Bile salt biotransformations by human intestinal bacteria

Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7α/β-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion. Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7α/β-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion. The human large intestine harbors a complex microbial flora (1Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Diversity of the human intestinal flora.Science. 2005; 308: 1635-1638Google Scholar). Bacterial density in the human colon is among the highest found in nature, approaching 1012 bacteria/g wet weight of feces (2Savage D.C. Microbial ecology of the gastrointestinal tract.Annu. Rev. Nutr. 1977; 31: 107-133Google Scholar, 3Whiteman W.B. Coleman D.C. Wiebe W.J. Prokaryotes: the unseen majority.Proc. Natl. Acad. Sci. USA. 1998; 95: 6578-6583Google Scholar). In contrast, the host suppresses significant bacterial colonization of the small intestine by a variety of mechanisms, including rapid transit times, antimicrobial peptides, proteolytic enzymes, and bile (4Wilson M. Microbial Inhabitants of Humans. 1-47. Cambridge University Press, Cambridge, UK2005: 375-392Google Scholar). Failure of these mechanisms leads to bacterial overgrowth of the small intestine, resulting in malabsorption as bacteria compete with the host for nutrients. Under normal conditions, bacterial fermentation in the colon represents an important salvage mechanism. Complex carbohydrates, which are intrinsically indigestible or which escape digestion and absorption in the proximal gut, are fermented by colonic bacteria to yield short-chain fatty acids. It has been estimated that these short-chain fatty acids constitute 3–9% of our daily caloric intake (4Wilson M. Microbial Inhabitants of Humans. 1-47. Cambridge University Press, Cambridge, UK2005: 375-392Google Scholar). Colonic bacteria also contribute to the salvage of bile salts that escape active transport in the distal ileum. The major bile salt modifications in the human large intestine include deconjugation, oxidation of hydroxy groups at C-3, C-7, and C-12, and 7α/β-dehydroxylation (Fig. 1). Deconjugation and 7α/β-dehydroxylation of bile salts increases their hydrophobicity and their Pka, thereby permitting their recovery via passive absorption across the colonic epithelium. However, the increased hydrophobicity of the transformed bile salts also is associated with increased toxic and metabolic effects. High concentrations of secondary bile acids in feces, blood, and bile have been linked to the pathogenesis of cholesterol gallstone disease and colon cancer (5McGarr S.E. Ridlon J.M. Hylemon P.B. Diet, anaerobic bacterial metabolism and colon cancer risk: a review of the literature.J. Clin. Gastroenterol. 2005; 39: 98-109Google Scholar). We present here a current review of the microbiology of bile acid metabolism in the human GI tract, focusing on understanding the biochemical mechanisms and physiological consequences of such metabolism on both the bacterium and the human host. Bile acids are saturated, hydroxylated C-24 cyclopentanephenanthrene sterols synthesized from cholesterol in hepatocytes. The two primary bile acids synthesized in the human liver are cholic acid (CA; 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (CDCA; 3α,7α-dihydroxy-5β-cholan-24-oic acid). Bile acids are further metabolized by the liver via conjugation (N-acyl amidation) to glycine or taurine, a modification that decreases the Pka to ∼5. Thus, at physiological pH, conjugated bile acids are almost fully ionized and may be termed bile salts (6Vlahcevic Z.R. Heuman D.M. Hylemon P.B. Physiology and pathophysiology of enterohepatic circulation of bile acids.in: Zakim D. Boyer T. Hepatology: A Textbook of Liver Disease. Vol 1. 