Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.     

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.     

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.     

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.     

Characterization of aBacillus subtilis germination mutant with pleiotropic alterations in spore coat structure

One class of spore germination mutants ofBacillus subtilis produces lysozymesensitive spores with altered surface structure. These mutations were pleiotropic in that the pattern of soluble and insoluble spore coat proteins was extensively changed with the virtual absence of a major 12kd polypeptide. Reversion to the lysozyme-resistant phenotype (and wild-type spore coat profile) at or near the site of the original mutation occurred at a frequency consistent with an initial point mutation.The 12kd protein was also absent from extracts of sporulating cells of the mutant although antigens of 14kd and 32kd protein cross-reacting with antibody to the 12kd polypeptide were detected. The 32kd antigen was also present in extracts of sporulating cells but not in the extracts of the spore coat of the wild type and is probably a precursor. Improper processing of such a precursor could account for the extensive alterations of coat structure.     

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.     

Mutations affecting glutamine synthetase activity in Salmonella typhimurium.

A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity.     

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.     

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.     

Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component.

Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.     

Bacillus subtilis Spore Coat

In response to starvation, bacilli and clostridia undergo a specialized program of development that results in the production of a highly resistant dormant cell type known as the spore. A proteinacious shell, called the coat, encases the spore and plays a major role in spore survival. The coat is composed of over 25 polypeptide species, organized into several morphologically distinct layers. The mechanisms that guide coat assembly have been largely unknown until recently. We now know that proper formation of the coat relies on the genetic program that guides the synthesis of spore components during development as well as on morphogenetic proteins dedicated to coat assembly. Over 20 structural and morphogenetic genes have been cloned. In this review, we consider the contributions of the known coat and morphogenetic proteins to coat function and assembly. We present a model that describes how morphogenetic proteins direct coat assembly to the specific subcellular site of the nascent spore surface and how they establish the coat layers. We also discuss the importance of posttranslational processing of coat proteins in coat morphogenesis. Finally, we review some of the major outstanding questions in the field.     

Effects of thermoradiation on bacteria.

A 60Co source was used to determine the effects of thermoradiation on Achromobacter aquamarinus, Staphylococcus aureus, and vegetative and spore cells of Bacillus subtilis var. globigii. The rate of inactivation of these cultures, except vegetative-cell populations of B. subtilis, was exponential and in direct proportion to temperature. The D10 (dose that inactivates 90% of the microbial population) value for A. aquamarinus was 8.0 Krad at 25 degrees C and 4.9 Krad at 35 degrees C. For S. aureus, D10 was 9.8 and 5.3 Krad at 35 and 45 degrees C, respectively. Vegetative cells of B. subtilis demonstrated a rapid initial inactivation followed by a steady but decreased exponential rate. The D10 at 25 degrees C was 10.3 Krad, but at 35 and 45 degrees C this value was 6.2 and 3.8 Krad, respectively. Between 0 and 95 Krad, survival curves for B. subtilis spores at 75 degrees C showed slight inactivation, increasing in rat at and above 85 degrees C. The D10 values for spores at 85 and 90 degrees C were 129 and 92 Krad, respectively. Significant synergism between heat and irradiation was noted at 35 degrees C for A. aquamarinus and 45 degrees C for S. aureus. The presence of 0.1 mM cysteine in suspending media afforded protection to both cultures at these critical temperatures. On the other hand, cysteine sensitized B. subtilis spores at radiation doses greater than 100 Krad. The combined effect of heat and irradiation was more destructive to bacteria than either method alone.     

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.     

Isolation and Characterization of Microsporum gypseum Lysosomes: Role of Lysosomes in Macroconidia Germination

Three types of lysosomes containing either acid protease, alkaline protease, or phosphodiesterase were isolated from a Microsporum gypseum macroconidial homogenate on Ficoll gradients. The acid protease was contained in an assimilative lysosome since its activity was affected by the complexity of the exogenous nitrogen source. Ultracentrifugation and electron microscopy revealed that the alkaline protease-containing vesicles were associated with the spore coat material prior to macroconidial germination. During macroconidial germination, zones of spore coat hydrolysis were seen surrounding these vesicles. Other larger vesicles, believed to contain the phosphodiesterase, were also observed in the spore coat during macroconidial germination.     

Effect of storage time and temperature and the variation among replicate tests (on different days) on the performance of spore disks and strips.

Laboratory-prepared spore disks were stored for 96 weeks at 22 degrees C with 50% relative humidity (RH) and at 4 degrees C with less than 1% RH. At the same time commercial spore strips were stored for 64 weeks at 22 degrees C with 50% RH. The spore count per unit and the heat resistance were measured at the beginning of the experiment and after 16, 32, 48, 64, 80, and 96 weeks of storage. The laboratory-prepared spore disks stored at 4 degrees C with less than 1% RH showed less change in numbers of spores per disks and decrease in the survival time than did the disks stored at 22 degrees C with 50% RH. Both the laboratory-prepared spore disks and the commercial spore strips stored at 22 degrees C with 50% RH decreased in survival times with increased storage time. The relative change in the survival times with storage was less for the commercial spore strips than for the laboratory-prepared spore disks.     

Thermal analysis of the spores of Bacillus cereus with special reference to heat activation.

The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.     

Antagonistic Role of CotG and CotH on Spore Germination and Coat Formation in Bacillus subtilis

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.     

Mutants of Bacillus subtilis affected in spore outgrowth.

Six mutants of Bacillus subtilis 168 that are temperature-sensitive in spore outgrowth were isolated. The outgrowth process proceeds normally at 35 degrees C, but at the non-permissive temperature (47 degrees C) it is arrested at a specific stage characteristic for each mutant strain. The mutants are not altered in vegetative growth whether at 35 degrees C or at 47 degrees C. They were characterized for their ability to synthesize RNA, proteins and DNA during outgrowth. A mutant defective in spore germination was also isolated; less than 5% of its spores can germinate at any of the temperatures tested. The mutations were mapped by means of transduction and transformation. The isolation of a number of outgrowth mutants which map at different loci and which affect outgrowth at different times is discussed in relation to the regulation of this process.     

Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability

A bank of pTV32 (Tn 917 lacZ) - generated Bacillus subtilis mutants were examined on milk agar for the ability to produce proteases at 48 degrees C. A single mutant, BUL786, was isolated, which could hydrolyze casein after overnight incubation at 48 degrees C. This mutant secreted protease 10 fold more at 48 degrees C when compared to 37 degrees C, and part of the activity appears to be 48 degrees C-specific. At high temperatures, other strains of B. subtilis, including hyperprotease secretors, were unable to secrete protease to any significant degree. The BUL786 strain is missing the 97K major heat shock protein. Since a number of other proteins also appear to be secreted at 48 degrees C, this mutant may be a hypersecretor of exported proteins at temperatures greater than 45 degrees C.