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Genetics and Molecular Biology

Bile Salt Hydrolase of Bifidobacterium longum—Biochemical and Genetic Characterization

Hiroshi Tanaka, Honoo Hashiba, Jan Kok, Igor Mierau
Hiroshi Tanaka
Snow Brand European Research Laboratories, 9747 AN Groningen, and
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Honoo Hashiba
Snow Brand European Research Laboratories, 9747 AN Groningen, and
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Jan Kok
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands
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Igor Mierau
Snow Brand European Research Laboratories, 9747 AN Groningen, and
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DOI: 10.1128/AEM.66.6.2502-2512.2000
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  • Fig. 1.
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    Fig. 1.

    Genetic and transcriptional organization of thebsh site (A) and RT-PCR analysis of the transcript of thebsh region (B and C). Pbsh, putativebsh promoter; A through E, primers A through E used for RT-PCR (Table 2); MWM, molecular weight marker; DNA, control reaction; +AMV, first-strand reaction with AMV; −AMV, first-strand reaction without AMV. (B) First-strand cDNA synthesis with primer E. (C) First-strand cDNA synthesis with primer C. In the lanes in which primers A and C were used and in the lanes in which primers A and F were used three times as much RT-PCR product was applied in order to obtain bands whose intensity was similar to that of the DNA control.

  • Fig. 2.
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    Fig. 2.

    Purification and immunostaining of B. longumBSH. (A) SDS-PAGE (10% polyacrylamide) after silver staining. Lane MW, molecular weight marker; lane CFE, cell extract; lanes Q, S, and GF, BSH active fraction after MonoQ chromatography, MonoS chromatography, and gel filtration, respectively. (B) Immunostaining with anti-C. perfringens BSH antibodies. Lane CFE, cell extract; lanes Q, S, and GF, BSH active fraction after MonoQ chromatography, MonoS chromatography, and gel filtration, respectively; lanes P and N, cell extract of E. coli with and withoutC. perfringens BSH, respectively.

  • Fig. 3.
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    Fig. 3.

    Alignment of BSH of various bacteria, of a hypothetical protein of Bacillus subtilis, and PVA of Bacillus sphaericus. Asterisks, identical amino acids; dots, similar amino acids. The boxes indicate amino acids involved in the active site of PVA, including C-1, D-20, Y-82, N-175, and R-228 (positions based on the B. sphaericus DNA sequence). The amino acid sequence which was determined with purified BSH is indicated by boldface type. The enzymes examined were the BSH of B. longum (BSHBLO),L. johnsonii BSH (BSHLBJ), L. plantarum (BSHLBP), and C. perfringens (BSHCLP); the hypothetical protein ofBacillus subtilis (HYPBSU); and Bacillus sphaericus PVA (PENABSP).

  • Fig. 4.
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    Fig. 4.

    Plasmid maps of plasmids pBH13 and pBH16, which confer BSH activity, and two subclones. Heavy lines, plasmid pUK21; boldface restriction sites, sites present in the insert; Plac,E. coli lacZ promoter.

  • Fig. 5.
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    Fig. 5.

    Nucleotide sequence of the bsh gene region ofB. longum. IR, inverted repeat; DR, direct repeat; −10, putative −10 promoter region; SD, putative SD sequence. The N-terminal amino acid sequence determined for purified BSH is underlined. Mutations are indicated above the nucleotide sequence by boldface letters which indicate the exchanged or inserted nucleotides. The amino acids of the supposed active site (C-1, D-20, N-81, N-172, and R-225) are double underlined.

Tables

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  • Table 1.

    Strains and plasmids

    Strain or plasmidPropertiesaReference or source
    Strains
     B. longumSBT2928Wild type, isolated from human fecesSnow Brand Milk Products Co., Ltd., Kawagoe, Japan
     B. longum SBT2928 mutant A3Spontaneous BSH-negative mutantThis study
     B. longum SBT2928 mutant 2001UV-induced BSH-negative mutantThis study
     B. longum SBT2928 mutant 2503UV-induced BSH-negative mutantThis study
     E. coli TOP10F−mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139Δ(ara-leu)7697 galU galK rpsL endA1 nupGInvitrogen Corp., Carlsbad, Calif.
     E. coli NM522supE thi Δ(lac-proAB) Δhsd5 (r− m−) [F′ proAB lacIqZΔM15]14
    Plasmids
     pUK21Kmr, multipurpose cloning vector55
     pCR-BluntKmr, blunt end cloning vectorInvitrogen Corp., Carlsbad, Calif.
     pBH13Kmr, 2.7-kb insert of the chromosome ofB. longum SBT2928, confers BSH activityThis study
     pBH16Kmr, 8-kb insert of the chromosome ofB. longum SBT2928, confers BSH activityThis study
     pBH13221.2-kb fragment of pBH13 cloned in pCR-BluntThis study
     pBH13511.5-kb fragment of pBH13 cloned in pCR-Blunt, confers BSH activityThis study
     pCR-A3bshregion obtained by PCR of bsh of B. longumSBT2928 mutant A3 cloned into pCR-BluntThis study
     pCR-2001bsh region obtained by PCR ofbsh of B. longum SBT2928 mutant 2001 cloned into pCR-BluntThis study
     pCR-2503bsh region obtained by PCR of bsh of B. longum SBT2928 mutant 2503 cloned into pCR-BluntThis study
     pCA-1pBH1351 with Cys-1–to–Ala mutationThis study
    • ↵a Kmr, kanamycin resistance.

  • Table 2.