3rd edition. Saunders, Philadelphia, PA1996: 376-417Google Scholar). Bile salts are secreted actively across the canalicular membrane and are carried in bile to the gallbladder, where they are concentrated during the interdigestive period. After a meal, release of cholecystokinin from the duodenum stimulates the gallbladder to contract, causing bile to flow into the duodenum (7Hofmann A.F. The continuing importance of bile acids in liver and intestinal disease.Arch. Intern. Med. 1999; 159: 2647-2658Google Scholar). Bile salts are highly effective detergents that promote solubilization, digestion, and absorption of dietary lipids and lipid-soluble vitamins throughout the small intestine. High concentrations of bile salts are maintained in the duodenum, jejunum, and proximal ileum, where fat digestion and absorption take place. Bile salts are then absorbed through high-affinity active transport in the distal ileum (6Vlahcevic Z.R. Heuman D.M. Hylemon P.B. Physiology and pathophysiology of enterohepatic circulation of bile acids.in: Zakim D. Boyer T. Hepatology: A Textbook of Liver Disease. Vol 1. 3rd edition. Saunders, Philadelphia, PA1996: 376-417Google Scholar). Upon entering the bloodstream, bile salts are complexed to plasma proteins and returned to the liver. Upon reaching the liver, they are cleared efficiently from the circulation by active transporters on the sinusoidal membrane of hepatocytes and rapidly secreted into bile. This process is known as the enterohepatic circulation. Figure 2 depicts the enterohepatic circulation in the context of the gastrointestinal anatomy and also indicates the relative numbers and genera of the predominant bacteria inhabiting each section of the GI tract. During the enterohepatic circulation, bile salts encounter populations of facultative and anaerobic bacteria of relatively low numbers and diversity in the small bowel. Bile salt metabolism by small bowel microbes consists mainly of deconjugation and hydroxy group oxidation. Ileal bile salt transport is highly efficient (∼95%), but approximately 400–800 mg of bile salts escapes the enterohepatic circulation daily and becomes substrate for significant microbial biotransforming reactions in the large bowel (6Vlahcevic Z.R. Heuman D.M. Hylemon P.B. Physiology and pathophysiology of enterohepatic circulation of bile acids.in: Zakim D. Boyer T. Hepatology: A Textbook of Liver Disease. Vol 1. 3rd edition. Saunders, Philadelphia, PA1996: 376-417Google Scholar). Comparison of bile acid composition in the gallbladder and feces illustrates the extent of microbial bile acid metabolism in the large intestine (Fig. 3). The secondary bile acids deoxycholic acid (DCA; 3α,12α-dihydroxy-5β-cholan-24-oic acid) and lithocholic acid (LCA; 3α-hydroxy-5β-cholan-24-oic acid) are produced solely by microbial biotransforming reactions in the human large intestine. DCA accumulates in the bile acid pool (LCA to a much lesser extent) as a result of passive absorption through the colonic mucosa and the inability of the human liver to 7α-hydroxylate DCA and LCA to their respective primary bile acids. LCA is sulfated in the human liver at the 3-hydroxy position, conjugated at C-24, and excreted back into bile (6Vlahcevic Z.R. Heuman D.M. Hylemon P.B. Physiology and pathophysiology of enterohepatic circulation of bile acids.in: Zakim D. Boyer T. Hepatology: A Textbook of Liver Disease. Vol 1. 3rd edition. Saunders, Philadelphia, PA1996: 376-417Google Scholar). The resultant bile acid sulfate is poorly reabsorbed from the gut. Even though 3-sulfo-LCA glycine and taurine conjugates are deconjugated and to some extent desulfated by intestinal bacteria, 3-sulfo-LCA/LCA is lost in feces and does not normally accumulate in the enterohepatic circulation (8Cowen A.E. Korman M.G. Hofmann A.F. Cass O.W. Coffin S.B. Metabolism of lithocholate in healthy man. II. Enterohepatic circulation.Gastroenterology. 