Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708

Abstract

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.

The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase

“…The baiB gene from Eubacterium sp. strain VPI 12708 is contained in a 2.4-kb chromosomal EcoRI fragment that was initially cloned in E. coli Y1090 by using a Xgtll vector (20). This 2.4-kb fragment was subsequently subcloned in E. coli DH5a by using a pUC19 vector (8).…”

“…The genes (baiE and baiF) encoding two other verified bile acid-inducible polypeptides are also included among the six or more open reading frames present in this operon. Most of the enzymes involved in bile acid 7ao-dehydroxylation are thought to be encoded from this operon (20). It has been suggested that the 7-dehydroxylation reaction may provide an ancillary electron acceptor for bacteria capable of expressing this activity (15).…”

“…One of the purified polypeptides, a 27,000-Mr species, is encoded by three separate genes (baiAl, baiA42, and baiA3) (5,6,13,39). One of these three gene copies (baiA2) resides on a large bile acid-inducible operon (20). The genes (baiE and baiF) encoding two other verified bile acid-inducible polypeptides are also included among the six or more open reading frames present in this operon.…”

“…strain VPI polypeptides all involve the ATP-dependent linking of AMP or coenzyme A (CoA) to cyclic carboxylated compounds. It has therefore been suggested (20) that the baiB gene encodes an enzyme that activates bile acids as the initial step in the 7a-dehydroxylation pathway. In this study, we expressed the cloned baiB gene in Escherichia coli and analyzed its expressed activities.…”

“…This bacterium synthesizes several new polypeptides after cholic acid induction (24,36). Three of these inducible polypeptides have been purified, and the genes encoding them have been cloned and sequenced (5,6,20,38). One of the purified polypeptides, a 27,000-Mr species, is encoded by three separate genes (baiAl, baiA42, and baiA3) (5,6,13,39).…”