Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat

Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component. cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.     

Contributions of crust proteins to spore surface properties in Bacillus subtilis

Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.     

Biochemical analysis of the Bacillus subtilis 1604 spore germination response

Germination at 37 degrees C of spores of Bacillus subtilis 1604 in the L-alanine and potassium phosphate (ALA) and the glucose, fructose, L-asparagine, potassium chloride (GFAK) germinant systems was triggered following heat activation at 70 degrees C for 1 h. In these conditions, 50% of the spore population became committed to germinate after exposure for 10 min and 14 min to ALA and GFAK, respectively, at which time 38% and 30% losses of OD600 had taken place. Dipicolinic acid (DPA) release, loss of heat resistance and release of soluble hexosamine-containing fragments occurred after commitment and were closely associated with loss of refractility in both the ALA and GFAK pathways. Net ATP synthesis could not be detected until 3-4 min after initiation of germination in both ALA and GFAK, by which time greater than 20% of the spore population was committed to germinate. The ALA and GFAK germination pathways were greater than 99% inhibited by 3 and 1 mM-HgCl2, respectively, as measured by OD600 loss. Reversible post-commitment HgCl2-sensitive sites were present in the ALA and GFAK pathways which were 50% inhibited by 0.125 mM and 0.05 mM-HgCl2, respectively. A pre-commitment HgCl2-sensitive site was identified in the ALA pathway which was 55% inhibited by 6 mM-HgCl2. At 3 mM-HgCl2, 70% of the spore population became committed to germinate in the ALA pathway, whereas less than 5% OD600 loss occurred. In this system, loss of heat resistance was associated with commitment, whereas OD600 loss and DPA release were identified as post-commitment events. The ALA and GFAK pathways were insensitive to a variety of metabolic inhibitors. Protease inhibitors had different effects on the ALA and GFAK pathways: phenylmethanesulphonyl fluoride (PMSF) solely inhibited ALA germination at a pre-commitment site and had little effect on GFAK germination, whereas N alpha-p-tosyl-L-arginine methyl ester (TAME) inhibited both the ALA and GFAK pathways at pre- and post-commitment sites. These results are discussed in relation to a recently proposed model for the triggering of Bacillus megaterium KM spore germination.     

Sporulation in Bacillus subtilis. Antigenic changes during spore formation

1. Antisera, prepared against extracts of cells and spores of Bacillus subtilis, were used in immunoelectrophoretic studies of the changes occurring in cell extracts during the course of spore formation. 2. At least 15 antigens could be detected in vegetative-cell extracts by the antiserum prepared against cell extracts and at least seven could be demonstrated in spore extracts by the homologous antiserum. 3. Cross-absorption studies showed that two of these antigens were probably completely specific for vegetative-cell extracts and that one was probably completely specific for spore extracts. The remainder were probably present in very small quantities in the heterologous extract. 4. In extracts of cells sporulating in an ;exhaustion medium' those antigens characteristic of the spore began to appear about 1hr. after the end of exponential growth. 5. In cells sporulating in a resuspension medium, spore antigens were detected at 4hr., and by 7hr. a decrease in vegetative-cell antigens was observed. 6. In an asporogenous mutant blocked early in sporulation there was neither an increase in spore antigens nor a decrease in vegetative-cell antigens. 7. In an asporogenous mutant blocked later in sporulation, there was an increase in spore antigens similar to that which occurred in the sporogenous strain.     

Effect of various growth conditions on spore formation and bacillomycin L production in Bacillus subtilis

Bacillomycin L is produced by Bacillus subtilis NCIB 8872 in the stationary phase; it is excreted into the culture medium, without prior accumulation in the bacterial cells. The production of bacillomycin L is largely dependent on the composition of the culture medium. The action of specific inhibitors of sporulation, netropsin and diethyl malonate, on antibiotic synthesis is dependent on the composition of the culture medium. Although they occurred at the same time, there appears to be no direct correlation between sporulation and antibiotic synthesis.     

