Spore development and stress resistance in Bacillus subtilis are governed by the master transcription factors Spo0A and B , respectively. Here we show that the coding genes for both regulatory proteins are dramatically induced, during logarithmic growth, after a temperature downshift from 37 to 20°C. The loss of B reduces the stationary-phase viability of cold-adapted cells 10-to 50-fold. Furthermore, we show that B activity is required at a late stage of development for efficient sporulation at a low temperature. On the other hand, Spo0A loss dramatically reduces the stationary-phase viability of cold-adapted cells 10,000-fold. We show that the requirement of Spo0A for cellular survival during the cold is independent of the activity of the key transition state regulator AbrB and of the simple loss of sporulation ability. Furthermore, Spo0A, and not proficiency in sporulation, is required for the development of complete stress resistance of cold-adapted cells to heat shock (54°C, 1 h), since a loss of Spo0A, but not a loss of the essential sporulation transcription factor F , reduced the cellular survival in response to heat by more than 1,000-fold. The overall results argue for new and important roles for Spo0A in the development of full stress resistance by nonsporulating cells and for B in sporulation proficiency at a low temperature.The exposure of bacteria to diverse growth-limiting conditions induces the synthesis of a large set of proteins (called general stress proteins) that protect the cell against internal (metabolic) or external (environmental) stresses (22,23,29,32,33). In the gram-positive, endospore-forming bacterium Bacillus subtilis, the general stress response is controlled mainly by B , the alternative transcription factor of the RNA polymerase that brings about a special physiological state which significantly enhances bacterial survival (11,20,22,23,29,32,33,37). It is estimated that over 200 genes (5% of the coding capacity of the genome) are directly or indirectly under B control, and the loss of B function leads to multiple-stress sensitivity, compromising the survival of the B null mutant strain (23,29,32). Besides having this very important, rapid, reversible, and plastic adaptive response (22,29), B. subtilis is also able to differentiate into dormant spores when nutritional conditions become so extreme that the B -dependent response would not be adequate to guarantee the survival of the cell (19,21,24,30,31). While B is the key regulatory protein involved in the reversible adaptive stress response of vegetative cells, the master transcription factor Spo0A is the key regulator responsible for the decision of a vegetative cell to differentiate into a dormant and highly resistant new cell, i.e., the spore (31). It is accepted that these responses, general stress adaptation and sporulation, are important for the survival of B. subtilis in its natural environment, i.e., soil (29-33). Furthermore, high levels of expression of general stress proteins provide stressed or starved cells with mu...

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (P i ), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of P i -supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that P i was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that P i acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.Clostridium perfringens is a gram-positive, anaerobic, endospore-forming bacterium causing gastrointestinal and histotoxic infections in humans and animals (2, 6, 9, 17). The virulence of this bacterium largely results from its prolific ability to produce at least 15 different toxins (18). In addition, enterotoxigenic C. perfringens isolates produce a 35-kDa enterotoxin (C. perfringens enterotoxin [CPE]), whose synthesis is under a strict positive control of sporulation (3,5,6,9,17). In C. perfringens, the production of CPE is confined to the large compartment (mother cell) of the sporangium where cpe transcription is believed to be driven from the mother cell-specific forms of the RNA polymerase, RNA-E and RNA-K (30). The copious amount of CPE (as much as 10% or more of the total protein of the developing sporangium) is accumulated probably only in the cytoplasm of the mother cell compartment until its release when the mother cell lyses at the completion of sporulation to liberate the mature spore (17). The released CPE rapidly binds to protein receptors present on the apical surface of enterocytes and induces cell permeabilization with the concomitant appearance of the symptoms of enterotoxaemia, intestinal cramping, and diarrhea (2,17,18).Despite the key role of spores in CPE synthesis and in the dissemination and developing of clostridial diseases, very little is known at the molecular level about the regulatory mechanisms governing the formation of spores in clostridia (6,9,11,13,20,23). Although from genome sequence analyses it can be assumed that the mechanism of spore formation in Bacillus and Clostridium is conserved (21,24,25), the main differences reside at the level of the initiation of the sporulation process (24, 25). While orthologs for spo0A and the genes activated by Spo0AϳP, along with most of the spo genes that are subsequently expressed during the morphogenesis of the spore,...