Correlation between spore structure and spore properties in Bacillus megaterium

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.     

Synthesis and deposition of spore coat proteins during sporulation of Bacillus megaterium

Rabbit (anti-spore coat protein) IgG was prepared by immunization with coat proteins extracted with sodium dodecyl sulfate and dithiothreitol from isolated spore coats of Bacillus megaterium ATCC 12872. Coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fraction by sandwich enzyme immunoassay using this antibody. The proteins in the forespore coat protein fraction increased from t3 and reached a plateau at t10. Immunoblot analysis for the coat proteins in sporulating cells revealed the sequential synthesis of various proteins in the mother cell cytoplasmic fraction and simultaneous deposition of the same proteins as in the forespore coat fraction. These results suggest that turnover of precursor proteins of the spore coat is very rapid if precursor proteins are produced and they are proteolytically processed to produce mature proteins. Specific antibody to the 48,000-dalton protein, which is a major protein, did not cross-react with any other major (36,000, 22,000, 19,500, and 17,500-dalton) proteins. Specific antibody to the 22,000-dalton protein did not cross-react with the 48,000, 36,000, 19,500, 17,500, and 16,000-dalton proteins, but did cross-react with the 44,000, 25,000, and 12,000-dalton proteins.     

Peptide anchoring spore coat assembly to the outer forespore membrane in Bacillus subtilis

Spore formation in Bacillus subtilis involves the formation of a thick, proteinaceous shell or coat that is assembled around a specialized membrane known as the outer forespore membrane. Here we present evidence that the assembling coat is tethered to the outer forespore membrane by a 26-amino-acid peptide called SpoVM, which is believed to form an amphipathic helix. We show that proper localization of SpoVM is dependent on SpolVA, a morphogenetic protein that forms the basement layer of the spore coat, and conversely, that proper localization of SpoIVA is dependent on SpoVM. Genetic, biochemical and cytological evidence indicates that this mutual dependence is mediated in part by contact between an amino acid side-chain located near the extreme C-terminus of SpoIVA and an amino acid side-chain on the hydrophilic face of the SpoVM helix. Evidence is also presented that SpoVM adheres to the outer forespore membrane via hydrophobic, amino acid side-chains on the hydrophobic face of the helix. The results suggest that the SpoVM helix is oriented parallel to the membrane with the hydrophobic face buried in the lipid bilayer.     

RNA synthesis during spore germination in Bacillus subtilis.

Summary We have analyzed the RNA synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [³H]uridine into RNA. A large increase in incorporation into RNA was found at 45–60 min into germination which was in part due to increases in the specific activity of the UTP pool. When corrected for specific activity changes, the instantaneous rate of RNA synthesis showed a seven to tenfold increase between 30 and 45 min of germination. Polyacrylamide gel electrophoresis studies showed that the RNA synthesized during germination appeared very similar to the RNA made during vegetative growth. DNA-RNA hybridization studies indicated that mRNA and rRNA were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of RNA synthesized during vegetative growth.In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine     

Germination-specific cortex-lytic enzyme is activated during triggering of Bacillus megaterium KM spore germination

Antibodies were raised against purified germination-specific cortex-lytic enzyme (GSLE) from spores of Bacillus megaterium KM which neutralized the ability of GSLE to germinate permeabilized spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE demonstrated that GSLE is spore-specific and that greater than 90% of the GSLE is associated with the dormant spore cortex peptidoglycan as a phosphorylated 63kD pro-form, which could only be visualized after lysozyme digestion of the peptidoglycan. During germination, the 63kD pro-form of GSLE is processed to release the active enzyme, which had an apparent molecular weight of 30kD. Inhibitor studies demonstrated that GSLE activation occurs as part of the commitment reaction and thus represents the first-identified enzymatic event to occur during germination triggering. Proteins that cross-react with anti-GSLE sera are present in spore fractions of other species.     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.     

Role of outer coat in resistance of Bacillus megaterium spore

The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed of galactosamine-6-phosphate (GalN-P) polymers with polypeptides and calcium. OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer of GalN-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experiments revealed that the outer coat limited the entry of compounds with a molecular weight of more than 2,000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of OC-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainly of GalN-P polymers, and the molecular sieving effect.     

Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis

Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.     

Protein catabolism in growing Bacillus megaterium during adaptation to salt stress

Elevated concentration of NaCl in liquid medium caused a concentration-dependent growth delay (adaptation lag) and decrease in the maximal growth rate of Bacillus megaterium. The adaptation to salt stress was accompanied by transformation of some otherwise stable (long-lived; LLP) cell proteins into quickly degraded (short-lived; SLP) ones. Exposure to the strongly growth-reducing 1 M NaCl increased the size of the SLP 'pool' of intracellular proteins from about 5 to about 15% of total protein. The major intracellular proteolytic capacity of B. megaterium is represented by intracellular serine proteinases (ISP). Paradoxically, their specific activity was lowered or masked during the adaptation phase marked by increased catabolism of short-lived and/or destabilized proteins by the stress. This documents that intracellular proteolytic activity cannot be a key regulator of protein catabolism during adaptation to stress.     

Protein composition of cell and forespore membranes of Bacillus subtilis

A sporulation mutant of Bacillus subtilis 168 was isolated and characterized. The mutant, designated SB-23, releases viable forespores at the end of the developmental period. Forespores were isolated on linear Renografin gradients and used as a source of forespore membranes. The protein composition of forespore membranes was found to differ from the protein composition of vegetative cell membranes by discgel electrophoresis. The results are discussed in relationship to morphological and physiological differentiation during bacterial sporulation.     

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation.

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.