The Alternative Sigma Factor σBofBacillus cereus: Response to Stress and Role in Heat Adaptation

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Abstract
A gene cluster encoding the alternative sigma factor σB, three predicted regulators of σB (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 σB revealed that there was 20.1-fold activation of σB after a heat shock from 30 to 42°C. Osmotic upshock and ethanol exposure also upregulated σB, 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 σB levels were observed, revealing that stress due to ATP depletion is not an important factor in σB activation in B. cereus. Analysis of transcription of the sigB operon by Northern blotting and primer extension revealed the presence of a σB-dependent promoter upstream of the first open reading frame (rsbV) of the sigB operon, indicating that transcription of sigB is autoregulated. A second σB-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, σB 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°C by a preadaptation to 42°C than the parent strain was, resulting in a more-than-100-fold-reduced survival of the mutant after 40 min at 50°C.