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.     

SpoIVB has two distinct functions during spore formation in Bacillus subtilis

The Bacillus subtilis SpoIVB protein is a critical component of the intercompartmental signal-transduction pathway that activates the sigma factor, σ^K, in the mother cell of the sporulating cell. SpoIVB, synthesized in the forespore chamber, must act across two layers of phospholipid membrane to facilitate proteolytic processing of inactive pro-σ^K to active σ^K. We have used a genetic approach to dissect SpoIVB function and found that this protein has two distinct developmental functions. One function is that of intercompartmental signalling of pro-σ^K processing. The other role is essential to spore formation and is illustrated by mutations of SpoIVB which allow cell–cell signalling of pro-σ^K processing but prevent the formation of viable spores. Using localized and site-specific mutagenesis we have identified a functional domain of SpoIVB that is involved in its non-signalling role.     

SpoVID guides SafA to the spore coat in Bacillus subtilis

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.     

Acid-soluble spore proteins of Bacillus subtilis

Acid-soluble spore proteins (ASSPs) comprise about 5% of the total protein of mature spores of different Bacillus subtilis strains. They consist of three abundant species, alpha, beta, and gamma, four less abundant species, and several minor species, alpha, beta, and gamma make up about 18, 18 and 36%, respectively, of the total ASSPs of strain 168, have molecular weights of 5,900, 5,9000, and 11,000, respectively, and resemble the major (A, C, and B) components of Bacillus megaterium ASSPs in several respects, including sensitivity to a specific B. megaterium spore endopeptidase. However, they have pI's of 6.58, 6.67, and 7.96, all lower than those of any of the B. megaterium ASSPs. Although strains varied in the proportions of different ASSPs, to overall patterns seen on gel electrophoresis are constant. ASSPs are located interior to the cortex, presumably in the spore cytoplasm, and are synthesized during sporulation and degraded during germination.     

Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis

We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.     

Proteinases in growth and spore formation of Bacillus thuringiensis]

Some serine proteases and leucine aminopeptidases were detected inside and outside the cells during the analysis of three crystalline and two acrystalline strains of Bac. thuringiensis var. galleriae. The data obtained on the protease formation during growth and sporulation and the level of their activity are indicative of intracellular proteases involvement in spore- and crystal formation. The enzymes isolated from the culture medium do not probably take part in these processes. The intracellular enzymes may account for the different crystal protein composition of various strains due to limited proteolysis of crystal proteins in the course of biosynthesis.     

Properties of the Bacillus subtilis spore coat.

About 70% of the protein in isolated Bacillus subtilis spore coats was solubilized by treatment with a combination of reducing and denaturing agents at alkaline pH. The residue, consisting primarily of protein, was insoluble in a variety of reagents. The soluble proteins were resolved into at least seven bands by sodium dodecyl sulfate gel electrophoresis. About one-half of the total was four proteins of 8,000 to 12,000 daltons. These were relatively tyrosine rich, and one was a glycoprotein. There was also a cluster of proteins of about 40,000 daltons and two or three in the 20,000- to 25,000-dalton range. The insoluble fraction had an amino acid composition and N-terminal pattern of amino acids very similar to those of the soluble coat proteins. A major difference was the presence of considerable dityrosine in performic acid-oxidized preparations of insoluble coats. Coat antigen including a 60,000-dalton protein not present in extracts of mature spores was detected in extracts of sporulating cells by immunoprecipitation. This large antigen turned over in a pulse-chase experiment. Antibodies to either the array of 8,000- to 12,000-dalton coat polypeptides or to the larger coat proteins reacted with this 60,000-dalton species, suggesting a common precursor for many of the mature coat polypeptides. Spore coats seem to be assembled by processing of proteins and by secondary modifications including perhaps dityrosine formation for cross-linking.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.