SummaryDuring sporulation in Bacillus subtilis, the committedcell undergoes substantial membrane rearrangements to generate two cells of different sizes and fates: the mother cell and the forespore. Here, we demonstrate that the master transcription factor Spo0A reactivates lipid synthesis during development. Maximal Spo0A-dependent lipid synthesis occurs during the key stages of asymmetric division and forespore engulfment. Spo0A reactivates the accDA operon that encodes the carboxylase component of the acetylCoA carboxylase enzyme, which catalyses the first and rate-limiting step in de novo lipid biosynthesis, malonyl-CoA formation. The disruption of the Spo0A-binding box in the promoter region of accDA impairs its transcriptional reactivation and blocks lipid synthesis. The Spo0A-insensitive accDA 0A cells were proficient in planktonic growth but defective in sporulation (s E activation) and biofilm development (cell cluster formation and water repellency). Exogenous fatty acid supplementation to accDA 0A cells overcomes their inability to synthesize lipids during development and restores sporulation and biofilm proficiencies. The transient exclusion of the lipid synthesis regulon from the forespore and the known compartmentalization of Spo0A and ACP in the mother cell suggest that de novo lipid synthesis is confined to the mother cell. The significance of the Spo0A-controlled de novo lipid synthesis during B. subtilis development is discussed.

SummaryCompartmentalized gene expression during sporulation is initiated after asymmetric division by cell-specific activation of the transcription factors s s s s F and s s s s E . Synthesis of these s s s s factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the antianti-s s s s F SpoIIAA as inhibitor of Spo0A activation. This effect did not require s s s s F activity, and it was abolished by expression of the phosphorelay-independent form Spo0A-Sad67 indicating that SpoIIAA directly interfered with Spo0A ~ P generation. IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA-P. Furthermore, expression of the non-phosphorylatable mutant SpoIIAAS58A (SpoIIAA-like), but not SpoIIAAS58D (SpoIIAA-P-like), completely blocked Spo0A-dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of s s s s F and s s s s E during development.

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...