1975; 69: 67-76Google Scholar). Deconjugation refers to the enzymatic hydrolysis of the C-24 N-acyl amide bond linking bile acids to their amino acid conjugates. This reaction is substrate-limiting and goes to completion in the large bowel. Bile salt hydrolases (BSHs) are in the choloylglycine hydrolase family (EC 3.5.1.24) and have been isolated and/or characterized from several species of intestinal bacteria (Table 1). The importance of the position, charge, shape, and chirality of various analogs of taurine/glycine conjugates on the rate of hydrolysis by BSHs has also been investigated (9Heijghebaert S.M. Hofmann A.F. Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase on human fecal cultures.J. Lipid Res. 1986; 27: 742-752Google Scholar). BSHs differ in subunit size and composition, pH optimum, kinetic properties, substrate specificity, gene organization, and regulation. BSHs do, however, share in common several conserved active site amino acids [cysteine 2 (Cys2), arginine 18 (Arg18), aspartic acid 21 (Asp21), asparagine 175 (Asn175), and arginine 228 (Arg228)] and share a high degree of amino acid sequence similarity with the penicillin V amidase of Bacillus sphaericus (Fig. 4). The conservation of tyrosine 82 (Tyr82) in penicillin V amidase and Asn82 in BSH are likely a result of differing steric requirements for their respective substrates (10Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google Scholar). Recently, a bsh from Clostridium perfringens was crystallized both in the apoenzyme form and in complex with taurodeoxycholate (TDCA; hydrolyzed product) at resolutions of 2.1 and 1.7 Å, respectively (11Rossocha M. Schultz-Heienbrok R. von Moeller H. Coleman J.P. Saenger W. Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product.Biochemistry. 2005; 44: 5739-5748Google Scholar). The structure revealed that the Cys2 residue is in position for nucleophilic attack of the N-acyl amide bond. Site-directed mutagenesis of the Cys2 residue from the BSH of Bifidobacterium longum and Bi. bifidum (10Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google Scholar, 18Kim G.B. Miyamoto C.M. Meighen E.A. Lee B.H. Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.Appl. Environ. Microbiol. 2004; 70: 5603-5612Google Scholar) as well as sulfhydryl inhibition of several BSHs have shown the importance of this residue in catalysis (10Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google Scholar, 13Gopal-Srivastava R. Hylemon P.B. Purification and characterization of bile salt hydrolase from Clostridium perfringens.J. Lipid Res. 1988; 29: 1079-1085Google Scholar, 14Grill J.P. Schneider F. Crociani J. Ballongue J. Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536.Appl. Environ. Microbiol. 1995; 61: 2577-2582Google Scholar). Alignment of amino acid sequences from BSHs shows that the Cys2 residue is conserved in all BSHs characterized to date (Fig. 4). The broad substrate specificities reported (Table 1) are potentially a function of a lack of conservation observed in residues making up the substrate binding pocket of the conjugated bile acid hydrolase gene product of C. perfringens (CBAH-1) and the corresponding residues predicted in amino acid multiple sequence alignment with other BSHs (Fig. 4). The sterol moiety is bound primarily through hydrophobic interactions in the CBAH-1 (residues highlighted in gray in Fig. 4) as well as hydrogen bonds to the carboxylate group. Although the crystal structure of CBAH-1 did not reveal specific recognition of the taurine/glycine moiety, kinetic data from several BSHs suggest that the conjugates are important in substrate specificity (Table 1). Therefore, additional crystallization and site-directed mutagenesis (preferably with mutagenesis of Cys2) of BSHs from different species will be helpful in explaining the kinetic observations of substrate specificity.TABLE 1.Characteristics of BSHs from human intestinal bacteriaOrganismNative Molecular MassSubunit Molecular MassApparent KmTCATCDCATDCAGCAGCDCAGDCApH OptimumReference(s)kDamMBacteroides fragilis25032.50.450.290.170.350.260.24.2–4.525Stellwag E.J. Hylemon P.B. Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.Biochim. Biophys. Acta. 1976; 452: 165-176Google ScholarBacteroides vulgatus14036+++−−−ND163Kawamoto K. Horibe I. Uchida K. Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus.J. Biochem. (Tokyo). 1989; 106: 1049-1053Google ScholarClostridium perfringens MCV 185250563733.53.6141.25.8–6.413Gopal-Srivastava R. Hylemon P.B. Purification and characterization of bile salt hydrolase from Clostridium perfringens.J. Lipid Res. 1988; 29: 1079-1085Google ScholarClostridium perfringens 1314736.1+ND++NDND4.5–5.515Lactobacillus johnsonii 100-10042(α), 38(β)12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar, 17Elkins C.A. Savage D.C. Identification of genes encoding conjugated bile salt hydrolase and transport in Lactobacillus johnsonii 100-100.J. Bacteriol. 1998; 180: 4344-4349Google Scholar, 23Elkins C.A. Savage D.C. CbsT2 from Lactobacillus johnsonii 100-100 is a transport protein of the major facilitator superfamily that facilitates bile acid antiport.J. Mol. Microbiol. Biotechnol. 2003; 6: 76-87Google ScholarIsozyme A115420.76+++ND+4.2–4.5Isozyme B10542, 380.95+++ND+4.2–4.5Isozyme C9542, 380.45NDNDNDNDND4.2–4.5Isozyme D80380.37NDNDNDNDND4.2–4.5Lactobacillus plantarum 80ND37.1aValue derived from the Protparam program (http://www.expasy.ch/tools/protparam.html) using the deduced amino acid sequence.TRTRTR+++4.7–5.528De Boever P. Verstraete W. Bile salt deconjugation by Lactobacillus plantarum 80 and its implication for bacterial toxicity.J. Appl. Microbiol. 1999; 87: 345-352Google ScholarLactobacillus acidophilusND35.0aValue derived from the Protparam program (http://www.expasy.ch/tools/protparam.html) using the deduced amino acid sequence.+NDND+NDNDND12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar, 27Corzo G. Gilliland S.E. Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts.J. Dairy Sci. 1999; 82: 466-471Google ScholarBifidobacterium longum BB536250400.8751.610.5161.331.41.375.5–6.513Gopal-Srivastava R. Hylemon P.B. Purification and characterization of bile salt hydrolase from Clostridium perfringens.J. Lipid Res. 1988; 29: 1079-1085Google ScholarBifidobacterium longum SBT2928125–130351.120.330.790.160.130.285.0–7.010Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google ScholarBifidobacterium bifidum ATCC 1186315035++++ ++ ++ +ND18Kim G.B. Miyamoto C.M. Meighen E.A. Lee B.H. Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.Appl. Environ. Microbiol. 2004; 70: 5603-5612Google ScholarBifidobacterium adolescentisND35aValue derived from the Protparam program (http://www.expasy.ch/tools/protparam.html) using the deduced amino acid sequence.NDNDNDNDNDNDND19Kim G.B. Brochet M. Lee B.H. Cloning and characterization of a bile salt hydrolase (bsh) from Bifidobacterium adolescentis.Biotechnol. Lett. 2005; 27: 817-822Google ScholarListeria monocytogenesND36.8aValue derived from the Protparam program (http://www.expasy.ch/tools/protparam.html) using the deduced amino acid sequence.NDNDNDNDND+ND20Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. et al.Comparative genomics of Listeria species.Science. 2001; 294: 849-852Crossref Scopus (34) Google Scholar, 164Sue D. Boor K.J. Wiedmann M. σB-dependent expression patterns of compatible solute transporter genes opuCA and Imo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes.Microbiology. 2003; 149: 3247-3256Google ScholarBSH, bile salt hydrolase; GCA, glycocholate; GCDCA, glycochenodeoxycholate; GDCA, glycodeoxycholate; ND, not determined; TCA, taurocholate; TCDCA, taurochenodeoxycholate; TDCA, taurodeoxycholate; TR, trace of activity; +, activity detected; −, no activity detected.a Value derived from the Protparam program (http://www.expasy.ch/tools/protparam.html) using the deduced amino acid sequence. Open table in a new tab Fig. 4.Multiple sequence alignment of cholylglycine hydrolases. Protein sequences were obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Alignments were made with the ClustalW program (http://www.ebi.ac.uk/clustalw/) using the GONNET 250 matrix. Residues highlighted in yellow are predicted active site amino acids based on the crystal structure of the BSH from C. perfringens (11Rossocha M. Schultz-Heienbrok R. von Moeller H. Coleman J.P. Saenger W. Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product.Biochemistry. 2005; 44: 5739-5748Google Scholar) as well as on site-directed mutagenesis and biochemical data (10Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google Scholar, 12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar, 13Gopal-Srivastava R. Hylemon P.B. Purification and characterization of bile salt hydrolase from Clostridium perfringens.J. Lipid Res. 1988; 29: 1079-1085Google Scholar, 14Grill J.P. Schneider F. Crociani J. Ballongue J. Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536.Appl. Environ. Microbiol. 1995; 61: 2577-2582Google Scholar). Residues highlighted in gray correspond to residues involved in substrate binding in the BSH from C. perfringens (11Rossocha M. Schultz-Heienbrok R. von Moeller H. Coleman J.P. Saenger W. Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product.Biochemistry. 2005; 44: 5739-5748Google Scholar). The secondary structural elements, which are based on the conjugated bile acid hydrolase from C. perfringens (CBAH-1) crystal structure, are shown above the alignment. The α and β designations of Lactobacillus johnsonii refer to the two isoforms of the genes found in this bacterium.View Large Image Figure ViewerDownload (PPT) BSH, bile salt hydrolase; GCA, glycocholate; GCDCA, glycochenodeoxycholate; GDCA, glycodeoxycholate; ND, not determined; TCA, taurocholate; TCDCA, taurochenodeoxycholate; TDCA, taurodeoxycholate; TR, trace of activity; +, activity detected; −, no activity detected. Genes encoding BSHs have been cloned from C. perfringens (15Coleman J.P. Hudson L.L. Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens.Appl. Environ. Microbiol. 1995; 61: 2514-2520Google Scholar), Lactobacillus plantarum (16Christiaens H. Leer R.J. Pouwels P.H. Verstraete W. Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.Appl. Environ. Microbiol. 1992; 58: 3792-3798Google Scholar), La. johnsonii (12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar, 17Elkins C.A. Savage D.C. Identification of genes encoding conjugated bile salt hydrolase and transport in Lactobacillus johnsonii 100-100.J. Bacteriol. 1998; 180: 4344-4349Google Scholar), Bi. longum (10Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google Scholar), Bi. bifidum (18Kim G.B. Miyamoto C.M. Meighen E.A. Lee B.H. Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.Appl. Environ. Microbiol. 2004; 70: 5603-5612Google Scholar), Bi. adolescentis (19Kim G.B. Brochet M. Lee B.H. Cloning and characterization of a bile salt hydrolase (bsh) from Bifidobacterium adolescentis.Biotechnol. Lett. 2005; 27: 817-822Google Scholar), and Listeria monocytogenes (20Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. et al.Comparative genomics of Listeria species.Science. 2001; 294: 849-852Crossref Scopus (34) Google Scholar, 21Dussurget O. Cabanes D. Dehoux P. Lecuit M. Buchrieser C. Glaser P. Cossart P. and the European Listeria Genome ConsortiumListeria monocytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in the intestinal and hepatic phases of listeriosis.Mol. Microbiol. 2002; 45: 1095-1106Google Scholar). Homologs and putative bsh genes have also been identified recently through microbial genome analysis. The organization and regulation of genes encoding BSH differ between species and genera. Monocistronic BSH genes have been reported in La. plantarum (16Christiaens H. Leer R.J. Pouwels P.H. Verstraete W. Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.Appl. Environ. Microbiol. 1992; 58: 3792-3798Google Scholar), La. johnsonii (12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar), Li. monocytogenes (21Dussurget O. Cabanes D. Dehoux P. Lecuit M. Buchrieser C. Glaser P. Cossart P. and the European Listeria Genome ConsortiumListeria monocytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in the intestinal and hepatic phases of listeriosis.Mol. Microbiol. 