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 (P A ) is sigma A dependent, and the other (P M ) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the P M 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 g per ml), and by heat shock of 50°C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 M paraquat, but after a 15-to 30-min delay. There was no induction by alkali (pH 9), 5 mM H 2 O 2 , the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 M 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 g 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.Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related gram-positive pathogens, this response is governed by the sigma B transcription factor. B. subtilis also encodes seven potential extracytoplasmic function (ECF) RNA polymerase sigma factors (9) that also contribute to stress resistance, but in a rather different fashion. There is evidence that all ( M , V , W , X , Y , Z , and YlaC) are expressed in B. subtilis (12), and several have been shown to contribute to stress resistance.The best-characterized ECF sigma factors of B. subtilis are W and X . Sigma W is expressed maximally late in growth (12) and switches on a large regulon (3, 15), including a large fraction of the genes that are most strongly induced in response to alkali shock (29). The sigX gene is also switched on in late logarithmic phase, and sigX mutants are impaired in the ability to survive at high temperature and oxidative stress (14).The sigM gene is cotranscribed with yhdL and yhdK, which negatively regulate SigM activ...

Promoters of nine Bacillus subtilis genes (bcrC, yacK, ydaH, yfnI, yjbD, ypbG, ypuA, yraA, and ysxA), all responsive to artificially induced increases in the stress-responsive extracytoplasmic function sigma factor, SigM, were mapped by rapid amplification of cDNA ends-PCR. The resulting promoter consensus suggests that overlapping control by SigX or SigW is common.

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 M), inosine germination was completely blocked in the mutant, while the rate of germination in 50 M L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.Under certain nutrient stresses Bacillus species undergo a complex differentiation process resulting in the formation of highly resistant endospores, which subsequently, when favorable growth conditions return, germinate back to vegetative cells and grow and divide. The molecular genetics of sporulation (7, 25) and germination (14, 15) of Bacillus species have been reviewed. Analysis of Bacillus subtilis mutants which are defective in response to one or both of their germinants-L-alanine or asparagine with glucose, fructose, and K ϩ (AGFK)-suggests that the germinants interact with separate germinant-specific receptor complexes within the spore (14). Mutations within the gerA operon of B. subtilis specifically block germination initiated by L-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded by the gerA operon suggest that they may be membrane associated, and they are the most likely candidates for the germinant receptor for L-alanine. Mutations in the gerB operon (18), responsible for AGFK germination in B. subtilis, allowed recognition of the novel germinant D-alanine; this strongly reinforces the argument that such gerA homologues encode the germinant receptor complexes.B. cereus endospores have been shown to germinate in response to inosine and L-alanine; a combination of these germinants elicits the most rapid response (32). Operons encoding putative receptor complexes for the germinants have been named gerI (6) and gerL (3), respectively, and these operons are members of the gerA family identified in B. subtilis. How the germinant receptors are activated and how this leads to the global changes of spore germination have, however, not...

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