy-dependent bile acid uptake activity. Primary bile acids were preferentially transported into E. coli cells expressing this gene, with at least sevenfold and fourfold increases in the uptake of cholic acid and chenodeoxycholic acid, respectively, over control reactions. Less transport activity was observed with cholylglycine, 7-oxocholic acid, and deoxycholic acid. The transport activity was inhibited by the proton ionophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and nigericin but not by the potassium ionophore valinomycin, suggesting that the transport is driven by the proton motive force across the cell membrane. In summary, we have cloned, sequenced, and expressed a bile acid-inducible bile acid transporter from Eubacterium sp. strain VPI 12708. To our knowledge, this is the first report of the cloning and expression of a gene encoding a procaryotic bile acid transporter.Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses bile acid 7␣-and 7␤-dehydroxylation activity (18,40). The bile acid 7␣-dehydroxylation activity is induced by unconjugated C 24 bile acids which possess a 7␣-OH group and is accompanied by the appearance of several new polypeptides on gels of soluble protein extracts from this bacterium (30, 39). The genes encoding several of these induced polypeptides have been previously mapped to a large bile acid-inducible (bai) operon in Eubacterium sp. strain VPI 12708 (10, 24). The bai operon contains at least nine open reading frames ( Fig. 1) and is assumed to encode a majority of the proteins involved in the bile acid 7␣-dehydroxylation pathway. The complete DNA sequences of baiB, baiC, baiE, baiA2, baiF, and baiH have been reported (10,24,41,42).In the 7␣-dehydroxylation pathway (5), the primary bile acids cholic acid and chenodeoxycholic acid are 7␣-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. The pathway is initiated by conjugation of the primary bile acid to coenzyme A (22). The bile acid-coenzyme A conjugate is sequentially oxidized to a 3-oxo (23) and then a 3-oxo-⌬ 4 bile acid intermediate. Following...

We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp. strain VPI 12708 that encode 27,000-M(r) polypeptides. Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products. We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity. Thin-layer chromatography of 14C-labeled bile acid products from reactions using cell-free extracts revealed a 3 alpha-hydroxysteroid dehydrogenase activity for the BaiA1 protein. The BaiA1 protein could utilize both NAD+ and NADP+, and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid. These results show that the BaiA proteins are novel 3 alpha-hydroxysteroid dehydrogenases.

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