Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

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.     

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.     

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.     

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.     

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.     

Changes in penicillin-binding proteins during sporulation of Bacillus subtilis

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined in samples collected at various times from sporulating cultures and compared with the PBPs in a presporulation sample. Large increases in vegetative PBPs 2B and 3 and the appearance of at least one new PBP (42,000 daltons) occurred at reproducible times during sporulation. In some strains a second new PBP (60,000 daltons) was also produced. By comparing the PBP activities in sporulating cells and two spo0 mutants we have classified these changes as sporulation-related events rather than the consequences of stationary-phase aging. The other vegetative PBPs (PBPs 1, 2A, 4, and 5) decreased during sporulation, but not in sufficient amount or at the appropriate time to account for the appearance of the new proteins. A possible connection between specific PBP changes and the penicillin-sensitive stages of sporulation is suggested.     

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.