Department of Microbiology, M409 Walters Life Sciences Bldg, University of Tennessee, Knoxville, TN 37996-0845, USA1
Author for correspondence: Dwayne C. Savage. Tel: +1 865 974 4015. Fax: +1 865 974 4007. e-mail: Dsavage1{at}utk.edu
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ABSTRACT |
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Keywords: intestinal microflora, probiotics, major facilitator superfamily, horizontal gene transfer, lactic acid bacteria
Abbreviations: BSH, bile salt hydrolase; EF, extracellular factor; LDGW, long-distance genome walking; LPEA, long primer extension and amplification; MFS, major facilitator superfamily
The GenBank accession numbers for the sequences determined in this work are AF054971, AF091248 (sequences of the complete BSH operons in L. johnsonii strain 100-100 and L. acidophilus strain KS-13, respectively) and AF297873 (sequence of cbsH).
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INTRODUCTION |
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The class of microbial enzymes that catalyse deconjugation have been collectively named conjugated bile salt hydrolases (BSH, EC 3 . 5 . 1 . 24). These enzymes can have demonstrable specificity for tauro- versus glyco-conjugates (Aries & Hill, 1970 ; Gilliland & Speck, 1977
), and with the exception of one expressed by Clostridium perfringens (Kishinaka et al., 1994
), are soluble cytosolic proteins (Stellwag & Hylemon, 1976
; Gopal-Srivastava & Hylemon, 1988
; Kawamoto et al., 1989
; Lundeen & Savage, 1990
; Grill et al., 1995
; Tanaka et al., 2000
). The molecular fate of the amino acid released by deconjugation in the bacterial cell and the benefit that the organisms may derive from such activity is unclear (De Smet et al., 1995
). The deconjugated product, or primary bile acid, is a toxic hydrophobic molecule that can disaggregate the cytoplasmic membrane (Thanassi et al., 1997
). These molecules are not metabolized and leave the cell passively or through some unidentified bile acid export mechanism. At the physiological pH of the intestinal lumen, deconjugates can be actively transported through the epithelium (Wong et al., 1994
; Hylemon & Harder, 1999
) and into the bloodstream of the host, or are precipitated in the faeces and excreted (Baron & Hylemon, 1997
).
BSH enzymes have been purified and characterized from Bacteroides vulgatus VI-31 (Kawamoto et al., 1989 ), Bacteroides fragilis subsp. fragilis (Stellwag & Hylemon, 1976
), Bifidobacterium longum BB536 (Grill et al., 1995
) and SBT2928 (Tanaka et al., 2000
), C. perfringens MCV 815 (Gopal-Srivastava & Hylemon, 1988
) and Lactobacillus johnsonii strain 100-100 (Lundeen & Savage, 1990
, 1992a
). Genes encoding BSH activity have been cloned from Lactobacillus plantarum 80 (Christiaens et al., 1992
), C. perfringens 13 (Coleman & Hudson, 1995
), L. johnsonii strain 100-100 (Elkins & Savage, 1998
) and Bif. longum SBT2928 (Tanaka et al., 2000
). Genetic characterization of these loci reveals different architectures: the L. plantarum 80 BSH transcript is monocistronic (Christiaens et al., 1992
); the L. johnsonii strain 100-100 BSH gene is arranged in tandem with at least two other functionally related genes (Elkins & Savage, 1998
); and the Bif. longum SBT2928 BSH gene is coordinately regulated with at least one other gene that shares a high level of amino acid homology with glutamine synthetase adenylyltransferase (glnE; Tanaka et al., 2000
). The extent of the operon in Bif. longum SBT2928 and the putative operon in L. johnsonii strain 100-100 is not known (Elkins & Savage, 1998
; Tanaka et al., 2000
); DNA sequence flanking the C. perfringens 13 BSH gene has not been characterized (Coleman & Hudson, 1995
).
