Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.     

Relationship between cortex content and properties of Bacillus sphaericus spores.

The muramic lactam content of spores of Bacillus sphaericus mutants defective in meso-diaminopimelic acid synthesis increases almost linearly with an increase of meso-diaminopimelic acid concentration in the medium. Since muramic lactam content is a measure of cortex content, the amount of cortex in spores of the mutants can be easily varied by changing the meso-diaminopimelic acid concentration in the medium. Characteristic properties were tested in spores containing different amounts of cortex. Critical amounts of cortex were associated with different spore properties. Refractility and dipicolinic acid accumulation in the spores both required about 20% of the maximum cortex content (although refractility is independent of dipicolinic acid content). For xylene octanol resistance, about 25% of the maximum cortex content was required.     

The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization

Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.     

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.     

Immunoelectron microscopic localization of one of the spore germination proteins, GerAB, in Bacillus subtilis spores.

Ultrastructural localization of GerAB, one of the proteins of Bacillus subtilis spores related to L-alanine-initiated germination, was investigated by immunoelectron microscopy with antipeptide (residues 61 to 80 of GerAB) antiserum and a colloidal gold-immunoglobulin G complex. Immunogold particles were visualized in the boundary region between the cortex and coat of dormant spores, and they were broadly dispersed into the cortex region after germination.     

Noninvolvement of the spore cortex in acquisition of low-molecular-weight basic proteins and UV light resistance during Bacillus sphaericus sporulation

Two major low-molecular weight, acid-soluble proteins (termed A and B proteins) were purified from Bacillus sphaericus spores and had properties similar to those of the analogous proteins from spores of other Bacillus species. These proteins were accumulated late in sporulation, when the developing spores became resistant to UV light, and were degraded during spore germination by a spore protease. A mutant of B. sphaericus unable to make spore cortex because of a block in diaminopimelic acid (DAP) biosynthesis accumulated and maintained levels of the A and B proteins similar to those in the DAP+ parent or the DAP- strain in which cortex formation was restored by growth with DAP. In addition, the DAP- strain grown without DAP acquired a level of UV light resistance identical to that of wild-type spores and at the time of appearance of the A and B proteins. These findings indicate that formation of little, if any, spore cortex is required for acquisition of UV light resistance or maintenance of high levels of A and B proteins. The data provide further support for a role of the A and B proteins in the spore's UV light resistance.     

Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species

AIMS: The aim of this work was to compare the chemical structure of the spore cortex of a range of species, and to determine any correlation between cortex structure and spore resistance properties. METHODS AND RESULTS: The fine chemical structure of the cortex of Bacillus subtilis, Bacillus megaterium, Bacillus cereus and Clostridium botulinum was examined by muropeptide analysis using reverse phase HPLC. There is a conserved basic structure between peptidoglycan of these species, with the only difference being the level of de-N-acetylation of an amino sugar. In order to determine if an alteration in cortex structure correlates with heat resistance properties, the peptidoglycan structure and properties of B. subtilis spores prepared under different conditions were compared. Peptidoglycan from spores prepared in Nutrient Broth (NB) showed reduction in single L-alanine substituted muramic acid to only 13.9% compared with 20.6% in CCY-grown spores. NB-prepared spores are also unstable, with 161-fold less heat resistance (60 min, 85 degrees C) and 43 times less Mn(2+) content than CCY-grown spores. Addition of MnCl(2) to NB led to a peptidoglycan profile similar to CCY-grown spores, sevenfold more heat resistance (60 min, 85 degrees C) and an 86-fold increase in Mn(2+) content. Addition of CCY salts to NB led all parameters to be comparable with CCY-grown spore levels. CONCLUSION: It has been shown that peptidoglycan structure is conserved in four spore-forming bacteria. Also, spore heat resistance is multifactorial and cannot be accounted for by any single parameter. SIGNIFICANCE AND IMPACT OF THE STUDY: Endospores made by diverse species most likely have common mechanisms of heat resistance. However, the molecular basis for their resistance remains elusive.     

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Effect of the internal water activity of bacterial spores on their heat resistance in water

The water contents and effective a_w of the core, cortex, and coat of spores in water, as well as the masses of the core, cortex, and coat plus exosporium in the dry state, are calculated from volumes, dry densities, and water absorption isotherms of the sporal components. From data presented here for spores ofBacillus subtilis var.niger andBacillus cereus T, and from previously published data forBacillus stearothermophilus, the logarithm of the heat resistance of the spores in water is linearly related to the effective a_w of their core and cortex.     

