Heat-shock proteins during growth and sporulation of Bacillus subtilis

Four major heat-shock proteins (hsps) with apparent molecular masses of 84, 69, 32 and 22 kDa were detected in exponentially growing stationary phase and sporulating cells of Bacillus subtilis heat-shocked from 30 to 43 degrees C. The most abundant, hsp69, is probably analogous to the E. coli groEL protein. These proteins were transiently inducible by heat-shock. Partial purification of RNA polymerase revealed several other minor hsps. One of these, a 48 kDa polypeptide probably corresponds to sigma 43. The synthesis of this polypeptide and at least two other proteins appeared to be under sporulation and heat-shock regulation and was affected by the SpoOA mutation.     

Two class A high-molecular-weight penicillin-binding proteins of Bacillus subtilis play redundant roles in sporulation.

The four class A penicillin-binding proteins (PBPs) of Bacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamed pbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic delta-lactam and L-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.     

Peptidoglycan transformations during Bacillus subtilis sporulation

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross‐linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high‐resolution electron cryotomographic imaging. Three‐dimensional reconstructions of whole cells in near‐native states revealed a thin PG‐like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram‐negative‐like PG layers as well as its thick, archetypal Gram‐positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.     

Dual localization pathways for the engulfment proteins during Bacillus subtilis sporulation

Engulfment in Bacillus subtilis is mediated by two complementary systems, SpoIID, SpoIIM and SpoIIP (DMP), which are essential for engulfment, and the SpoIIQ-SpoIIIAGH (Q-AH) zipper, which provides a secondary engulfment mechanism and recruits other proteins to the septum. We here identify two mechanisms by which DMP localizes to the septum. The first depends on SpoIIB, which is recruited to the septum during division and provides a septal landmark for efficient DMP localization. However, sporangia lacking SpoIIB ultimately localize DMP and complete engulfment, suggesting a second mechanism for DMP localization. This secondary targeting pathway depends on SpoIVFA and SpoIVFB, which are recruited to the septum by the Q-AH zipper. The absence of a detectable localization phenotype in mutants lacking only SpoIVFAB (or Q-AH) suggests that SpoIIB provides the primary DMP localization pathway while SpoIVFAB provides a secondary pathway. In keeping with this hypothesis, the spoIIB spoIVFAB mutant strain has a synergistic engulfment defect at septal thinning (which requires DMP) and is completely defective in DMP localization. Thus, the Q-AH zipper both provides a compensatory mechanism for engulfment when DMP activity is reduced, and indirectly provides a compensatory mechanism for septal localization of DMP when its primary targeting pathway is disrupted.     

Developmental modulation of deoxyribonucleic acid-binding proteins of Bacillus subtilis during sporulation stages

The isolation of deoxyribonucleic acid (DNA)-binding proteins from various stages of growth and sporulation of Bacillus subtilis is described. After adsorption and elution from phosphocellulose, the proteins were fractionated according to their ability to adsorb to denatured calf thymus DNA-cellulose or native B. subtilis DNA-cellulose. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and purification was monitored by a nitrocellulose filter binding assay. Approximately 1% of the proteins in the crude extract adsorbed to denatured calf thymus DNA-cellulose and 0.1% adsorbed to native B. subtilis DNA-cellulose. Each class of proteins varied qualitatively and quantitatively as sporulation proceeded. Several proteins from the exponential phase of growth that bound to denatured DNA were lost by T(0), whereas at T(5) new polypeptides appeared. Fewer changes in the profile of proteins with affinity for native DNA were observed between exponential phase and T(0); however, the dominant species in these eluates were clearly different.     

Autolysins during sporulation of Bacillus subtilis 168

Conditions for zymographic detection of a 41-kDa spore cortex hydrolysis-specific autolysin, A6, from Bacillus subtilis 168 were optimised. A6 was present during sporulation from stages II–IV and remained active in the dormant spore. Its expression was controlled by the mother cell-specific early-sporulation sigma factor σ^E. The characteristic muramic acid δ-lactam of spore cortical peptidoglycan was not necessary for cortex hydrolysis by A6, but it may be important in the inability of the major vegetative autolysin LytC to digest wild-type cortex. Two other minor autolysins were also observed during sporulation. The possible physiological significance of these observations is discussed.     

Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis.

Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation. At least six proteins were labeled in vitro by using [gamma-32P]ATP and extracts of vegetative cells. In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes.     

Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilis

Bacterial cell shape is determined by a rigid external cell wall. In most non-coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin-binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild-type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP-PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.     

Stability and synthesis of the penicillin-binding proteins during sporulation

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined after incubation of vegetative and sporulating cultures with chloramphenicol, an inhibitor of protein synthesis. The results indicate that the sporulation-specific increases in vegetative PBPs 2B and 3 and the appearance of two new PBPs, 4* and 5*, depend on concurrent protein synthesis, which is most likely to be de novo synthesis of the PBPs rather than synthesis of an activator or processing enzyme. It was also learned that in vivo the PBPs differ in their individual stabilities, which helps to explain some of the quantitative changes that occur in the PBP profile during sporulation. All the membrane-bound PBPs, except possibly PBP 1, were found to be stable in the presence of crude extracts of sporulating cells that contained proteolytic activity.     

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.     

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.     

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.