Expression of dnaK and groESL operons during sporulation of Bacillus megaterium

Expression of the dnaK and groEL genes during sporulation was assayed by determination of their mRNA levels by Northern blotting and compared with the relative level and rate of synthesis of the corresponding proteins. The ability of sporulating cells to respond to a heat shock by an increase in dnaK and groEL expression was determined at the same time. Synthesis of DnaK and GroEL encoding mRNAs during sporulation in non-shocked cells was low suggesting that this kind of cytodifferentiation was not accompanied by enhanced synthesis of these chaperones. Also the ability of sporulating cells to respond to a heat shock by stimulating their synthesis substantially decreased during the reversible and dropped to negligible values during the irreversible sporulation phase. Nevertheless, some dependence of the heat shock response on sporulation exists because sporulation suppression by mutation or by netropsin treatment further decreased the cells' capacity to respond to a heat shock.     

巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用,它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸,６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...     

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.     

Calcium accumulation during sporulation of Bacillus megaterium KM.

Accumulation of Ca2+ in Bacilli occurs during stages IV to VI of sporulation. Ca2+ uptake into the sporangium was investigated in Bacillus megaterium KM in protoplasts prepared in stage III of sporulation and cultured to continue sporulation. These protoplasts and whole cells exhibit essentially identical Ca2+ uptake, which is compared with that of forespores isolated in stage V of sporulation. Ca2+, uptake into both sporangial protoplasts and isolated forespores occurs by Ca2+-specific carrier-mediated processes. However, protoplasts exhibit a Km value of 31 micrometer, and forespores have a Km value of 2.1 mM. Sporangial protoplasts accumulate Ca2+ against a concentration gradient. In contrast, Ca2+ uptake into isolated forespores is consistent with downhill transfer in which both rate and extent of uptake are affected by the external Ca2+ concontration. Dipicolinic acid has no effect on Ca2+ uptake by isolated forespores, apart from decreasing the external Ca2+ concentration by chelation. A model for sporulation-specific Ca2+ accumulation is proposed, in which Ca2+ is transported into the sporangium, resulting in a concentration of 3--9 mM in the mother-cell cytoplasm. This high concentration of Ca2+ enables carrier-mediated transfer down a concentration gradient into the forespore compartment, where a low free Ca2+ concentration is maintained by complexing with dipicolinic acid.     

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.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.     

Chromosome age and segregation during sporulation of Bacillus megaterium.

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.     

Expression of a P-type Ca(2+)-transport ATPase in Bacillus subtilis during sporulation

The open reading frame designated yloB in the genomic sequence of Bacillus subtilis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca(2+)-transporters, located respectively in the SERCA and the Golgi apparatus. The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca(2+)-binding sites in SERCA. Sporulating but not vegetative B. subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca(2+)-dependent phosphorylated intermediate. Half-maximal activation of phosphointermediate formation occurred at 2.5 microM Ca(2+). Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45Ca accumulation during sporulation. However, spores from knockouts were less resistant to heat and showed a slower rate of germination. It is concluded that the P-type Ca(2+)-transport ATPase from B. subtilis is not essential for survival, but assists in the formation of resistant spores. The evolutionary relationship of the transporter to the eukaryotic P-type Ca(2+)-transport ATPases 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.     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Characterization of the Bacillus subtilis sporulation gene spoVK.

The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.     

Energy-dependence of calcium accumulation during sporulation of Bacillus megaterium KM.

Ca2+ accumulation and endogenous respiration of sporulating Bacillus megaterium are inhibited to the same extent by electron-transport of inhibitors and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting that Ca2+ is accumulated by an active transport process. Forespores isolated in stage V of sporulation demonstrated Ca2+-specific carrier-mediated Ca2+ uptake, consistent with downhill transfer [Hogarth & Ellar (1978) Biochem. J. 176, 197-203]. In the present studies forespore Ca2+ uptake was unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by concentrations of respiratory inhibitor that inhibited forespore endogenous respiration by 85%. These data suggest that Ca2+ enters the isolated forespore by facilitated diffusion. Ca2+ uptake into sporulating protoplasts was completely inhibited by concentrations of respiratory inhibitors that had no effect on either Ca2+ uptake or respiration of stage-V forespores, but which resulted in inhibition of mother-cell membrane NADH oxidase. These results indicate that the mother-cell membrane is a site for active transport of Ca2+ into the sporulating cell. The effects of the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide on mother-cell membrane adenosine triphosphatase, NADH oxidase and protoplast Ca2+ uptake were examined.