In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of H , which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.Initiation of sporulation in Bacillus subtilis is regulated by a signal transduction pathway, the phosphorelay, which is a multicomponent phosphotransfer system that is switched on in response to environmental, cell cycle, and metabolic signals (3, 36). The processing and integration of these signals by the phosphorelay control the level of phosphorylation of the transcription factor, Spo0A. Environmental and cellular signals that favor sporulation activate autophosphorylation of the sensor kinases KinA, KinB, KinC, and KinD, leading to input of a phosphate group into the phosphorelay (1,2,12,13,18,20,33,37,46). In this relay, the phosphate group is subsequently transferred to a response regulator, Spo0F. The resulting molecule, Spo0F-P, serves as a substrate for the Spo0B protein, a phosphotransferase which finally activates Spo0A by transferring the phosphate to the Spo0A protein (2). Spo0A-P, the activated form of Spo0A, indirectly controls the transcription of a number of genes by regulating the level of other transcription regulators. Spo0A-P directly activates transcription of the genes for many regulatory proteins and sigma factors required for cell-type-specific gene expression. Spo0A-P is also known to stimulate axial filament formation and asymmetric polar septation, which give rise to two unequal cells, a larger mother cell and a forespore cell (21, 38).Just after septation, gene expression is controlled by the RNA polymerase sigma factors, F in the forespore and E in the mother cell. Later in sporulation, when the forespore has become engulfed by the mother cell, F and E are r...

We have identified the following events during the late stage in the mother cell in Bacillus subtilis: spore detachment from the polar site of the mother cell, membrane rupture, cell wall collapse, and release of the free spore. The membrane rupture was followed by mother cell lysis. Moreover, we found that NucB, an extracellular nuclease, is involved in DNA degradation after mother cell lysis.Programmed cell death is a phenomenon of widespread biological importance and is recognized to play a role in morphogenesis by eliminating unnecessary cells. Most studies on cell death have been carried out with vertebrates (5), and little attention has been given to the process of programmed cell death in unicellular organisms, although cell suicide responses have been reported to occur in prokaryotic and unicellular eukaryotic cells (6).The gram-positive bacterium Bacillus subtilis possess two characteristics which make it an attractive microorganism for the study of cell death at the prokaryote level: first, like Escherichia coli, it continuously divides by binary fission into two functionally and structurally identical daughter cells; second, it has a simple developmental cycle called sporulation (1, 2). In response to nutrient deprivation, B. subtilis forms dormant and environmentally resistant spores. The spore attains a state of dormancy, which is maintained until the return to a favorable environment induces spore germination and further growth. Formation of a spore takes about 8 h at 37°C and is controlled by a genetically regulated process. During sporulation, the timing of spore maturation is coordinated by continuous close collaboration between two cells, the mother cell and forespore. The mother cell engulfs the future daughter cell and eventually actively lyses prior to release of the spore. However, previous studies, which have focused on cell wall lysis in the mother cell death process, have provided only a limited understanding of the process of mother cell death during sporulation. Therefore, the initial goal of this study was to characterize the mother cell death process by cytological studies using time-lapse fluorescence microscopy.Mother cell death observed in liquid culture. The morphological characteristics of mother cell death in B. subtilis 168 were observed when the cells were allowed to develop in shaking liquid culture at the late stage of sporulation. At 3 to 18 h after initiation of sporulation (T 3 to T 18 ), the cultures were collected, and then the membranes and DNA were stained with dyes FM4-64 (a membrane-impermeative fluorescent lipophilic styryl dye) (2.5 g/ml for 10 min) and DAPI (4Ј,6Ј-diamidino-2-phenylindole) (a membrane-permeative fluorescent dye) (1 g/ml for 10 min), respectively (11). Cells with asymmetric septa and cells with engulfing sporangia within the rod-shaped mother cells at T 3 and T 6 were visualized using the fluorescence of FM4-64 ( Fig. 1B and E). The forespores in sporangia that had completed engulfment were not stained by FM4-64. However, fluorescence in the outl...

