SigM, an Extracytoplasmic Function Sigma Factor ofBacillus subtilis, Is Activated in Response to Cell Wall Antibiotics, Ethanol, Heat, Acid, and Superoxide Stress

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Abstract
The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (PA) is sigma A dependent, and the other (PM) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the PM fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 μg per ml), and by heat shock of 50°C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 μM paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H2O2, the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 μM monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 μg of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival—a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.

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