Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains

Abstract

Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.

FIG. 1.

(A) Activity staining on a nondenaturing polyacrylamide gel. Lane 1, BSH enzyme from L. acidophilus ATCC 53546 (as a positive control); lane 2, soluble fraction of B. bifidum ATCC 11863 from sonication treatment; lane 3, purified native BSH; lane 4, soluble fraction of IPTG-induced E. coli BL21(DE3) containing pBSH36b from B-PER treatment. (B) SDS-PAGE analysis of cell extracts at each purification step. Lanes 1 through 4: active fractions from B. bifidum ATCC 11863 (lane 1, cell extract; lane 2, HIC column; lane 3, anion-exchange column; lane 4, anion-exchange column plus HIC); lane M, marker proteins (molecular masses in kilodaltons are indicated on the right).

FIG. 2.

Map of plasmids pBSH14, pBSH27, and pBSH274. Black boxes show cloning vectors pBR322 and pUC19, grey boxes indicate the position of the bsh gene, and white boxes are the cloned sequences outside the bsh gene. The arrows indicate the direction of the bsh gene. Restriction enzyme sites used for mapping the clones are shown above the maps (S, Sau3A; Nc, NcoI; B, BamHI; N, NdeI; E, EcoRI; and P, PstI).

FIG. 3.

Nucleotide sequence of the bsh gene. The N-terminal amino acid sequence determined from the purified BSH is underlined. The amino acid sequences of the proposed active site residues (C-1, D-20, N-81, N-172, and R-225) are in double-underlined boldface type. Variable regions used for the B. bifidum-specific primer set are shaded.

FIG. 4.

Multiple alignment of BSHs of various bacteria and PVAs of B. sphaericus based on the sequences around the proposed active sites of B. sphaericus PVA. Abbreviations for BSHs: bbi, B. bifidum; blo, B. longum; ljb, L. johnsonii BSH-β; efci, Enterococcus faecium; efca, Enterococcus faecalis; lmo, L. monocytogenes; lpa, L. plantarum; lga, Lactobacillus gasseri; lja, L. johnsonii BSH-α; cpe, C. perfringens; pva, B. sphaericus PVA. Conserved amino acids are highlighted in black boxes, and similar amino acids are in grey boxes. Arrows indicate the amino acids proposed to be the key residues in the active sites of B. sphaericus PVA, including C-1, D-20, Y-82, N-175, and R-228. Positions are based on the PVA of B. sphaericus, starting with the Cys residues at the N-terminal end of mature proteins (BSH and PVA).

FIG. 5.

Characterization of the bsh transcript and bsh promoter. (A) Identification of the 5′ terminus of the bsh transcript by primer extension. The TSP (+1; indicated by an arrow) is shown in the right lane. The DNA sequence is shown on the left and +1 is indicated by an asterisk. (B) Schematic organization of the bsh promoter. The putative −10 and −35 motifs of the bsh promoter are underlined, and the proposed RBS and start codon (ATG) of bsh are indicated in boldface. The TSP (+1) is enlarged. An inverted repeat sequence in the −35 region is indicated by arrows. (C) A promoter sequence predicted with NNPP software, version 2.2. The consensus −35 (TTGACA) and −10 (TATAAT) sequences are presented under the arrows, and the proposed TSP is shown in enlarged type. Score, the fitness value to the consensus promoter sequences. (D) Northern hybridization of total RNA with the probe prepared with the primers BBI-F and BBI-R. The 1.2-kb transcript corresponding to the bsh transcript is indicated by an arrow.

FIG. 6.

Map of plasmids pDB150, pUCB150, pDB095, and pUCB095. Black boxes indicate the vectors pDrive and pUC19, grey arrows indicate the position and orientation of the bsh gene (950 bp), and white boxes are for the upstream region (550 bp) of the bsh gene. P_lac, lac promoter in the vectors. Restriction enzyme sites used for the cloning in the opposite direction are HindIII (H) and KpnI (K).

FIG. 7.

Substrate specificity of B. bifidum ATCC 11863 BSH. Six major human bile salts are shown: taurocholic acid (TC), taurodeoxycholic acid (TDC), taurochenodeoxycholic acid (TCDC) glycocholic acid (GC), glycodeoxycholic acid (GDC), and glycochenodeoxycholic acid (GCDC). The relative activity was calculated using GC as a standard at 100%.

FIG. 8.

PCR products of B. bifidum strains with primers BSH-F and BSH-R (A) and BIF-F and BIF-R (B). Lanes: 1, B. bifidum ATCC 11863; 2, B. bifidum ATCC 35914; 3, B. bifidum ATCC 15696; 4, B. bifidum ATCC 29521; 5, B. bifidum KL 301; 6, B. bifidum KL 306; 7, B. longum ATCC 15707; 8, B. adolescentis ATCC 15703; 9, B. infantis ATCC 15697; and 10, B. breve ATCC 15700; M, 1-kb DNA ladder (New England Biolabs). PCR amplicons of 970 bp (A) were obtained with DNA extracted from B. bifidum strains, and amplicons of 495 bp (B) were obtained with DNA extracted from B. bifidum and B. adolescentis strains.