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. 2004 Jan;186(2):316-25.
doi: 10.1128/jb.186.2.316-325.2004.

The Alternative Sigma Factor sigmaB of Bacillus Cereus: Response to Stress and Role in Heat Adaptation

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Free PMC article

The Alternative Sigma Factor sigmaB of Bacillus Cereus: Response to Stress and Role in Heat Adaptation

Willem van Schaik et al. J Bacteriol. .
Free PMC article

Abstract

A gene cluster encoding the alternative sigma factor sigma(B), three predicted regulators of sigma(B) (RsbV, RsbW, and RsbY), and one protein whose function is not known (Orf4) was identified in the genome sequence of the food pathogen Bacillus cereus ATCC 14579. Western blotting with polyclonal antibodies raised against sigma(B) revealed that there was 20.1-fold activation of sigma(B) after a heat shock from 30 to 42 degrees C. Osmotic upshock and ethanol exposure also upregulated sigma(B), albeit less than a heat shock. When the intracellular ATP concentration was decreased by exposure to carbonyl cyanide m-chlorophenylhydrazone (CCCP), only limited increases in sigma(B) levels were observed, revealing that stress due to ATP depletion is not an important factor in sigma(B) activation in B. cereus. Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a sigma(B)-dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated. A second sigma(B)-dependent promoter was identified upstream of the last open reading frame (orf4) of the sigB operon. Production of virulence factors and the nonhemolytic enterotoxin Nhe in a sigB null mutant was the same as in the parent strain. However, sigma(B) was found to play a role in the protective heat shock response of B. cereus. The sigB null mutant was less protected against the lethal temperature of 50 degrees C by a preadaptation to 42 degrees C than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50 degrees C.

Figures

FIG. 1.
FIG. 1.
Diagram of the organization of the sigB gene cluster of B. cereus ATCC 14579. The large arrows represent open reading frames and indicate their orientations and sizes. The code numbers of these open reading frames in the B. cereus genome database (20) are RZC05131, RZC05126, RZC05124, RZC05127, and RZC05128. The predicted sizes of the encoded proteins (in amino acids [aa]) are also indicated. Predicted terminators downstream of orf4 and rsbY are indicated by stem-loop structures. σB-dependent promoters identified in this study are indicated by small arrows. The levels of amino acid identity with homologues of the open reading frames in B. anthracis Ames (39), B. cereus ATCC 10987 (http://www.tigr.org), B. thuringiensis subsp. israelensis ATCC 35646 (http://www.ergo-light.com), and B. subtilis 168 (26) are indicated at the bottom. Note that in B. subtilis 168 the gene encoding the RsbY homologue (rsbP) is not located directly downstream of the sigB operon like rsbY in the B. cereus group.
FIG. 2.
FIG. 2.
Overproduction and purification of σB. (Left panel) SDS-PAGE of protein extracts from E. coli BL21-Codonplus-(DE3)-RIL carrying either pET28-b or pMT01. (Right panel) Immunodetection of σB with anti-σB antiserum. Ten micrograms of protein was loaded for each sample. Lane 1, crude protein extract from E. coli carrying pET28-b; lane 2, crude protein extract from E. coli carrying pMT01; lane 3, crude protein extract from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 4, inclusion bodies isolated from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 5, representative fraction of purified σB after elution from an Ni2+ affinity column. The arrow indicates the position of the overproduced σB protein.
FIG. 3.
FIG. 3.
Stress-induced activation of σB in B. cereus. (A) Cellular σB levels upon exposure to stress. Protein extracts from mid-logarithmic-phase B. cereus cells (lane 1) and stressed B. cereus cells were prepared as described in Materials and Methods. B. cereus cells in the mid-logarithmic growth phase were exposed to 4% (vol/vol) ethanol (lane 2), pH 5.2 (the pH was adjusted by addition of HCl) (lane 3), 2.5% (wt/vol) NaCl (lane 4), 50 μM H2O2 (lane 5), 1 mM diamide (lane 6), and 42°C for 30 min (lane 7). Proteins were also extracted from an overnight culture (lane 8). Forty micrograms of protein of each sample was loaded on the SDS-PAGE gel. Immunoblotting was performed with the sample by using the σB antiserum. (B) Relative amounts of σB. The signal intensities from the Western blot shown in panel A were quantified by using the Gel-Pro Analyzer software package (Media Cybernetics). The value for the mid-logarithmic-phase culture was defined as 1.
FIG. 4.
FIG. 4.
Effects of CCCP on growth, the intracellular concentration of ATP, and σB expression of B. cereus. (A) Growth (circles) and intracellular ATP concentration (bars) in B. cereus cells from a mid-logarithmic-phase culture (ML) after exposure to 1, 2, 10, and 50 μM CCCP (left panel) and after a heat shock (HS) at 42°C for 30 min (right panel). Growth of the culture was monitored by determining the increase in the OD600 during the 30 min of exposure to CCCP or 42°C. The increase in the OD600 over 30 min for an untreated culture was defined as 100%, and growth in the CCCP-treated and heat-shocked cultures was related to this value. Intracellular ATP concentrations were determined by the firefly luciferase assay as described in Materials and Methods. (B) Relative levels of σB in CCCP-treated B. cereus cells (lanes 1 to 5 contained 0, 1, 2, 10, and 50 μM CCCP, respectively) and heat-shocked B. cereus cells (lane 6). The cellular levels of σB were estimated by immunoblotting with anti-σB antiserum and quantification of the signal by the Gel-Pro Analyzer software package as described in the text. Forty micrograms of protein from each sample was loaded on the SDS-PAGE gel used for Western blotting.
FIG. 5.
FIG. 5.
Analysis of transcription of the sigB operon in B. cereus. (A) Northern blot analysis of transcription of the sigB operon. Total RNA was extracted from B. cereus ATCC 14579 and B. cereus FM1400 cells during mid-logarithmic growth in BHI medium (lanes 1 and 3, respectively) and after a 10-min exposure to 42°C (lanes 2 and 4, respectively). 32P-labeled internal PCR products of rsbV and orf4 were used as probes. Hybridization of the probe with target RNA was visualized by exposure to a Phosphoscreen and scanning with a Storm scanner. Transcript sizes are indicated by arrowheads. (B) Primer extension analysis of promoters 5′ of rsbV and orf4. For all reactions 50 μg of RNA was used. Total RNA was extracted from B. cereus ATCC 14579 and B. cereus FM1400 cells during logarithmic growth in BHI medium (lanes 1 and 3, respectively) and after a 10-min exposure to 42°C (lanes 2 and 4, respectively). Mapped transcriptional start sites are indicated by arrowheads. Lanes A, C, G, and T contained the corresponding sequencing ladders for localization of the transcripts. (C) Promoter sequence alignment for the σB-dependent promoters 5′ of rsbV and orf4. The positions of identified −35 and −10 regions are indicated. The nucleotides in boxes in the −35 and −10 regions fit the σB promoter consensus sequence of B. subtilis (18). Uppercase letters in the B. subtilis σB promoter consensus sequence indicate highly conserved residues, and lowercase letters indicate less conserved residues (R = A or G, W = A or T). Transcriptional start sites and ATG start codons are indicated by boldface type. Putative Shine-Dalgarno sequences are underlined.
FIG. 6.
FIG. 6.
Survival of of B. cereus ATCC 14579 cells (circles) and B. cereus FM1400 cells (squares) in the mid-logarithmic growth phase at 30°C (open symbols) and after pretreatment at 42°C for 30 min before exposure to 50°C (solid symbols). The averages of three independent experiments are shown. The error bars indicate standard deviations.

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