Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Precursor processing during the maturation of a spore-coat protein in Bacillus megaterium KM.

ABSTRACT: A protein of apparent mol.wt. 35000 that is extractable from the purified coat fraction of Bacillus megaterium KM spores is synthesized during sporulation as a precursor protein from which a 12-13 amino acid peptide is removed. Cleavage of this small peptide is delayed until 60-90 min after precursor synthesis and is concomitant with the morphological appearance of stage VI. The addition of chloramphenicol, subsequent to precursor synthesis, prevents the appearance of this late processing event. Two-dimensional non-equilibrium pH-gradient gel electrophoresis of the integument extract of forespores isolated at stage V from sporangia pulse-labelled with L-[35S]methionine 1 h before isolation, revealed both unprocessed and processed components. Similar analysis of total protein from the corresponding mother cells revealed only the unprocessed component in relatively small amounts, suggesting that, although the protein may be synthesized in the mother-cell compartment, processing may be restricted to the forespore. Peptide analysis by limited proteolysis was used to examine the relationship between the 35000- and a 17500-mol.wt. coat protein. The possible implications of limited proteolytic processing to maturation of the spore coat are discussed.

SUBMITTER: Stewart GS

PROVIDER: S-EPMC1154239 | biostudies-other | 1983 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Characterization, purification and synthesis of spore-coat protein in Bacillus megaterium KM.

Project description:The spore-coat fraction from Bacillus megaterium KM, when prepared by extraction of lysozyme-digested integuments with SDS (sodium dodecyl sulphate) and urea, contains three N-terminal residues and a major component of apparent mol.wt. 17500. Electron microscopy of this fraction shows it to consist of an ordered multilamellar structure similar to that which forms the coat region of intact spores. The 17500-dalton protein, which has been purified to homogeneity, has an N-terminal methionine residue, has high contents of glycine, proline, cysteine and acidic amino acids and readily polymerized even in the presence of thiol-reducing agents. It is first synthesized between late Stage IV and early Stage V, which correlates with the morphological appearance of spore coat. Before Stage VI the 17500-dalton protein is extractable from sporangia by SDS in the absence of thiol-reducing reagents. Between Stage VI and release of mature spores the protein becomes resistant to extraction by SDS unless it is supplemented by a thiol-reducing reagent. In addition to that of the spore-coat protein, the timing of synthesis of all the integument proteins was analysed by SDS/polyacrylamide-gel electrophoresis and non-equilibrium pH-gradient electrophoresis. Several integument proteins are conservatively synthesized from as early as 1h after the end of exponential growth (t1), which may reflect protein incorporation into the spore outer membrane.

| S-EPMC1158096 | biostudies-other

The use of inhibitors to identify early events during Bacillus megaterium KM spore germination.

Project description:The germination response of spores of Bacillus megaterium KM, as measured by loss of A600, is more than 95% inhibited by 1 mM-HgCl2. Two Hg2+-sensitive sites (referred to as 'sites I and II') have been identified during germination. Site I represents a pre-commitment event and can be protected from HgCl2 by 50 mM-D-alanine, whereas site II represents a post-commitment event and is not D-alanine-protectable. At 1 mM-HgCl2, 25% of the spore population becomes committed to germinate, but an A600 loss of less than 5% occurs. In this system, loss of heat resistance was associated with commitment, whereas selective cortex hydrolysis, release of pyridine-2,6-dicarboxylic acid, Zn2+ and soluble peptidoglycan, as well as loss of refractility, were identified as post-commitment events. The commitment event was reversibly inhibited by several proteinase inhibitors and a membrane bulking agent. A model of spore germination based on these results is presented.

| S-EPMC1147068 | biostudies-other

Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination.

Project description:Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.

| S-EPMC1153977 | biostudies-other

Calcium accumulation during sporulation of Bacillus megaterium KM.

Project description: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.

| S-EPMC1186221 | biostudies-other

Searching for protein-protein interactions within the Bacillus subtilis spore coat.

Project description:The capability of endospores of Bacillus subtilis to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.

| S-EPMC2687167 | biostudies-literature

Structure-function analysis of the Bacillus megaterium GerUD spore germinant receptor protein.

Project description:Germination of Bacillus spores is triggered by the interaction of germinant molecules with specialized receptor proteins localized to the spore inner membrane. Germinant receptors (GRs) are comprised typically of three interacting protein subunits, each of which is essential for receptor function. At least some GRs appear to have a fourth component, referred to as a D-subunit protein. A number of D-subunit proteins were shown previously to be capable of modulating the activity of associated GRs. Here, we investigate the topology and structure-function relationships of the Bacillus megaterium QM B1551 GerUD protein, which is associated with the GerU GR. The presented data demonstrate that GerUD can be subjected to relatively extensive structural modifications while retaining function. Indeed, the presence of either of the two transmembrane spanning domains is sufficient to modulate an efficient GerU-mediated germinative response. The precise function of D-subunit proteins has yet to be established, although they may act as molecular chaperones within the spore inner-membrane environment.

| S-EPMC4661057 | biostudies-literature

Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology.

Project description:Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis, the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called Cotbeta, which is encoded only in the genomes of the Bacillus cereus group. We found that Cotbeta is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotbeta null mutant in the Sterne strain reveals that Cotbeta has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotbeta null mutation has no effect on virulence in a murine model of B. anthracis infection.

| S-EPMC9972873 | biostudies-literature

Teichoic acid and lipid metabolism during sporulation of Bacillus megaterium KM.

Project description:The biochemistry of teichoic acid and lipid metabolism has been studied during sporulation of Bacillus megaterium KM. Measurements of cell-wall and membrane teichoic acid have shown that net synthesis of these polymers ceases at the onset of sporulation. Pulse-labelling studies show that the period of asymmetric septation and forespore engulfment is marked by an initiation of turnover of membrane teichoic acid but not of wall teichoic acid. This is reflected in the presence of inner-membrane teichoic acid and the virtual absence of wall teichoic acid in dormant spores. The total amount of lipid phosphorus in the sporulating cell increases by 70% as a result of asymmetric septation and subsequent engulfment of the forespore. The phosphorus requirement for this synthesis is derived from a pool formed during exponential growth, which is not exchangeable with extracellular Pi during sporulation. These results suggest that during sporulation a proportion of the glycerol 3-phosphate produced by preferential degradation of membrane teichoic acid formed during exponential growth is used for phospholipid synthesis during sporulation.

| S-EPMC1158131 | biostudies-other

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

Project description: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.

| S-EPMC1186563 | biostudies-other

Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation.

Project description:A major Bacillus anthracis spore coat protein of 13.4 kDa, designated Cot alpha, was found only in the Bacillus cereus group. A stable ca. 30-kDa dimer of this protein was also present in spore coat extracts. Cot alpha, which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a sigma(K)-regulated gene. On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium. In addition, disruption of the gene encoding Cot alpha resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs. The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme. They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate. In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the cot alpha gene into the mutant. Since Cot alpha is an abundant outer spore coat protein of the B. cereus group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of B. anthracis spores.

| S-EPMC412170 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data