Multiple physiological and environmental signals are needed to initiate endospore formation in Bacillus subtilis. One key event controlling sporulation is activation of the Spo0A transcription factor. Spo0A is a member of a large family of conserved regulatory proteins whose activity is controlled by phosphorylation. We have isolated deletion mutations that remove part of the conserved amino terminus of Spo0A and make the transcription factor constitutively active, indicating that the amino terminus normally functions to keep the protein in an inactive state. Expression of an activated gene product is sufficient to activate expression of several sporulation genes in the absence of signals normally needed for initiation of sporulation. Our results indicate that nutritional, cell density, and cell-cycle signals are integrated through the phosphorylation pathway that controls activation of Spo0A.
The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn9171ac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn9l7 mutation was also found to be an allele of aid. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spoOA, spoOH, spoOK, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.Cells of Bacillus subtilis can differentiate into dormant heat-resistant endospores under appropriate environmental conditions. Regulatory events during the initiation of sporulation lead to the formation of an asymmetric cell division septum, generating two distinct cell types. The smaller cell, or forespore, develops into the mature spore after being engulfed by the larger cell, the mother cell (reviewed in reference 10). Eventually, the mother cell lyses, releasing the mature heatresistant endospore.Dramatic changes in gene expression, physiology, and metabolism underlie the morphological changes associated with spore formation. A variety of regulatory circuits and genes required for sporulation have been characterized. In addition, many genes that are expressed during sporulation are not essential for development (10). While much work has focused on gene expression and regulation, relatively little is known about metabolism and the generation of energy for synthesis of new products required for the morphological development that occurs during sporulation. The tricarboxylic acid cycle seems to play a role in the generation of energy during sporulation, as mutants that are defective in tricarboxylic acid cycle enzymes are defective in sporulation (13,18,56). Protein turnover is known to increase during sporulation (30, 47) and probably plays a role in generating substrates (peptides and amino acids) for further metabolism and new macromolecular synthesis.Previously, we described the isolation and characterization of mutations that caused a defect in sporulation but could be partially suppressed by the presence or overproduction of the histidine protein kinase encoded by kinA (19,20,37). These mutations were called ski (pronounced "sky," for suppressed by kinase) and include spoOK (37) and bofA (20,36). Kin...
The development of competence in Bacillus subtilis is regulated by growth conditions and several regulatory genes. In complex media competence development is poor, and there is little or no expression of late competence genes. mec mutations permit competence development and late competence gene expression in complex media, and bypass the requirements for many of the competence regulatory genes. In this paper we describe the cloning and characterization of mecA. The mecA gene product acts negatively in the development of competence. Null mutations in mecA allowed expression of a late competence gene comG, under conditions where it is not normally expressed, including in complex media and in cells mutant for several competence regulatory genes. Overexpression of MecA from a multicopy plasmid resulted in inhibition of comG transcription. The DNA sequence of mecA was determined and the predicted gene product showed no significant similarity to any protein in the database. Expression of a mecA-lacZ translational fusion was constitutive during growth and did not vary significantly in the different media tested. The role of mecA in competence development and other stationary phase phenomena is discussed.
spoOA and spoOH are needed for the initiation of sporulation and for the development of genetic competence in Bacillus subtilis. Transcription of spoOA initiates from two promoters, Pv and Ps. Pv is active during vegetative growth and is recognized by RNA polymerase containing oA. Expression from Ps increases during sporulation and depends on cH, the spoOH gene product. A deletion mutation, spoOAAPs, that removes the promoter controlled by frH blocked sporulation but had no detectable elfect on competence. These results indicate that expression of spoOA from Ps is necessary for sporulation and that the requirement for spoOH in competence development is not due to its role in expression of spoOA.Under conditions of nutrient deprivation, cells of Bacillus subtilis enter into a developmental pathway that leads to the production of dormant, heat-resistant endospores. Sporulation involves the production of two distinct cell types with different developmental fates (reviewed in reference 11). Sporulation is not the only form of development in B. subtilis. When cells are grown in minimal glucose medium, a fraction of the cells in a culture differentiate to become competent to take up exogenous DNA (reviewed in reference 9).Both sporulation and competence development are controlled in part by the spoOA gene product, a transcription factor that functions as an activator and a repressor, depending on the location of the target binding site. SpoOA is a response regulator of the large family of two-component regulatory systems (23). The activity of SpoOA is controlled by phosphorylation (6), and the phosphorylated (activated) form has a higher affinity for DNA than does the nonphosphorylated form (4,5,31,32). Several proteins are required for the phosphorylation of SpoOA, including the histidine protein kinases KinA (3, 24), KinB (33), and KinC (19) and the phosphotransfer proteins SpoOF and SpoOB. One or more kinases autophosphorylate on a histidine residue and serve as substrate to donate phosphate to SpoOF. SpoOB then acquires the phosphate from SpoOF-P, and finally phosphate is transferred from SpoOB-P to SpoOA (6). A threshold concentration of SpoOA-P appears to be necessary for the initiation of sporulation (8).Transcription of spoOA is controlled by two promoters, Pv and Ps (7). Pv is expressed during vegetative growth and is transcribed by RNA polymerase containing the major sigma factor OA. Ps is induced during sporulation and requires (u as well as SpoOA-P for expression (26,30). ( H, the spoOH gene product, is also needed for the development of competence and the expression of competence genes (2, 17).Previous work had characterized the role of Pv in sporulation (7). Pv is not needed for sporulation under normal conditions in the absence of glucose. However, mutations that * Corresponding author. Phone: (617) Construction of spoOAAPs. To test the role of spoOAPs in competence and sporulation, we constructed a deletion mutation that removed the sporulation promoter, Ps. This mutation is contained in plasmid pSK5 ...
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