The long range goal of these studies is to increase our knowledge of bile salt metabolism in the body. I propose to use specific intestinal bacteria as model systems to help us address several basic questions in the arena of Bile Acid Molecular Biology. For example, how does a bile acid molecule activate or repress a gene? What constitutes a bile acid binding site or domain on a protein? The specific aims of this proposal are: 1) Elucidate the biochemical mechanism of bile acid 7-dehydroxylation in the intestinal Eubacterium sp. VPI 12708; 2) clone, characterize and determine the nucleotide sequence of a cholic acid inducible operon that appears to code for the polypeptides that are involved in bile acid 7-dehydroxylation; 3) purify and characterize the cholic acid inducible NADH:flavin oxidoreductase and cholic acid binding activities; 4) clone and determine the nucleotide sequence of the bile salt hydrolase gene from Clostridium perfringens. The mechanism of 7-dehydroxylation will be elucidated by demonstrating the proposed intermediates in this pathway and by using custom synthesized radiolabeled bile acids as substrates for 7- dehydroxylation will be elucidated by demonstrating the proposed intermediates in this pathway and by using custom synthesized radiolabeled bile acid metabolism will be cloned into lambda gt-11 or lambda-DASH bacteriophage vectors, detected using synthetic DNA probes and sequenced by the Sanger dideoxy technique. NADH:flavin oxidoreductase and cholic acid binding activities will be purified using DEAE-cellulose, gel filtration, cibacron blue and DEAE-HPLC techniques. In regard to health relatedness, the gut flora generates 20 to 25% of the biliary bile acid pool (secondary bile acids). We have shown in the rat that the hydrophobic secondary bile acids may be important regulators of hepatic cholesterol and bile acid metabolism. Dissolution of cholesterol gallstones by chenodeoxycholic acid and ursodeoxycholic acid is complicated by intestinal bile acid 7-dehydroxylation. If our proposed new mechanism of 7-dehydroxylation is correct, it should allow for a more rational synthesis of bile acid analogs that are resistant to 7-dehydroxylation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040986-03
Application #
3241516
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1989-01-01
Project End
1993-12-31
Budget Start
1991-01-01
Budget End
1991-12-31
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Mallonee, D H; Hylemon, P B (1999) Use of a short A/T-rich cassette for enhanced expression of cloned genes in Escherichia coli. Mol Biotechnol 11:27-35
Doerner, K C; Takamine, F; LaVoie, C P et al. (1997) Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes. Appl Environ Microbiol 63:1185-8
Mallonee, D H; Hylemon, P B (1996) Sequencing and expression of a gene encoding a bile acid transporter from Eubacterium sp. strain VPI 12708. J Bacteriol 178:7053-8
Dawson, J A; Mallonee, D H; Bjorkhem, I et al. (1996) Expression and characterization of a C24 bile acid 7 alpha-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli. J Lipid Res 37:1258-67
Baron, S F; Hylemon, P B (1995) Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli. Biochim Biophys Acta 1249:145-54
Mallonee, D H; Lijewski, M A; Hylemon, P B (1995) Expression in Escherichia coli and characterization of a bile acid-inducible 3 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. Curr Microbiol 30:259-63
de Prada, P; Setchell, K D; Hylemon, P B (1994) Purification and characterization of a novel 17 alpha-hydroxysteroid dehydrogenase from an intestinal Eubacterium sp. VPI 12708. J Lipid Res 35:922-9
Franklund, C V; Baron, S F; Hylemon, P B (1993) Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708. J Bacteriol 175:3002-12
Mallonee, D H; Adams, J L; Hylemon, P B (1992) The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase. J Bacteriol 174:2065-71
Baron, S F; Franklund, C V; Hylemon, P B (1991) Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. J Bacteriol 173:4558-69

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