L. johnsonii strain 100-100 expresses high levels of BSH activity (Lundeen & Savage, 1990 ). In contrast to other bacteria studied, the activity in strain 100-100 involves four enzymes that exist as homo- and heterotrimers of two antigenically distinct proteins, designated
and ß (Lundeen & Savage, 1992a
). A 2977 bp EcoRI genomic clone, pIN-BSH2, that expresses hydrolase activity in Escherichia coli cells has been identified and shown to encode the ß peptide (ORF3, hereafter referred to as cbsHß; Elkins & Savage, 1998
). A 651 nt partial ORF1 (hereafter referred to as cbsT1) and a 1353 nt complete ORF2 (hereafter referred to as cbsT2) were encoded on the fragment 5' of cbsHß. These ORFs share similarity with transport proteins of the major facilitator superfamily (MFS; Saier et al., 1999
) and approximately 80% predicted amino acid sequence similarity to each other. They increase uptake of conjugated bile acids by as much as threefold over control levels when expressed in E. coli cells that were exposed to an extracellular factor (EF; Lundeen & Savage, 1990
, 1992b
) produced by strain 100-100 (Elkins & Savage, 1998
).
We have characterized this locus and report here that cbsT1, cbsT2 and cbsHß probably consitute a BSH operon. In addition, we report a second locus that encodes BSH. We have also assayed a collection of 50 Lactobacillus spp. for BSH activity and the cbsHß locus. We speculate that BSH activity was acquired horizontally and implicate the activity as an important factor for colonization by lactobacilli of the gastrointestinal tract.
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METHODS |
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Oligonucleotide primers.
Primers used in this study (Table 1) were synthesized by Gibco-BRL Custom Primers Division under standard conditions.
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Recombinant DNA methods.
Plasmid vector pSportI (Gibco-BRL) was used in all recombinant DNA procedures. Three constructs that harbour strain 100-100 genomic loci encoding cbsT1-cbsT2-cbsHß, cbsT2-cbsHß or cbsHß were cloned under control of the pSportI lac-inducible promoter. Primers containing engineered KpnI sites, T1strt, T2strt and Hßstrt, were constructed 5' of cbsT1, cbsT2 and cbsHß respectively and used with a primer containing an engineered BamHI site 3' of cbsHß, Hßend. Genomic DNA was amplified by PCR, digested with KpnI and BamHI (Promega), and cloned into identically digested vector DNA with T4 DNA ligase (Promega). The chimeras were designated pT1T2Hß, pT2Hß and pHß, respectively, and transformed into E. coli DH5 cells. Constructs were identified by blue/white colony screening on LuriaBertani (LB) medium containing 100 µg ampicillin (Amp) ml-1, 0·5 mM IPTG and 40 µg X-Gal ml-1 (LB-Amp-IPTG-X-Gal). Plasmid DNA for each construct was purified (Wizard Plus Minipreps DNA Purification Kit) and sequenced to verify content and orientation. To clone and express the predicted BSH gene from L. acidophilus strain KS-13, chromosomal DNA was purified from strain KS-13 cells and PCR-amplified with primers La-Hßa and La-Hßb. The fragment was cloned at the KpnI site of pSportI. Transformants were screened for BSH activity.
Generation of an L. johnsonii strain 100-100 genomic library.
A genomic library of strain 100-100 was created with pSportI and screened for BSH activity. Chromosomal DNA was purified from strain 100-100 cells and partially digested with Sau3AI (Promega). The fragments were cloned at the BamHI site of pSportI in E. coli DH5 cells. The library was screened for clones expressing BSH activity.
DNA sequencing and analysis.
DNA generated from LDGW and LPEA PCR, direct amplified DNA, and cloned DNA was sequenced by the ABI Prism automated fluorescent method (Molecular Biology Resource Facility, University of Tennessee, Knoxville). Direct amplified DNA and cloned DNA were sequenced bidirectionally using universal forward and reverse primers and/or synthesized oligonucleotide walking primers. DNA sequence was analysed for start and stop codons and translated into predicted amino acid sequence with MacVector. ORFs were putatively identified from similarity with published sequences using the advanced basic local alignment search tool (BLAST; NIH website).
Assay for BSH activity.
Transformed E. coli DH5 cells were screened for the ability to hydrolyse conjugated bile acids with an agar plate assay (Dashkevicz & Feighner, 1989
). Cultures were streaked or replica plated onto MRS agar containing 0·5% taurodeoxycholic acid (Sigma), a conjugated bile acid, 100 µg Amp ml-1 and 0·5 mM IPTG (MRS-TDCA-Amp-IPTG). Cultures were incubated anaerobically for 2448 h. BSH activity was detected when a halo of deoxycholic acid precipitated around colonies in the medium.
Screen for cbsHß.
