An efficient expression and secretion system based on Bacillus subtilis phage φ105 and its use for the production of B. cereus β-lactamase I

Thornewell, Susan J.; East, Alison K.; Errington, Jeff

doi:10.1016/0378-1119(93)90223-p

Cited by 27 publications

(21 citation statements)

References 21 publications

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“…In this construct, the coding region of the FZB45 phyA gene is preceded by strong Bacillus phage φ105 holin promoter (Leung & Errington, 1995). After linearizing with ScaI the recombinant plasmid was used to transform competent B. subtilis MU331 containing defective prophage φ105 sequence (Thornewell et al, 1993). The defective MU331 prophage has been deleted in a region needed for cell lysis, and contains a ts mutation in the phage immunity repressor allowing induction by a shift in temperature without concomitant cell lysis (Gibson & Errington, 1992).…”

Section: Methodsmentioning

confidence: 99%

“…To overexpress phytase in B. subtilis the phytase gene was subcloned into the integrative Bacillus expression vector pSG1112, a derivative of pSG703 (Thornewell et al, 1993) using the NdeI and BamHI cloning sites of the vector plasmid. In this construct, the coding region of the FZB45 phyA gene is preceded by strong Bacillus phage φ105 holin promoter (Leung & Errington, 1995).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect a aThe GenBank accession numbers for the sequences determined in this work are AY055219 to AY055226.

et al. 2002

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect a aThe GenBank accession numbers for the sequences determined in this work are AY055219 to AY055226.

et al. 2002

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“…Water for all solutions was purified using a Milli-Q water purification system (Millipore were replaced by cysteine, were constructed in the phagemid vector pKS ca tPl (obtained from T. Leung, Sir William Dunn School of Pathology Oxford) using the uracil template protocol [9] and expressed in Bacillus subtilis [10] with the bacteriophage vector pSG703. In this expression system the enzyme is secreted into the medium after heat shock and 6 h growth.…”

Section: Methodsmentioning

confidence: 99%

Site‐specific introduction of an electroactive label into a non‐electroactive enzyme (β‐lactamase I)

et al. 1997

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A cysteine residue was introduced close to the active site of β-lactamase I by site-directed mutagenesis to replace tyrosine-105 and was subsequently modified with an electroactive SH-speciflc reagent, 7V-(2-ferrocene-ethyl)maleimide. The resulting modified enzyme became electroactive, showing good quasireversible electrochemistry which was characteristic of the attached ferrocene moiety while retaining its specific enzymatic activity. In the presence of a suicide substrate, 6P-iodopenicillanic acid, the redox potential shifted +20 mV suggesting that the label was sensitive to changes in the active site of the enzyme.

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“…At least 28 regions were found to align with the consensus sequence GWCGKRAA TWCMAK (W ¼ A or T, K ¼ G or T, R ¼ A or G, M ¼ A or C), including the O R 1-O R 6 (data not shown). In the thermo-inducible expression system, 14,15 the gene of interest is cloned downstream of a strong holin gene promoter. 14 However, no such consensus sequence was identified at the 2 35 or 2 10 RNA recognition regions of the holin gene promoter.…”

Section: Sequence Analysis Of Mtcf105 and Orf4mentioning

confidence: 99%

“…10,11 A thermo-inducible expression system based on phage f105 has been established in Bacillus subtilis. 14,15 Unlike other Bacillus expression systems that employed plasmid-based, constitutive promoters, this prophage expression system is capable of driving recombinant protein expression in a thermo-inducible manner. The complete suppression of heterologous protein expression before thermo-induction is desirable for expressing toxic products.…”

Section: Introductionmentioning

confidence: 98%

Interaction of a Putative Transcriptional Regulatory Protein and the Thermo-inducible cts-52 Mutant Repressor in the Bacillus subtilis Phage φ105 Genome

Chan

Lim

2003

Journal of Molecular Biology

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A 144 amino acid residue cts-52 mutant repressor (mtc phi 105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage phi 105 is involved in the control mechanism of a thermo-inducible expression system. Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor. On the basis of the protein sequence alignment, a DNA-binding alpha helix-beta turn-alpha helix (HTH) motif was identified in the N-terminal region (residues 18-37) of the repressor as well as in the polypeptide of ORF4 (residues 22-41). In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. To study their DNA binding properties, the wild-type repressor (wtc phi 105) and the mutant repressor mtc phi 105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays. Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR). Our data showed that a temperature shift does not influence the wtc phi 105-operators' binding affinity, while the binding of mtc phi 105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtc phi 105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures. mRNA levels monitored by real-time RT-PCR indicated a suppression of mtc phi 105 expression, but a stimulation of ORF4 transcription after thermo-induction. Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P(M) and P(R) within the immF region.

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An efficient expression and secretion system based on Bacillus subtilis phage φ105 and its use for the production of B. cereus β-lactamase I

Cited by 27 publications

References 21 publications

Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect a aThe GenBank accession numbers for the sequences determined in this work are AY055219 to AY055226.

Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect a aThe GenBank accession numbers for the sequences determined in this work are AY055219 to AY055226.

Site‐specific introduction of an electroactive label into a non‐electroactive enzyme (β‐lactamase I)

Interaction of a Putative Transcriptional Regulatory Protein and the Thermo-inducible cts-52 Mutant Repressor in the Bacillus subtilis Phage φ105 Genome

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