MinCD-dependent regulation of the polarity of SpoIIIE assembly and DNA transfer

During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore. The direction of DNA translocation is controlled by the specific assembly of SpoIIIE in the mother cell and subsequent export of DNA into the forespore. We present evidence that the MinCD heterodimer, which spatially regulates cell division during vegetative growth, serves as a forespore-specific inhibitor of SpoIIIE assembly. The deletion of minCD increases the ability of forespore-expressed SpoIIIE to assemble and translocate DNA, and causes otherwise wild-type cells to reverse the direction of DNA transfer, producing anucleate forespores. We propose that two distinct mechanisms ensure the specific assembly of SpoIIIE in the mother cell, the partitioning of more SpoIIIE molecules into the larger mother cell by asymmetric cell division and the MinCD-dependent repression of SpoIIIE assembly in the forespore. Our results suggest that the ability of MinCD to sense positional information is utilized during sporulation to regulate protein assembly differentially on the two faces of the sporulation septum.     

Levels of Small Molecules and Enzymes in the Mother Cell Compartment and the Forespore of Sporulating Bacillus megaterium

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.     

DNA replication during endospore development in Metabacterium polyspora

The guinea pig intestinal symbiont Metabacterium polyspora is an uncultured, endospore-forming member of the Firmicutes. Unlike most endospore-forming bacteria, sporulation is an obligate part of the M. polyspora life cycle when it is associated with a guinea pig. Binary fission is limited to a brief period in its life cycle, if exhibited at all. Instead, M. polyspora relies on the formation of multiple endospores for reproduction. Sporulation is initiated immediately after germination, which leaves little time for the cell to accumulate resources to support spore formation. Using immunolocalization of the nucleotide analogue bromodeoxyuridine (BrdU), we were able to follow replication dynamics in M. polyspora . BrdU was provided to cells within the guinea pig intestinal tract. BrdU was incorporated into DNA located within the forespores throughout development, at all stages prior to spore maturation. Our results suggest that in M. polyspora , DNA replication within the forespore is not suppressed during sporulation as it is in other endospore-forming bacteria. Replication within forespores would allow M. polyspora to maximize its reproductive potential and supply each endospore with at least one complete copy of the genome.     

Red pigment in Bacillus megaterium spores

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Chromosome strand segregation during sporulation in Bacillus subtilis

After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.     

Morphogenesis of the membrane-bound electron-transport system in sporulating Bacillus megaterium KM.

The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM?HAVE BEEN COMPARED WIth those of similar fractions prepared from exponential-phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a + a3, and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential-phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but DL-glycerol 3-phosphate and L-malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH oxidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five-fold increase in NADH-cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and L-malate oxidases; DL-glycerol 3-phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors.     

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

Characterization of a polysaccharide deacetylase gene homologue (pdaB) on sporulation of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases. Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID. The pdaB mutant was deficient in spore formation. Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods. The pdaB spores that had lost their viability were empty. Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant. This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells. Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.     

Effects of inactivation or overexpression of the sspF gene on properties of Bacillus subtilis spores.

Inactivation of the Bacillus subtilis sspF gene had no effect on sporulation, spore resistance, or germination in a wild-type strain or one lacking DNA protective alpha/beta-type small, acid-soluble proteins (SASP). Overexpression of SspF in wild-type spores or in spores lacking major alpha/beta-type SASP (alpha- beta- spores) had no effect on sporulation but slowed spore outgrowth and restored a small amount of UV and heat resistance to alpha- beta- spores. In vitro analyses showed that SspF is a DNA binding protein and is cleaved by the SASP-specific protease (GPR) at a site similar to that cleaved in alpha/beta-type SASP. SspF was also degraded during spore germination and outgrowth, and this degradation was initiated by GPR.     

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.     

Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis

Cells of Bacillus subtilis actively co-ordinate the initiation of sporulation with DNA replication and repair. Conditions that perturb replication initiation or replication elongation induce expression of a small protein, Sda, that specifically inhibits the histidine kinases required to initiate spore development. Previously, the role of Sda has been studied during chronic blocks to DNA replication. Here we show that induction of Sda is required to delay the initiation of sporulation when replication elongation is transiently blocked or after UV irradiation. During the recovery phase, cells efficiently sporulated, but this required the proteolysis of Sda. The rapid proteolysis of Sda required the ClpXP protease and the uncharged C-terminal sequence of Sda. Replacing the last two residues of Sda, both serines, with aspartic acids markedly stabilized Sda. Strains expressing sdaDD from the endogenous sda locus were unable to efficiently initiate sporulation after transient replication stress. We conclude that the Sda replication checkpoint is required to delay the initiation of sporulation when DNA replication is transiently perturbed, and that the intrinsic instability of Sda contributes to shutting off the pathway. The Sda checkpoint thus co-ordinates early events of spore development, including the polar cell division, with successful completion of chromosome replication.     

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene.

The product of the spoIIM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation. To investigate whether expression of spoIIM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts. The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature. This facilitates the use of a genetic technique employed by Illing et al to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment. The results of the integration/excision experiments using pKSV7 support the conclusion that spoIIM must be expressed in the forespore. Biochemical analysis of forespore and mother cell fractions suggests that spoIIM is also expressed in the mother cell. The conditional integrational vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of alpha-complementation and thus should be useful for other kinds of genetic manipulations in B subtilis.     

Characterization of the parB-Like yyaA Gene of Bacillus subtilis

We have characterized the yyaA gene of Bacillus subtilis, located near the origin of chromosome replication (oriC). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the yyaA gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of yyaA caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.     

c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.