Coleman & Hudson (1995) identified conserved amino acid motifs in cloned BSHs by aligning predicted protein sequences. A motif at the amino terminus and a motif centred around residue 225 were selected and used to construct primers Hß675a and Hß675b, respectively. The primers were engineered to cbsHß sequence in strain 100-100 and encompass a region of approximately 675 bp. The primers were used in standard PCR reactions to amplify genomic DNA from other lactobacilli.
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RESULTS |
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BSH operon in strain 100-100
LPEA produced long cDNA that extended to the 5' terminus of the mRNA species complementary to primer Hß-1. This cDNA was amplified with AP and nested primers, T1-1 and T1-2, in two successive nested LDGW PCR reactions. The first PCR reaction produced two visible bands of approximately 2 and 0·8 kbp when analysed by 1% agarose gel electrophoresis (Fig. 2a, lane 2). The second, nested reaction produced a single fragment of approximately 0·5 kbp (Fig. 2a
, lane 3). Considering the respective placement of T1-1 and T1-2, a decrease of approximately 0·3 kbp from 0·8 to 0·5 kbp is proportional to and consistent with genomic DNA sequence from strain 100-100. We believe that the fragment of 2 kbp (Fig. 2a
, lane 2) is an artifact. LDGW PCR is known to produce artifacts of unrelated sequence that can be eliminated, in our experience, in subsequent nested amplifications with SSPs. Alternatively, or in addition, the 2 kbp fragment could have been amplified from a similar primer-binding site for T1-1 that is found within cbsT2. Purified DNA from the second reaction was sequenced with T1-2 and matched identically the DNA sequence from the genome of strain 100-100 directly downstream of the T1-2 binding site (Fig. 2b
). The complement of the DNA sequence extended 78 nt upstream of cbsT1 and was tailed with a GS stretch. Sequences centred approximately -10 and -35 of the transcriptional start site, AATATAA and TTGATT respectively, were identified that resemble E. coli -10 and -35 consensus promoter sequences, TATAAT and TTGACA respectively, that are recognized by
70 (Fig. 2b
). Expression analysis of cbsHß was undertaken with three clones, pT1T2Hß, pT2Hß and pHß. All three clones expressed BSH activity when transformed into E. coli DH5
cells incubated anaerobically on MRS-TDCA-Amp-IPTG plates (data not shown).
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DISCUSSION |
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The BSH operon of strain 100-100 contains two genes, cbsT1 and cbsT2, that share a high level of similarity in their DNA and predicted amino acid sequence. This finding suggests that the genes are duplicates. Functional studies have shown that cbsT2 and partial cbsT1, when expressed in E. coli, increase uptake of conjugated bile acids (Elkins & Savage, 1998 ). These genes, while demonstrating putative topologies characteristic of MFS transporters (Saier et al., 1999
), do not display impressive sequence similarity, as identified by BLAST, to any known genes. Members of several subfamilies of the MFS provide the closest sequence similarity. In support of our observations, M. Saier and colleagues have phylogenetically classified cbsT1 and cbsT2 as members of a new subfamily of the MFS called the conjugated bile salt transporter family (BST) (Saier et al., 1999
; http://www-biology.ucsd.edu/~msaier/transport/titlepage.html).
We were able to produce and amplify cDNA from strain 100-100 using primers engineered to cbsHß and cbsT1. LPEA produced one fragment of 0·5 kbp that was identical in sequence to genomic DNA from the upstream end of cbsT1. This sequence extended 78 nt upstream of the start site of cbsT1. We conclude that cbsT1, cbsT2 and cbsHß are probably encoded polycistronically. The expression analysis with cbsHß supported that conclusion. When expressed under lac-inducible promotion, clones containing cbsT1-cbsT2-cbsHß or cbsT2-cbsHß produced precipitates of deoxycholic acid in the medium. Therefore, cbsT1 and cbsT2 probably do not contain mRNA termination sequences, at least not ones that are recognized by E. coli. However, further expression evidence and DNA motif analysis are necessary to convincingly demonstrate the polycistronic expression of the three genes.
