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.     

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.     

The source of the heat resistance of bacterial spores: Study of water in spores by NMR

It has been postulated that the heat stabilization of the essential macromolecules in the core of the spore may be produced by dehydration at two levels: (i) the spore is drier at high relative humidity than the vegetative cell and (ii) the core of the spore may be less hydrated than the cortex and the coat. The latter postulate was subjected to experimental testing by ¹H-NMR studies of the water signal in the five species of spores and coat and (coat + cortex) preparations. The transverse relaxation rate $\text{(}\text{1}\text{T}{}_2\text{)}$ was determined in samples equilibrated at constant relative humidity. To allow for the effect of paramagnetic ions on $\text{1}\text{T}{}_2$ a model system (wool keratin) was studied in the presence of known amounts of Ca(II), Mn(II), Cu(II), Ni(II) and Fe(III). Because of the dominant effect of Mn(II) on $\text{1}\text{T}{}_2$, the effect of small amounts of other metal ions in spores was neglected. The relaxation rate of water at a particular relative humidity and manganese concentration was consistently less for intact spores than for coat or coat + cortex, hence the water in the core is more mobile than in the outer integuments. Sorption isotherm studies have shown that at a particular relative humidity there is about as much water in the core as in the cortex and coat. These two results taken together indicate that the hypothesis that the core is drier than the cortex and coat is incorrect, hence the spore is not heat-stabilized in this way. A theory is proposed in which heat stabilization is attributed to immobilization of essential enzymes and nuclei acids by a solid support, calcium dipicolinate, in a similar fashion to the heat stabilization of enzymes in a charged polymer matrix. It is proposed that stabilization is effected by electrostatic and hydrogen bonds between the calcium dipicolinate and the essential macromolecules. Experiments in progress show that enzymes and DNA are heat-stabilized in vitro by calcium dipicolinate.     

Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers

A variant strain that produced spores lacking exosporium was isolated from a culture of Bacillus megaterium QM-B1551. Two additional spore morphotypes were obtained from the parent and variant strains by chemical removal of the complex of coat and outer membrane. Among the four morphotype spores, heat resistance did not correlate with total water content, wet density, refractive index, or dipicolinate or cation content, but did correlate with the volume ratio of protoplast to protoplast plus cortex. The divestment of integument layers exterior to the cortex had little influence on heat resistance. Moreover, the divestment did not change the response of either the parent or the variant spores to various germination-initiating agents, except for making the spores susceptible to germination by lysozyme. The primary permeability barrier to glucose for the intact parent and variant spores was found to be the outer membrane, whereas the barrier for the divested spores was the inner membrane.     

Effect of depletion of FtsY on spore morphology and the protein composition of the spore coat layer in Bacillus subtilis

Bacillus subtilis FtsY is a homolog of the alpha-subunit of mammalian signal recognition particle (SRP) receptor, and is essential for protein translocation and vegetative cell growth. An FtsY conditional null mutant (strain ISR39) can express ftsY during the vegetative stage but not during spore formation. Spores of ISR39 have the same resistance to heat and chloroform as the wild-type, while their resistance to lysozyme is reduced. Electron microscopy showed that the outer coat of spores was incompletely assembled. The coat protein profile of the ftsY mutant spores was different from that of wild-type spores. The amounts of CotA, and CotE were reduced in spore coat proteins of ftsY mutant spores and the molecular mass of CotB was reduced. In addition, CotA, CotB, and CotE are present in normal form at T(8) of sporulation in ftsY mutant cells. These results suggest that FtsY has a pivotal role in assembling coat proteins onto the coat layer during spore morphogenesis.     

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.     

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207-217. 1963.-It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates.     

Characterization of a Bacillus cereus protease mutant defective in an early stage of spore germination.

