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.     

Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus

• 1.1. Spore coat extracts from Bacillus thuringiensis subspecies kurstaki and israelensis and Bacillus cereus T and B. cereus NRRL 569 were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by amino acid analysis.
• 2.2. Both B. cereus spore coats had similar electrophoretic profiles.
• 3.3. The B. thuringiensis spore coats contained crystal proteins as major components as well as lower mol. wt proteins.
• 4.4. B. thuringiensis subsp. israelensis had a unique coat protein profile which was different from B. cereus and B. thuringiensis subsp. kurstaki coats.
• 5.5. Insecticidal activity of spores against the tobacco hornworm, Manduca sexta, and the mosquito, Aedes aegypti, also was determined.
• 6.6. B. thuringiensis subsp. kurstaki spores were lethally toxic to the tobacco hornworm (Lepidoptera) larvae, whereas spores of the other subspecies were not.
• 7.7. Except for subspecies israelensis, none of the spores was effective against the mosquito (Diptera) larvae.

     

Characterization of spore appendages from Bacillus cereus strains

AIMS: Further characterization and comparison of spore appendages from Bacillus cereus strains. METHODS AND RESULTS: Appendages were isolated from 10 B. cereus strains from the food industry and food-borne outbreaks. The appendage proteins were dissolved in sample buffer containing 2% SDS and 5% mercaptoethanol at 100 degrees C, and subjected to SDS-PAGE. None of the appendages showed identical protein patterns. Western blots, using antibodies raised against a 3.5 kDa appendage protein, showed that the majority of the appendage proteins reacted with the antibody. Removal of the appendages by sonic treatment of the spores did not alter their heat resistance. The appendages were digested by proteinase K, pepsin, and the enzymes in the detergent Paradigm 10, but not by trypsin or chymotrypsin. Spore adhesion to stainless steel was scarcely affected by removal of the appendages. Digestion of adhered intact spores (with appendages) with Paradigm 10 showed a high degree of variation. CONCLUSIONS: Spore appendages from B. cereus are complex proteinaceous structures that differ among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Information about spore appendages and their involvement in spore adhesion is crucial for improving cleaning methods used for control of bacterial spores in the food industry.     

Deletion of sigB in Bacillus cereus affects spore properties

In Bacillus cereus and other gram-positive bacteria the alternative sigma factor sigma(B) is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene in B. cereus on spore properties. In particular, spores of the sigB deletion mutant showed a defect in germination upon exposure to the germinants alanine and inosine.     

Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.     

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.     

Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.     

Patterns of spore locations in pairs of Bacillus cereus sporangia.

The location patterns, relative to the cross wall, of terminal-to-subterminal Bacillus cereus spores were determined in pairs of sporangia. The presence of three types of patterns suggests that spores are randomly located, but medium-dependent variability of the frequency ratios of the patterns strongly suggests that nonrandom localization cannot be discounted.     

Involvement of the spore coat in germination of Bacillus cereus T spores

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

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.     

Interaction of iodine with Bacillus subtilis spores and spore forms

Buffered solutions of iodine (pH 7.0) were effective against Bacillus subtilis spores, but concentrations and contact times for effective sporicidal action were relatively high. Concentrations of 500 to 1000 ppm available iodine with a contact time of 30–45 min were required to produce a 3–5 log reduction. Treatment of spores with agents which caused progressive extraction of coat protein and cortex hexosamine was associated with increased sensitivity to iodine. Treatment of spores with iodine produced extraction of spore coat protein which was potentiated in the presence of NaOH, but there was no evidence of breakdown of cortex hexosamines or release of dipicolinic acid, either from intact spores or spore protoplasts. Sporicidal concentrations of iodine stimulated the uptake of (³²P) phosphate over an initial period of 30–40 min, but phosphate then leaked from the cells; 1000 ppm available iodine produced total loss within 60 min. Results of this investigation are consistent with previous findings which suggest that the resistance of spores to biocides is related to the barrier properties of the spore outer layers and that the sporicidal action of halogen-releasing agents is related to their ability to cause coat and cortex degradation, leading to rehydration of the spore protoplast and allowing diffusion to their site of action on the underlying protoplast.     

Clostridium perfringens spore germination: characterization of germinants and their receptors

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.     

Bacillus cereus cell and spore properties as influenced by the micro-structure of the medium

Aim:  To investigate the effect of different growth conditions on Bacillus cereus cell and spore properties.
Methods and Results:  Bacillus cereus was grown on agar plates with different surface water conditions (wet and dry) or viscosity. Cell populations displayed different types of behaviour, and heterogeneity was manifested in cell motility and dimension. Spore populations were heterogeneous regarding their properties, namely size and thermal resistance. The smallest spores were produced from flagellated cells, which also displayed jet-motility, growing on the wettest agar. Cytometric analysis also revealed within the smallest spores a sub-population labelled by propidium iodide (PI), indicating that spore populations were partly damaged. Nonmotile cells grown on diffusion-limiting media were elongated and produced the least thermal-resistant spores.
Conclusions:  The micro-structural properties of the media were found to influence cell and spore properties. Abundant surface water enabled flagellar motility and resulted in a heterogeneous cell and spore population, the latter including small and damaged spores. High viscosity gave rise to filamentous cells and more heat-sensitive spores.
Significance and Impact of the Study:  This study provides useful information on conditions resulting in heterogeneous populations of damaged and heat-sensitive spores.     

Alteration by heat activation of enzymes localized in spore coats of Bacillus cereus

Heat activation (70 degrees C for 20 min) resulted in alteration in structural proteins and enzymes found in Bacillus cereus spore coats. The three notable changes were increased glycosylation of coat proteins, alteration in polypeptide pattern on sodium dodecyl sulfate - polyacrylamide gels, and an increase in free SH groups of proteins. About three polypeptides leaked out in small quantities from the spore coats during heat activation. The extraction of five spore coat associated enzyme activities was followed during the coat stripping procedures, which left the cortex and core intact. Two of these activities, L-alanine dehydrogenase and purine nucleoside hydrolase, were solubilized when the undercoat was extracted by 1,4-dithioerythritol (DTE) at pH 9.8. Three other activities, a protease, a corticolytic enzyme, and purine nucleoside phosphorylase, were solubilized by both DTE alone and DTE plus urea at pH 9.8. The DTE plus urea extraction removed the two more insoluble coat layers, the outer cross-patch, and the inner pitted layers. Mutants deficient in the cross-patch layer contained normal amounts of the protease, corticolytic, and purine nucleoside phosphorylase activities suggesting their association with the pitted layer. In intact spores all five enzymes were found to be stable to the heat activation treatment. However, extracted and partially purified preparations of protease, purine nucleoside phosphorylase, and L-alanine dehydrogenase were heat sensitive. Similar preparations of corticolytic enzyme and purine nucleoside hydrolase were stable to the heat activation conditions.