The genetic analysis of BSH activity supports protein characterization studies. Strain 100-100 produces two hydrolases, and ß, that combine to form homo- and heterotrimeric complexes. Purified
and ß hydrolases have estimated molecular masses of 42 and 38 kDa, respectively (Lundeen & Savage, 1992a
). The calculated molecular masses of the BSH from the Sau3AI genomic clone (complete ORF) and cbsHß are 36667 and 34916 Da, respectively. In addition to having the larger molecular mass, the BSH from the genomic clone had an amino-terminal sequence that matches, with only two differences, the first 25 amino acids of the
hydrolase (Lundeen & Savage, 1992a
). The observed differences in amino acid sequence are probably due to problems in the peptide sequencing. To prepare the
peptide for sequencing, it was hydrolysed with 6 M HCl and derivatized with phenylisothiocyanate as previously reported (Lundeen & Savage, 1992a
). Under such conditions, Cys cannot be resolved, and Glu and Gln cannot be distinguished during sequencing. Such problems undoubtedly explain the discrepancies between the chemical peptide sequence and the peptide sequence inferred from the nucleotide sequence. We conclude, therefore, that the complete 978 nt ORF from the Sau3AI genomic clone encodes BSH
and designate this ORF cbsH
. We also conclude that the known genetic elements responsible for BSH activity in strain 100-100 have been identified and cloned.
CbsH shares the highest amino acid sequence similarity with the BSH from L. plantarum 80 (Christiaens et al., 1992
). Similar to the BSH in strain 80, cbsH
is not arranged in tandem with other ORFs and contains an inverted repeat 3' of the stop codon. The BSH protein(s), however, has not been isolated or characterized from strain 80 (Christiaens et al., 1992
). We suggest that the strain may also contain a second hydrolase locus encoding additional functionally related genes if the activity was acquired horizontally. We also tentatively conclude that cbsH
, similar to the BSH gene from 80, is encoded monocistronically.
Our phenotypic and genetic screens suggest that BSH activity was acquired horizontally in lactobacilli. Lactobacilli within the species acidophilus, brevis, buchneri, fermentum, gasseri and plantarum express a variable BSH phenotype. PCR screening within L. acidophilus and L. delbrueckii subsp. bulgaricus also produced variable results. Nearly all (9/10) of the cbsHß-positive isolates were from human sources, which suggests that this locus is important for persistent colonization of the gastrointestinal tract. Taken in aggregate, these results suggest that the BSH phenotype and genotype is variable within a given species. It is not known whether BSH genes and related genes are encoded on mobile genetic elements. It is known, however, that the BSH gene from Bif. longum SBT2928 is flanked by inverted repeats (Tanaka et al., 2000 ). It is interesting to note, furthermore, that strain 100-100 encodes a maturase, mat, downstream of cbsHß. Although primary literature on bacterial maturases is scarce, the enzymes have endonuclease and reverse transcriptase activity that faciliates movement and splicing of cDNA into the genome in a process known as retrohoming (Edgell et al., 2000
). Group II maturases are encoded by self-contained ORFs with independent promoters. Most group II maturases are inserted in or associated with mobile genetic elements (Edgell et al., 2000
). The putative maturase in strain 100-100 is located complementary to but in close proximity with DNA encoding the BSH operon.
A BSH operon identical in genomic architecture to L. johnsonii strain 100-100 is conserved in L. acidophilus strain KS-13. DNA sequence identity to strain 100-100 ends immediately upstream of the putative promoter and downstream of cbsHß. We suggest that this locus forms a genomic mobile element. L. acidophilus is one of only two organisms that respond to an EF of unknown composition produced by strain 100-100 (Lundeen & Savage, 1992b ). Unlike other BSH systems, hydrolase activity in strain 100-100 is increased by as much as three- to fivefold within 20 min after conjugated bile acids are added to suspensions of stationary-phase cells. The increase is due to induction of a soluble extracellular molecule and not enzymic or regulatory proteins (Lundeen & Savage, 1992b
). EF also enhances uptake of conjugated bile acids in E. coli cells expressing cbsT2 and partial cbsT1 from strain 100-100 (Elkins & Savage, 1998
). Therefore, the transporters in strain KS-13 are related to the bacteriums ability to respond to EF.
The conserved DNA sequence upstream of cbsT1 in strain KS-13 supported our data on the putative promoter for the cbsHß operon in strain 100-100. A region of approximately 135 nt upstream from cbsT1 should be conserved in strain KS-13 if it is important for promotion and transcription of the genes. Lundeen & Savage (1990) demonstrated at a physiological level that induction of BSH expression corresponds with entry of strain 100-100 into stationary phase. LPEA analysis supported this conclusion as well. There are no conserved operator/enhancer elements since sequence identity between strain KS-13 and 100-100 ends upstream of the -35 promoter element. Thus, the RNA analysis supports simple sigma-dependent stationary-phase induction of BSH activity.