The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.Bacillus subtilis undergoes transformation during nutrient starvation from an actively growing vegetative cell into a metabolically dormant and environmentally resistant spore capable of withstanding a wide range of environmental stresses, including heat, UV radiation, and noxious chemicals (6). The cortex, a layer of peptidoglycan contributes greatly to this almost indestructible property of the spore. Besides providing this high level of protection, the spore has the capacity to respond to germinants in the presence of nutrients. Extensive cortex modifications involving glycosylation, proteolysis, and cross-linking enhance the integrity of the cortex which is vital for the stability and subsequent efficient germination of the spore (2,20,21). However, the mechanisms involved in the establishment of cortex integrity still remain unclear.We have identified dgkA, the structural gene for sn-1,2-diacylglycerol (DAG) kinase as a sporulation gene that is essential for the maintenance of spore stability and viability in B. subtilis. This kinase was identified and studied in Escherichia coli long ago (11,22). Studies by Loomis et al. (12) indicated that the secondary structure of DAG kinase, which is a membrane-bound protein, consists of three transmembrane alphahelical segments, an amphipathic helix, and an alpha-helix with the helical segments comprising more than 75% of the polypeptide. DAG kinase phosphorylates DAG, an important intermediate in phospholipid biosynthesis and breakdown, generating phosphatidic acid which enters the main pathway of phospholipid biosynthesis (17). The lipid biosynthetic pathway has been extensively researched in E. coli (3,4,8,10). In all organisms, the acylation of sn-glycerol-3-phosphate to phosphatidic acid indicates initiation of the pathway (5). In bacteria, the pathway proceeds with the conversion of phosphatidic acid to CDP-diacylglycerol, which is a precursor of the common bacterial lipid phosphatidylethanolamine. Several closely related genes that perform the reactions of this pathway have been characterized in B. subtilis (1,13,19). W...

In the course of the Bacillus subtilis functional genomics project, an open reading frame called ycbG whose product is classified as a transcriptional regulatory protein with a helix-turn-helix motif in the putative D-glucarate/galactarate utilization operon (ycbCDEFGHJ) was initially screened as the gene disruptant that exhibits a defect that blocked the early stage of sporulation. However, the transcription of ycbCDEFG was extremely highly induced in response to nutrient exhaustion by the disruption of ycbG, but inactivation of the transcription from upstream ycbC in the ycbG mutant restored the sporulation efficiency, suggesting that the inappropriate over-production of the ycbCDEFG gene products inhibits efficient sporulation. We further analyzed the role of the ycbCDEFGHJ cluster and found that (i) a unit of ycbCDEFGHJ was induced by either D-glucarate or D-galactarate, and (ii) the cell growth was inhibited by the mutation of the ycbF and ycbH genes, that respectively encode the putative proteins, D-glucarate dehydratase and D-galactarate dehydratase on plates supplemented with D-glucarate and D-galactarate, respectively, as the sole carbon source. Our results indicate that the ycbCDEFGHJ genes are involved in the utilization of D-glucarate and D-galactarate in B. subtilis.

Insertional mutagenesis with mini-Tn10 was performed to identify new genes involved in sporulation of Bacillus subtilis. Here, we report on the characterization of the ybdA locus, which encodes a putative ATP-binding cassette transporter. The ybdA gene is the 6th cistron of the putative ybcOPQST-ybdABDE operon. A deletion mutation in ybdA and an insertional mutation in ybdB exhibited highly oligosporogenous phenotypes and led to a decrease in the transcription controlled by Spo0A, which is a key response regulator required for the initiation of sporulation. We further observed that the transcription of this operon was strongly induced after the end of the exponential growth phase in the wild-type strain, but not in a spo0A null mutant. Our data suggest that the YbdA and YbdB proteins are able to affect incorporation of nutrient signals during initiation of sporulation and may act as components of positive feedback systems of Spo0A activation.