    Primers used for PCR

    PrimerSequence (5′ to 3′)Use
    ACTG CCA CCT GTA TTC GAAmplification of mutantbsh and RT-PCR
    BAGC ACT GGT GTC CGT TTC TRT-PCR
    CGAC CTC GTC GAC GGA GTRT-PCR
    DGCC TCG TTC GTC CAC GART-PCR
    ETTG CTC AAT GCC ATA TCAmplification of mutantbsh and RT-PCR
    FCGG ACA CCA GTG GAC ATRT-PCR
    ALAGCC ACT GGT GTC CGT TTC TaCys-1–to–Ala mutation primer
    SALGAC CTC GTC GAC GGA GTbPrimer with SalI site for SDMd
    ATGCAT AAC GGA CTC CCT TCcPrimer with ATG for SDMd
    FWGTA AAA CGA CGG CCA GStandard −20 forward primer
    • ↵a The boldface type indicates nucleotide changes.

    • ↵b The boldface type indicates anSalI site.

    • ↵c The boldface type indicates a complementary sequence for the ATG start codon.

    • ↵d SDM, site-directed mutagenesis.

  • Table 3.

    Effect of DTT on BSH activity after sonication ofB. longum SBT2928 cells

    Length of sonication (min)Sonication conditionsaAssay conditionsaActivitybProtein concn (mg/ml)Sp actb
    U/ml%U/mg%
    3−DTT−DTT1.04120.891.1711
    −DTT+DTT4.6550.895.250
    +DTT−DTT9.81120.8311.8112
    +DTT+DTT8.71000.8310.5100
    9−DTT−DTT0.4741.610.34
    −DTT+DTT3.9371.612.435
    +DTT−DTT10.81031.576.9103
    +DTT+DTT10.51001.576.7100
    • ↵a −DTT, no DTT present; +DTT, 10 mM DTT present.

    • ↵b The level of activity in the presence of 10 mM DTT after sonication in the presence of 10 mM DTT was defined as 100%.

  • Table 4.

    Relative activity and Km values ofB. longum BSH with various bile salts

    Substrate (−OH positions)aRelative activity (%)Km (mM)
    GCA (3α, 7α, 12α)850.16
    GDCA (3α, 12α)910.28
    GCDCA (3α, 7α)1000.13
    TCA (3α, 7α, 12α)431.12
    TDCA (3α, 12α)290.79
    TCDCA (3α, 7α)510.33
    THDCA (3α, 6α)11NDb
    TUDCA (3α, 7β)19ND
    • ↵a GCA, glycocholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; THDCA, taurohyodeoxycholic acid; TUDCA, tauroursodeoxycholic acid.

    • ↵b ND, not determined.

  • Table 5.

    Effects of various ions and inhibitors on BSH activity

    SubstanceaConcn (mM)% Inhibition
    Iodoacetate387
    Periodic acid3100
    N-Ethylmaleimide167
    HgCl20.186
    pCMBA0.0187
    CuCl25100
    CaCl23094
    MgSO43084
    MgCl2300
    EDTA3018
    PMSF0.526
    NaCl50152b
    • ↵a pCMBA,para-chloromercuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride.

    • ↵b Percent activation.

  • Table 6.

    Alignment of the regions immediately upstream of the ATG start codons of six Bifidobacterium genes and B. longum BSH with the 16S rRNA 3′-terminal consensus sequence

    Embedded Image
    • a The sequence of the 3′ end ofBifidobacterium 16S ribosomal DNA is 3′ tcttcctccactagg 5′, shown in lowercase letters under each SD region. The box indicates the central AGG motif of the SD region in bifidobacteria.

    • b Distance (in nucleotides) between ATG and the core AGG sequence in the SD region.

    • c The numbers in parentheses are GenBank nucleotide sequence accession numbers.

    • d Possible alignment of the 3′ end of the 16S ribosomal DNA with the upstream region of bsh.

  • Table 7.

    Overview of bsh mutants

    MutantMutationActivity in B. longumActivity in E. coliProtein in B. longumaProtein in E. colia
    A3Spontaneous mutant, G-A transition creating a stop codon in place of amino acid 200 (Trp), deletion of 117 amino acidsNo activity detectedActivityNo protein detectedProtein of original size
    2503UV-induced T-C transition causing Leu80Ser exchangeNo activity detectedNo activity detectedProtein presentProtein present
    2001Insertion of G after GG of Gly-267, causing a frameshift and loss of the last 50 amino acidsNo activityNo activityNo proteinNo protein
    C1AbSite-directed mutagenesis of TG of Cys-1 to GC, resulting in Cys1Ala exchangeNo activityProtein present
    • ↵a The presence of BSH was determined by Western blotting with polyclonal antibodies.

    • ↵b Mutation present only in bshcloned in E. coli.

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Bile Salt Hydrolase of Bifidobacterium longum—Biochemical and Genetic Characterization
Hiroshi Tanaka, Honoo Hashiba, Jan Kok, Igor Mierau
Applied and Environmental Microbiology Jun 2000, 66 (6) 2502-2512; DOI: 10.1128/AEM.66.6.2502-2512.2000

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Bile Salt Hydrolase of Bifidobacterium longum—Biochemical and Genetic Characterization
Hiroshi Tanaka, Honoo Hashiba, Jan Kok, Igor Mierau
Applied and Environmental Microbiology Jun 2000, 66 (6) 2502-2512; DOI: 10.1128/AEM.66.6.2502-2512.2000
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KEYWORDS

Amidohydrolases
Bifidobacterium

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