Darrell H. Mallonee

Expression in Escherichia coli and characterization of a bile acid-inducible 3α-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708

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-Mr 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 3α-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 3α-hydroxysteroid dehydrogenases.

Use of a short A/T-rich cassette for enhanced expression of cloned genes in Escherichia coli.

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from abaiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.

Molecular Biology of Bile Acid 7α-Dehydroxylation in an Intestinal Eubacterium Species

The gastrointestinal tract of adult humans contains as many as 1014 bacteria, which may represent 400 to 500 different species (Moore and Holdeman, 1974; Holdeman et al., 1976). More than 99% of these bacteria are obligate anaerobes. The majority (two-thirds) belong to the following species: Bacteroides spp., Eubacterium aerofaciens, E. eligens, E. biforme, E. rectale, Fusobacterium prausnitzii, Bifidobacterium adolescente, B. longum, Peptostreptococcus productus, and Gemmiger formicilis (Moore and Holdeman, 1974; Holdeman et al., 1976). These bacteria are capable of carrying out a wide variety of biotransformations of both endogenous and exogenous compounds (Goldman, 1978). In contrast to oxidative and conjugative biotransformations that are carried out in the liver, the intestinal microflora tend to hydrolyze and reduce compounds that enter the gastrointestinal tract.