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

Abstract

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.

Phenolic Acid-Mediated Regulation of the padC Gene, Encoding the Phenolic Acid Decarboxylase of Bacillus subtilis

“…Relative expression levels were calculated using the comparative critical threshold (⌬⌬C T ) method RTL ϭ 2 ͑⌬CT of sample Ϫ ⌬CT of reference͒ (22). The 16S rRNA was chosen as an internal control to calculate the threshold cycles as (6). To calculate the relative transcript levels (RTL), the ⌬C T s of the genes were compared to the ⌬C T s of the same genes in the reference conditions.…”

High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

“…The generated standard curves were used to convert the Ct values of each amplified gene in the cDNA preparations to copy numbers of cDNA molecules. The estimated copy number was normalized to the value for 16 S rRNA, as previously described [44]. …”