Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.     

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.     

Immunological Detection of 22K Protein in Sporulating Cells of Bacillus megaterium ATCC 12872

The synthesis and deposition of 22,000-dalton (22K) spore coat protein were examined immunochemically on the sporulating cells of Bacillus megaterium ATCC 12872 using the antibody to purified 22K spore coat protein. This antibody cross-reacted with 44K and 25K proteins in immunoblot analysis of dormant spore coat proteins. Immunoblot analysis on the sporulating cells showed that 22K protein was detected from t₈ in forespore coat protein fractions. Sandwich enzyme immunoassay revealed that 22K protein in the spore coat protein fraction appeared at t₆ and reached a plateau at t₉, and 22K protein in the mother cell cytoplasmic fraction was detected at only t₇ and t₈ at a very low level.     

Coat protein synthesis during sporulation of Bacillus subtilis: immunological detection of soluble precursors to the 12,200-dalton spore coat protein.

Antibody specific to the 12,200-dalton spore coat protein of Bacillus subtilis was used to detect the synthesis of cross-reacting material during sporulation. Cross-reacting protein was first detected by immunoprecipitation after 4 h of development and represented at least 1 to 2% of the total soluble protein synthesis at 5.5 h. A polypeptide of 21,000 daltons was detected in immunoprecipitates by gel electrophoresis. This polypeptide did not accumulate in sporulating cells and was rapidly turned over at the time of coat deposition. In contrast, a 32,000-dalton polypeptide reacted with antibody when unlabeled cell protein was denatured with sodium dodecyl sulfate, separated by gel electrophoresis, and transferred to nitrocellulose paper. This polypeptide was not detected during cell growth or the first 3.5 h of development but was found to accumulate in sporulating cells at 5.5 h. The lack of detection of this polypeptide by immunoprecipitation of undenatured protein indicates that the antigenic sites which cross-reacted with antibody to the 12,200-dalton protein sequence were not exposed unless the molecular conformation was altered. The 32,000-dalton protein may be a primary translation product which is proteolytically processed into mature spore coat protein via a 21,000-dalton intermediate.     

Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A.

Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

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.     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

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.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.     

Properties of Bacillus cereus temperature-sensitive mutants altered in spore coat formation.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38 degrees C). The spores also germinated slowly when produced at 35 degrees C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 35 degrees C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.     

Immunoelectron microscopic studies on spore coat proteins of Bacillus megaterium

An immunochemical staining technique for the spore coat proteins of Bacillus megaterium ATCC 12872 was developed using colloidal gold as a second antibody. For reducing the non-specific immunogold binding and increasing the specific binding, the affinity-purified IgG was used as a first antibody. In sporulating cells at t10, gold particles were found not only in the spore coat but also in the mother cell cytoplasm, suggesting that some coat proteins were synthesized in the cytoplasm. Use of the specific affinity-purified antibody to 48K-protein demonstrated that this protein was one of the components of the outer coat.     

Synthesis of Bacillus cereus spore coat protein

The major structural protein of Bacillus cereus spore coats was synthesized, commencing 1 to 2 h after the end of exponential growth, as a precursor with a mass of ca. 65,000 daltons. About 40% of this precursor, i.e. 26,000 daltons, was converted to spore coat monomers of 13,000 daltons each, perhaps as disulfide-linked dimers. The rate of conversion varied, being initially slow, most rapid at the time of morphogenesis of the coat layers, and then slow again late in sporulation, coincident with a decrease in intracellular protease activity. There was a second major spore coat polypeptide of about 26,000 daltons that was extractable from mature spores in variable amounts. This protein had a peptide profile and a reactivity with spore coat protein antibody that were very similar to those of the 13,000-dalton monomers. It is probably a disulfide-linked dimer that is not readily dissociated.     

CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis

Bacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation-specific and heat-labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo-regulator CotH contributes to the switch between the coat of 25°C and that of 42°C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components.     

Purification and characterization of intracellular proteases of Clostridium perfringens type A

Five intracellular proteases from sporulating cells of Clostridium perfringens type A were identified and three could be separated by DEAE-Sephacel. Two, I-A and I-B, had caseinolytic activity and one, I-C, was only active on N-benzoyl-DL-arginine-p-nitroanilide. I-A and I-B could each be further separated by Sephacryl S-300 into I-A-1 and I-A-2 and I-B-1 and I-B-2, respectively. I-A-1, a chymotrypsin-like enzyme, was the major intracellular protease, constituting 74% of the intracellular caseinolytic activity. In addition to cytoplasmic proteases both trypsin and chymotrypsin-like enzyme activity was associated with the membrane fraction. I-A-1 had a molecular weight of 330,000, with subunits of 120,000 and 138,000. I-A-1 cleaved a 1200 molecular weight peptide from C. perfringens enterotoxin. Early sporulating cell extracts of C. perfringens contained three presumptive enterotoxin precursors, which disappeared following treatment with I-A. Such cells also contained at least 10 spore coat related proteins, only one (51,500 molecular weight) of which was sensitive to I-A-1. The results indicate a possible role for the major intracellular protease in the processing of C. perfringens enterotoxin and a less important role, if any, in spore coat formation.     

Amino acids in the Bacillus subtilis morphogenetic protein SpoIVA with roles in spore coat and cortex formation

Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In Bacillus subtilis, the attachment of the coat to the spore surface and the synthesis of the cortex both depend on the spore protein SpoIVA. To identify functionally important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of spoIVA that result in defects in coat and cortex formation. One mutant resembles the null mutant, as sporulating cells of this strain lack the cortex and the coat forms a swirl in the surrounding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulating cells of these strains, we frequently observed swirls of mislocalized coat in addition to a coat surrounding the spore, in the same cell. Using immunofluorescence microscopy, we found that in two of these mutants, SpoIVA fails to localize to the spore, whereas in the remaining strains, localization is largely normal. These mutations identify amino acids involved in targeting of SpoIVA to the spore and in attachment of the coat. We also isolated a large set of mutants producing spores that are unable to maintain the dehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered structure.