Article Figures & Data
Figures
- 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.
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.
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.
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.
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
- Table 1.
Strains and plasmids
Strain or plasmid Propertiesa Reference or source Strains B. longumSBT2928 Wild type, isolated from human feces Snow Brand Milk Products Co., Ltd., Kawagoe, Japan B. longum SBT2928 mutant A3 Spontaneous BSH-negative mutant This study B. longum SBT2928 mutant 2001 UV-induced BSH-negative mutant This study B. longum SBT2928 mutant 2503 UV-induced BSH-negative mutant This study E. coli TOP10 F−mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139Δ(ara-leu)7697 galU galK rpsL endA1 nupG Invitrogen Corp., Carlsbad, Calif. E. coli NM522 supE thi Δ(lac-proAB) Δhsd5 (r− m−) [F′ proAB lacIqZΔM15] 14 Plasmids pUK21 Kmr, multipurpose cloning vector 55 pCR-Blunt Kmr, blunt end cloning vector Invitrogen Corp., Carlsbad, Calif. pBH13 Kmr, 2.7-kb insert of the chromosome ofB. longum SBT2928, confers BSH activity This study pBH16 Kmr, 8-kb insert of the chromosome ofB. longum SBT2928, confers BSH activity This study pBH1322 1.2-kb fragment of pBH13 cloned in pCR-Blunt This study pBH1351 1.5-kb fragment of pBH13 cloned in pCR-Blunt, confers BSH activity This study pCR-A3 bshregion obtained by PCR of bsh of B. longumSBT2928 mutant A3 cloned into pCR-Blunt This study pCR-2001 bsh region obtained by PCR ofbsh of B. longum SBT2928 mutant 2001 cloned into pCR-Blunt This study pCR-2503 bsh region obtained by PCR of bsh of B. longum SBT2928 mutant 2503 cloned into pCR-Blunt This study pCA-1 pBH1351 with Cys-1–to–Ala mutation This study ↵a Kmr, kanamycin resistance.
- Table 2.
Primers used for PCR
Primer Sequence (5′ to 3′) Use A CTG CCA CCT GTA TTC GA Amplification of mutantbsh and RT-PCR B AGC ACT GGT GTC CGT TTC T RT-PCR C GAC CTC GTC GAC GGA GT RT-PCR D GCC TCG TTC GTC CAC GA RT-PCR E TTG CTC AAT GCC ATA TC Amplification of mutantbsh and RT-PCR F CGG ACA CCA GTG GAC AT RT-PCR ALA GCC ACT GGT GTC CGT TTC Ta Cys-1–to–Ala mutation primer SAL GAC CTC GTC GAC GGA GTb Primer with SalI site for SDMd ATG CAT AAC GGA CTC CCT TCc Primer with ATG for SDMd FW GTA AAA CGA CGG CCA G Standard −20 forward primer - Table 3.
Effect of DTT on BSH activity after sonication ofB. longum SBT2928 cells
Length of sonication (min) Sonication conditionsa Assay conditionsa Activityb Protein concn (mg/ml) Sp actb U/ml % U/mg % 3 −DTT −DTT 1.04 12 0.89 1.17 11 −DTT +DTT 4.6 55 0.89 5.2 50 +DTT −DTT 9.8 112 0.83 11.8 112 +DTT +DTT 8.7 100 0.83 10.5 100 9 −DTT −DTT 0.47 4 1.61 0.3 4 −DTT +DTT 3.9 37 1.61 2.4 35 +DTT −DTT 10.8 103 1.57 6.9 103 +DTT +DTT 10.5 100 1.57 6.7 100 - 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
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
Mutant Mutation Activity in B. longum Activity in E. coli Protein in B. longuma Protein in E. colia A3 Spontaneous mutant, G-A transition creating a stop codon in place of amino acid 200 (Trp), deletion of 117 amino acids No activity detected Activity No protein detected Protein of original size 2503 UV-induced T-C transition causing Leu80Ser exchange No activity detected No activity detected Protein present Protein present 2001 Insertion of G after GG of Gly-267, causing a frameshift and loss of the last 50 amino acids No activity No activity No protein No protein C1Ab Site-directed mutagenesis of TG of Cys-1 to GC, resulting in Cys1Ala exchange No activity Protein present