Temperature-sensitive sporulation mutants of Bacillus cereus were screened for intracellular protease activity that was more heat labile than that of the parental strain. One mutant grew as well as the wild type at 30 and 37 degrees C but sporulated poorly at 37 degrees C in an enriched or minimal medium. These spores germinated very slowly in response to alanine plus adenosine or calcium dipicolinate. During germination, spores produced by the mutant rapidly became heat sensitive, but released dipicolonic acid and mucopeptide fragments more slowly than the wild type and decreased only partially in density while remaining phase white (semirefractile). In freeze-etch electron micrographs, the mature spores were deficient in the outer cross-patched coat layer. During germination, the spore coat changes associated with wild-type germination occurred very slowly in this mutant. Although the original mutant was also a pyrimidine auxotroph, reversion to prototrophy did not alter any of the phenotypic properties discussed. Selection of revertants that germinated rapidly or sporulated well at 37 degrees C, however, resulted in restoratin of all wild-type properties (exclusive of the pyrimidine requirement) including heat-stable protease activity. The reversion frequency was consistent with an initial point mutation, indicating that a protease alteration resulted in production of spores defective in a very early stage of germination.     

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.     

The effect of sporulation temperature on sporal characteristics ofBacillus subtilis A

Spores ofBacillus subtilis A were produced at different temperatures (23°–49°C) and examined for a number of sporal characteristics. Spore heat resistance increased with sporulation temperature to 45°C, with spores grown at 49°C showing a dramatic reduction in resistance. Spore crops showed biphasic thermal death curves whether enumerated on germination medium with or without calcium dipicolinate. This strain produces both rough and smooth variants. Of the spores produced at 23°C, 99% were rough, had a density of 1.305, and an average core/core + cortex volume ratio of 0.1838. At 49°C, 99% were smooth, had a density of 1.275, and an average volume ratio of 0.3098. Between these temperatures both spore types were produced. There appeared to be no direct correlation with sporulation temperature, heat resistance, and dipicolinate content. There was an increase in both the magnesium and calcium contents to 45°C with a dramatic reduction at 49°C. The 1.305 density spores had higher calcium and dipicolinate contents than the 1.275 spores, although both spore types showed biphasic thermal death curves. The mechanisms involved in determining which spore type (rough/smooth) is produced at a specific growth temperature is unknown.Florida Agricultural Experiment Station Journal Series Number R-00312.     

Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.     

Ultrastructural Analysis During Germination and Outgrowth of Bacillus subtilis Spores

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.     

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

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.     

Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures

AIMS: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. METHODS AND RESULTS: Bacillus subtilis spores prepared at temperatures from 22 to 48 degrees C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. CONCLUSIONS: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.     

Changes of ultrastructure in spore coat of Bacillus thiaminolyticus during germination and outgrowth.

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Effect of trichloroacetic acid treatment on certain properties of spores of Bacillus cereus T.

Spores of Bacillus cereus T treated with trichloroacetic acid (6.1--61.2 mM) were compared with untreated spores, and as the concentration of the chemical increased, the following alterations in spore properties were found: (1) the extent of germination decreased irrespective of the germination medium used; (2) the spores became sensitive to sodium hydroxide (1 N) and hydrochloric acid (0.27 N), but not to lysozyme (200 micrograms/ml); (3) loss of dipicolinate increased on subsequent heating; and (4) the spores became more sensitive to heat. However, trichloroacetic acid-treated spores were still viable and there was no significant change in spore components. The mechanism of action of trichloroacetic acid is discussed.     

Characterization of spores of Bacillus subtilis that lack most coat layers

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.     

Germination properties of a spore coat-defective mutant of Bacillus subtilis.

The presence of the gerE36 mutation in strains of Bacillus subtilis 168 resulted in poor germination of their spores in a range of germinants, as measured by the fall in absorbance of spore suspensions. Although resistant to heat and organic solvents, spores were sensitive to lysozyme; electron microscopy revealed that their coat structure was incomplete. These spores responded to germinants by losing heat resistance and changing from phase bright to phase gray. The release of dipicolinic acid and the fall in absorbance of spore suspensions reached only 75 and 50% of wild-type levels, respectively, but followed the same time course as the loss of heat resistance. Although the germination response was incomplete, the concentration of L-alanine required to elicit it was the same for the mutant as for the wild type. The properties of mutant spores suggest that an intact spore coat is not required for the initial interaction between germinant and spore, but that the coat layers may contain molecules important in later stages of germination. In transduction with phage SPP1, the gerE36 mutation mapped between citF and ilvB and was 90% cotransduced with citF2. The gerE mutation identifies the location of a gene important for the progress of late stages of spore formation.