We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spoOH, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spoOH encodes a sigma factor of RNA polymerase, orH, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by (rH (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn9171ac, in the presence and absence of iFH. These genes had lower levels of expression in the absence of ifH than in the presence of cfH. Patterns of expression of the csh genes during growth and sporulation in wild-type and spoOH mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn9l7lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spoOH mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spoOH in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.Endospore formation in the gram-positive bacterium Bacillus subtilis involves extensive reprograming of gene expression and a series of complex morphological and physiological changes (14,25,32,37). Rapidly growing cells can be induced to differentiate upon nutrient deprivation, and all conditions of nutrient deprivation that cause efficient sporulation have been shown to cause a drop in the intracellular concentration of GDP and GTP (23,24). Furthermore, conditions that cause a drop in these nucleotides (for example, addition of the drug decoyinine [29]), even in the presence of excess nutrients, can cause efficient sporulation (15), provided that the cells are at relatively high densities (17,38). Thus, it is thought that a drop in the intracellular level of GDP and GTP is necessary and sufficient for efficient sporulation (14, 16).At least seven regulatory genes, called spoO genes, are required for the initiation of sporulation. Mutations in these genes prevent the earliest morphological change associated with sporulation, the formation of an asymmetric division septum following nutrient deprivation (26,32,33 (2,3,(45)(46)(47)(48). spoVG is normally expressed during vegetative growth, and expression increases ...

Phosphorylation of the transcription factor encoded by spo0A is required for the initiation of sporulation in Bacillus subtilis. Production and accumulation of Spo0AϳP is controlled by histidine protein kinases and the spo0 gene products. To identify additional genes that might be involved in the initiation of sporulation and production of Spo0AϳP, we isolated genes which when present on a multicopy plasmid could suppress the sporulation defect of a spo0K mutant. kinC was one gene isolated in this way. A multicopy plasmid containing kinC completely or partially suppressed the sporulation defect caused by mutations in spo0K, kinA, spo0F, and spo0B, indicating that at least when overexpressed, KinC is capable of stimulating phosphorylation of Spo0A independently of the normal phosphorylation pathway. The predicted product of kinC is 428 amino acids long and is most similar to KinA and KinB, the histidine protein kinases involved in the initiation of sporulation. In otherwise wild-type strains, kinC null mutations caused little or no defect in sporulation under the conditions tested. However, in the absence of a functional phosphorelay (spo0F or spo0B), KinC appears to be the kinase responsible for phosphorylation of the sof-1 and rvtA11 forms of Spo0A.Cells of the gram-positive soil bacterium Bacillus subtilis differentiate to form heat-resistant metabolically dormant spores under appropriate conditions. The initiation of sporulation depends upon activation of the Spo0A transcription factor by phosphorylation (20), and a threshold concentration of Spo0AϳP appears to be required (11). Spo0AϳP is involved in the transcriptional regulation of many sporulation genes. It directly activates transcription of spoIIA, spoIIE, and spoIIG, which are necessary for sporulation (4,6,48,49,57,62). Also, Spo0AϳP represses transcription of abrB (56), which encodes a repressor of several genes involved in sporulation (44,55,59,64).Spo0A receives phosphate, albeit indirectly, from KinA (2, 38) and KinB (58), histidine protein kinases that belong to a conserved family of proteins known as sensor kinases. Sensor kinases are generally involved in signal transduction; they autophosphorylate and donate phosphate to particular proteins in response to changes in the environment, modifying the activity of those proteins. The proteins that receive phosphate from sensor kinases belong to a conserved family of proteins known as response regulators, which are often involved in transcriptional regulation. Together, a sensor protein and its cognate response regulator form a two-component system. There are many examples of two-component systems involved in signal transduction in bacteria (1, 37), and similar systems in plants (10) and yeasts (36) have recently been identified.Although Spo0A belongs to the response regulator family of proteins, it does not normally obtain phosphate directly from a histidine protein kinase. Rather, Spo0A receives phosphate through a multicomponent phosphorelay (9). KinA (and other kinases) first donates phosphate to...

We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible promoter Pspac. Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0AϳP. These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.The initiation of sporulation in Bacillus subtilis is controlled, in part, by a multicomponent phosphorelay that results in the phosphorylation, and hence activation, of the transcription factor encoded by spo0A (4). Several histidine protein kinases are involved in the initiation of sporulation (1,20,22,29). Phosphate is transferred from the kinases to Spo0F, from Spo0F to Spo0B, and finally from Spo0B to Spo0A (4). A threshold mechanism controls the initiation of sporulation (5), and the phosphorelay functions to integrate many of the signals that control initiation, including nutrient deprivation, DNA replication, DNA damage, chromosome partitioning, and Krebs cycle signals (13)(14)(15)(16)(17)(18). In addition to the phosphorelay, several other proteins are known to regulate production and accumulation of Spo0AϳP. These proteins include the phosphatase encoded by spo0E (28, 31), the transcriptional regulator encoded by sinR (2, 25, 26), the oligopeptide permease encoded by spo0K (30,33), and the proteins encoded by soj and spo0J (16).One protein that has been postulated to control the phosphorelay is the G protein encoded by obg (42). The obg gene product has been purified and shown to bind GTP (40) and to have GTPase activity (42). obg is immediately downstream from and cotranscribed with spo0B (6), is essential for vegetative growth, and is required for efficient sporulation (21). Because of its association with spo0B, it has been tempting to imagine that Obg has a role in regulating the activity of the phosphorelay and perhaps of Spo0B (4,8,12).A conditional allele of obg. To test the effects of obg on growth and sporulation, we made a conditional allele of obg such that the only copy of the gene was under the control of the LacI-repressible, IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible promoter Pspac (11,45). We cloned obg after PCR amplification using primers that were designed on the basis of the published DNA sequence (3, 6). A 197-bp fragment of obg, extending from 70 bp upstream of the putative start codon to the AatII site at codon 43 (3, 6), was sequenced to be sure that there were no mutations introduced during the PCR amplification and cloning. This fragment was then cloned downstream of Pspac in the integrational vector pDH88 (11) to give pVS7 (Fig. 1). pVS7 was integrated into the chromosome of B. subtilis by selecting for chloramphenicol resistance in the presence of IPTG, allowing expression of the intact obg g...

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