2002; 45: 1095-1106Google Scholar), and Bi. bifidum (18Kim G.B. Miyamoto C.M. Meighen E.A. Lee B.H. Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.Appl. Environ. Microbiol. 2004; 70: 5603-5612Google Scholar). A gene encoding BSH (CBAH-1) cloned from C. perfringens (15Coleman J.P. Hudson L.L. Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens.Appl. Environ. Microbiol. 1995; 61: 2514-2520Google Scholar) differed significantly in size and amino acid sequence from a BSH purified from a different strain of C. perfringens (13Gopal-Srivastava R. Hylemon P.B. Purification and characterization of bile salt hydrolase from Clostridium perfringens.J. Lipid Res. 1988; 29: 1079-1085Google Scholar). The inactivation of the gene encoding CBAH-1 resulted in only partial reduction in BSH activity (BSH activity was 86% of that in the wild type), suggesting multiple BSH genes in C. perfringens. Furthermore, the crystal structure showed that the enzyme encoded by the CBAH-1 gene forms an active homotetramer (11Rossocha M. Schultz-Heienbrok R. von Moeller H. Coleman J.P. Saenger W. Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product.Biochemistry. 2005; 44: 5739-5748Google Scholar). These observations, coupled with the detection of both intracellular and extracellular BSHs, provide further evidence for multiple isoforms, although the organization and regulation of the bsh gene(s) from C. perfringens are not known at present (22Kishinaka M. Umeda A. Kuroki S. High concentrations of conjugated bile acids inhibit bacterial growth of Clostridium perfringens and induce its extracellular cholylglycine hydrolase.Steroids. 1994; 59: 485-489Google Scholar). Polycistronic operons encoding three genes involved in bile salt deconjugation (cbsT1, cbsT2, and cbsHβ) have been characterized in La. johnsonii and La. acidophilus (12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar). Genes cbsT1 and cbsT2 appear to be gene duplications that encode taurocholate/CA antiport proteins of the major facilitator superfamily, whereas cbsHβ encodes the BSH β-isoform (23Elkins C.A. Savage D.C. CbsT2 from Lactobacillus johnsonii 100-100 is a transport protein of the major facilitator superfamily that facilitates bile acid antiport.J. Mol. Microbiol. Biotechnol. 2003; 6: 76-87Google Scholar). In addition, an uncharacterized extracellular factor has been detected in La. johnsonii 100-100, which stimulates BSH activity and uptake of conjugated bile salts during the stationary growth phase (12Elkins C.A. Moser S.A. Savage D.C. Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.Appl. Environ. Microbiol. 2001; 147: 3403-3412Google Scholar, 24Lundeen S.G. Savage D.C. Multiple forms of bile salt hydrolase from Lactobacillus sp. strain 100-100.J. Bacteriol. 1992; 174: 7217-7220Google Scholar). BSH expression is also growth phase-dependent. Stationary phase expression has been reported in Bacteroides fragilis (25Stellwag E.J. Hylemon P.B. Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.Biochim. Biophys. Acta. 1976; 452: 165-176Google Scholar), and exponential phase expression was reported for Bi. longum (10Tanaka H. Hashiba H. Kok J. Mierau I. Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization.Appl. Environ. Microbiol. 2000; 66: 2502-2512Google Scholar). BSHs appear to enhance the bacterial colonization of the lower gastrointestinal tract of higher mammals. The physiological advantages of BSHs are not fully understood and may vary between bacterial species and genera. It has been hypothesized that deconjugation may be a mechanism of the detoxification of bile salts. De Smet et al. (26De Smet I. Van Hoorde L. Vande Woestyne M. Christiaens H. Verstraete W. Significance of bile salt hydrolytic activities of lactobacilli.J. Appl. Bacteriol. 1995; 79: 292-301Go