We have cloned the genetic determinants for BSH activity in L. johnsonii strain 100-100 and have extended the study into other Lactobacillius species. In strain 100-100, two genetic loci encode BSH activity. The cbsHß locus is an operon of three genes encoding two bile-acid-related functions: hydrolysis and transport. This locus is conserved in other Lactobacillus species, specifically human isolates. Within some species, however, BSH activity and the cbsHß locus were not constant traits. L. acidophilus strain KS-13 encoded the cbsHß locus but DNA sequences flanking the three-gene operon were not conserved. Our data support a hypothesis that BSH activity is important at some level for colonization of the gastrointestinal tract and that BSH genes were acquired horizontally. Bile acid transport by contrast could be studied. Is there a bile acid exporter or is export activity catalysed concomitantly with import? What is the function of EF in bile acid transport? Experiments designed to identify the energy source and kinetics of transport may provide functional information that supports the phylogenetic assignment of cbsT1 and cbsT2 into a new family of the MFS. This information, once obtained, may lead to a working model for BSH activity in enteric bacteria.
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ACKNOWLEDGEMENTS |
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This research was funded by the University of Tennessee and the National Dairy Council.
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REFERENCES |
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Aries, V. & Hill, M. J. (1970). Degradation of steroids by intestinal bacteria. Biochim Biophys Acta 202, 526-534.[Medline]
Baron, S. F. & Hylemon, P. B. (1997). Biotransformation of bile acids, cholesterol, and steroid hormones. In Gastrointestinal Microbiology, vol. I, Gastrointestinal Ecosystems and Fermentations , pp. 470-510. Edited by R. I. Mackie & B. A. White. New York:International Thomson Publishing.
Batta, A. K., Salen, G., Arora, R., Shefer, S., Batta, M. & Person, A. (1990). Side chain conjugation prevents bacterial 7-dehydroxylation of bile acids. J Biol Chem 265, 10925-10928.
Cheah, P. Y. (1990). Hypothesis for the etiology of colorectal cancer an overview. Nutr Cancer 14, 5-13.[Medline]
Christiaens, H., Leer, R. J., Pouwels, P. H. & Verstraete, W. (1992). Cloning and expression of a conjugated bile salt hydrolase gene from Lactobacillus plantarum by using a direct plate assay. Appl Environ Microbiol 58, 3792-3798.[Abstract]
Cole, C. B. & Fuller, R. (1974). Bile acid deconjugation and attachment of chicken gut bacteria: their possible role in growth depression. Br Poult Sci 25, 227-231.
Coleman, J. P. & Hudson, L. L. (1995). Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens. Appl Environ Microbiol 61, 2514-2520.[Abstract]
Dashkevicz, M. P. & Feighner, S. D. (1989). Development of a differential medium for bile salt hydrolase-active Lactobacillus spp. Appl Environ Microbiol 55, 11-16.[Abstract]
De Smet, I., Van Hoorde, L., De Saeyer, N., Vande Woestyne, M. & Verstraete, W. (1994). In vitro study of bile salt hydrolase (BSH) activity of BSH isogenic Lactobacillus plantarum 80 strains and estimation of cholesterol lowering through enhanced BSH activity. Microb Ecol Health Dis 7, 315-329.
De Smet, I., Van Hoorde, L., Vande Woestyne, M., Christiaens, H. & Verstraete, W. (1995). Significance of bile salt hydrolytic activities of lactobacilli. J Appl Bacteriol 79, 292-301.[Medline]
Edgell, D. R., Belfort, M. & Shub, D. A. (2000). Barriers to intron promiscuity in bacteria. J Bacteriol 182, 5281-5289.
Edwards, J. B. D. M., Ravassard, P., Icard-Liepkalns, C. & Mallet, J. (1995). cDNA cloning by RT-PCR. In PCR 2: a Practical Approach , pp. 89-118. Edited by M. J. McPherson, B. D. Hames & G. R. Taylor. New York:Oxford University Press.