Relation of the heat resistance of bacterial spores to chemical composition and structure II. Relation to cortex and structure

The relation between the amount of cortex, measured as total hexosamine, as diaminopimelic acid and as muramic lactam, and the heat resistance of spores of five different strains of Bacillus stearothermophilus was studied. Electron micrographs of thin sections of the spores were made to relate the structure of the spores to chemical and thermal characteristics. It was found that the amount of the cortex was significantly related to heat resistance of the spores. Strains with more electron-dense and better organized cortices were found to express higher heat resistance.     

Relation of the heat resistance of bacterial spores to chemical composition and structure. II. Relation to cortex and structure

The relation between the amount of cortex, measured as total hexosamine, as diaminopimelic acid and as muramic lactam, and the heat resistance of spores of five different strains of Bacillus stearothermophilus was studied. Electron micrographs of thin sections of the spores were made to relate the structure of the spores to chemical and thermal characteristics. It was found that the amount of the cortex was significantly related to heat resistance of the spores. Strains with more electron-dense and better organized cortices were found to express higher heat resistance.     

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.     

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Inhibition of cortex hydrolysis during spore germination by CdCl2

When the spores of Bacillus megaterium QM B1551 (ATCC 12872) were incubated with 5 mM CdCl2 at 30 C, they underwent the early germination events, such as loss of heat resistance and release of calcium dipicolinate, in the same way as when they were germinated by glucose + KNO3. However, germination by CdCl2 caused no increase in the reducing groups in the cortex and no excretion of glucosamine-containing materials due to the hydrolysis of the cortex peptidoglycan. Addition of CdCl2 at any time during germination by glucose + KNO3 inhibited the release of glucosamine-containing materials from the spores, whereas removal of cadmium from the CdCl2-germinated spores by treatment with cysteine restored the hydrolysis of peptidoglycan. These results suggested that CdCl2 caused the early events of spore germination but prevented the spores from undergoing the events following germination by inhibiting the enzymatic lysis of the cortex peptidoglycan. The conclusion from the study is that cortex degradation is not always required for the initiation of germination.     

Collapse of cortex expansion during germination of Bacillus megaterium spores

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast-plus-cortex, C/(P+C), of the CdCl2-germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose-induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.     

芽孢杆菌孢子萌发机理的研究进展

芽孢杆菌休眠孢子的萌发是孢子恢复到营养生长的第一个决定性步骤。孢子被营养性萌发剂和各种非营养信号诱导而萌发恢复到营养细胞状态。芽孢萌发后就丧失了对外界胁迫的抵抗力。该文主要从芽孢萌发信号传导、营养萌发受体、萌发中的离子通道、皮层溶解酶的功能、非营养诱导萌发和萌发途径等方面阐述芽孢杆菌孢子萌发机理的进展,并对其前景作了简要评述。     

Cortex content of asporogenous mutants of Bacillus subtilis.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.     

Permeability of gentamicin and polymyxin B into the inside of Bacillus subtilis spores

The penetration of gentamicin and polymyxin B into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold--immunoglobulin G (IgG) complex. The colloidal gold particles were located predominantly in the coat region of both gentamicin-treated and polymyxin B-treated spores and were hardly observed in the other regions, i.e., the cortex and core regions. When these antibiotic-treated spores were subsequently treated with CaCl2, the number of gold particles bound to the coat region was greatly decreased. These results suggest that these two antibiotics are able to penetrate into the spore coat but not into the cortex or core, that is, the primary permeability barrier to them exists between the coat and the cortex regions.     

Structural changes in spores under high temperature exposure]

The electron microscopy of ultrathin sections of B. cereus spores showed that no lysis and destructive changes occurred in the main structural components of the spores when heated to 99 degrees C (in distilled water). By the time 99% of the population were destroyed, the spores seemed to preserve the exosporium of the sporoderm, the cortex and the sporoblast intact. Even autoclaving at 120 degrees C for 15 min brought about no visible changes in the ultrastructure of the spores, though it killed the whole spore population.     

The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration.

Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.