Elkins, C. A. & Savage, D. C. (1998). Identification of genes encoding conjugated bile salt hydrolase and transport in Lactobacillus johnsonii 100-100. J Bacteriol 180, 4344-4349.
Eyssen, H. & deSomer, P. (1963). The mode of action of antibiotics in stimulating growth of chicks. J Exp Med 117, 127-138.
Gilliland, S. E. & Speck, M. L. (1977). Deconjugation of bile acids by intestinal lactobacilli. Appl Environ Microbiol 33, 15-18.[Medline]
Gopal-Srivastava, R. & Hylemon, P. B. (1988). Purification and characterization of a bile salt hydrolase from Clostridium perfringens. J Lipid Res 29, 1079-1085.[Abstract]
Grill, J.-P., Schneider, F., Crociani, J. & Ballongue, J. (1995). Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536. Appl Environ Microbiol 61, 2577-2582.[Abstract]
Hylemon, P. B. & Harder, J. (1999). Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems. FEMS Microbiol Rev 22, 475-488.
Kandell, R. L. & Bernstein, C. (1991). Bile salt/acid induction of DNA damage in bacterial and mammalian cells: implications for colon cancer. Nutr Cancer 16, 227-238.[Medline]
Kawamoto, K., Horibe, I. & Uchida, K. (1989). Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus. J Biochem 106, 1049-1053.[Abstract]
Kay, R. M. (1981). Effects of diet on the fecal excretion and bacterial modification of acidic and neutral steroids, and implications for colon carcinogenesis. Cancer Res 41, 3774-3777.[Abstract]
Kishinaka, M., Umeda, A. & Kuroki, S. (1994). High concentrations of conjugated bile acids inhibit bacterial growth of Clostridium perfringens and induce its extracellular cholylglycine hydrolase. Steroids 59, 485-489.[Medline]
Lundeen, S. G. & Savage, D. C. (1990). Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100. J Bacteriol 172, 4171-4177.[Medline]
Lundeen, S. G. & Savage, D. C. (1992a). Multiple forms of bile salt hydrolase from Lactobacillus sp. strain 100-100. J Bacteriol 174, 7217-7220.[Abstract]
Lundeen, S. G. & Savage, D. C. (1992b). Characterization of an extracellular factor that stimulates bile salt hydrolase activity in Lactobacillus sp. strain 100-100. FEMS Microbiol Lett 94, 121-126.
Min, G. & Powell, J. R. (1998). Long-distance genome walking using the long and accurate polymerase chain reaction. Biotechniques 24, 398-400.[Medline]
Moser, S. A. & Savage, D. C. (2001). Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli. Appl Environ Microbiol 67, 3476-3480.
Saier, M. H., Jr, Beatty, J. T., Goffeau, A. & 11 other authors (1999). The major facilitator superfamily. J Mol Microbiol Biotechnol 2, 257279.
Savage, D. C. (1977). Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 31, 107-133.[Medline]
Savage, D. C. (2000). Probiotic bacteria in the gastrointestinal environment: factors influencing their survival and colonization. Biosci Microflora 19, 9-14.
Savage, D. C., Lundeen, S. G. & OConnor, L. T. (1995). Mechanisms by which indigenous microorganisms colonise epithelial surfaces as a reservoir of the lumenal microflora in the gastrointestinal tract. Microecol Ther 21, 27-36.
Stellwag, E. J. & Hylemon, P. B. (1976). Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis. Biochim Biophys Acta 452, 165-176.[Medline]
Tanaka, H., Hashiba, H., Kok, J. & Mierau, I. (2000). Bile salt hydrolase of Bifidobacterium longum biochemical and genetic characterization. Appl Environ Microbiol 66, 2502-2512.
Thanassi, D. G., Cheng, L. W. & Nikaido, H. (1997). Active efflux of bile salts by Escherichia coli. J Bacteriol 179, 2512-2518.[Abstract]
Weber, B. A., Klein, J. R. & Henrich, B. (1998). The arbZ gene from Lactobacillus delbrueckii subsp. lactis confers to Escherichia coli the ability to utilize the ß-glucoside arbutin. Gene 212, 203-211.[Medline]
Wong, M. H., Oelkers, P., Craddock, A. L. & Dawson, P. A. (1994). Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter. J Biol Chem 269, 1340-1347.
Received 1 May 2001;
revised 15 August 2001;
accepted 20 August 2001.