Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.     

Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.     

Identification of germination gene of Bacillus megaterium

Glucose, KNO3, proline and leucine initiate the spore germination of B. megaterium ATCC 12872, but not of B. megaterium ATCC 19213. In order to isolate the gene concerning germination of B. megaterium ATCC 12872, we constructed its gene library in plasmid vector, and introduced into B. megaterium ATCC 19213. We obtained a transformant whose spores differed from those of the wild type strain with respect to germinability. Spores of this transformant could be germinated by glucose, proline or leucine. The recombinant plasmid prepared from this transformant was found to carry 2 kilobase pairs fragment of B. megaterium ATCC 12872 DNA. This fragment may contain the gene encoding the protein which plays an important role in germination.     

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.     

Role of outer coat in resistance of Bacillus megaterium spore

The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed of galactosamine-6-phosphate (GalN-P) polymers with polypeptides and calcium. OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer of GalN-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experiments revealed that the outer coat limited the entry of compounds with a molecular weight of more than 2,000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of OC-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainly of GalN-P polymers, and the molecular sieving effect.     

Germination of the decoated spores of Bacillus megaterium

Decoated spores of Bacillus megaterium ATCC 12872 were prepared by extracting the inner coat components with an alkaline solution containing sodium dodecyl sulfate and dithiothreitol (SDS-DTT) from outer coat-deficient mutant spores, which were produced from one of the mutants isolated and named MAE-05 by us. The decoated mutant spores germinated as well as the intact spores of the mutant and the parent, indicating that the outer and inner spore cats cannot be essential structures for the initiation of germination. When the SDS-DTT-treated MAE-05 spores were converted to H-spores by incubation in citrate-phosphate buffer (pH 3.5) at 30 C for 3 hr, they lost their germinability by glucose and KNO3. Ca-spores, prepared by treating H-spores with 10 mM calcium acetate at 37 C for 60 min, regained the germinability. Experiments on the interaction of 45Ca with the cortex and the inner membrane isolated from H-spores suggested that the calcium present in the inner membrane might be related to germinability.     

Spore-spheroplast transformation of Bacillus megaterium

A new method for transformation of Bacillus megaterium was developed by modification of Chang and Cohen's method. In our method, spore spheroplasts were used as recipient cells instead of the protoplasts of vegetative cells. Longer incubation (60 min) of spore spheroplasts and plasmid DNA before treatment with polyethylene glycol remarkably increased the efficiency of transformation. The frequency of transformation was about 10(4) per microgram of plasmid DNA. A shot-gun-type cloning of chromosome DNA of B. megaterium ATCC 12872 was available in B. megaterium ATCC 19213 strain by this transformation method.     

Occurrence in Bacillus megaterium of uridine 5''-diphospho-N-acetylgalactosamine and uridine 5''-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer.

We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography. From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine. They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface. These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium.     

Biochemical defects of outermost layer deficient mutants during sporulation of Bacillus megaterium.

To determine the regulation of morphogenesis of the outermost layer, the thick layer outside the inner coat, of the Bacillus megaterium spore, we isolated 15 outermost layer deficient mutants of B. megaterium using transposon Tn917. Three mutant strains lacked both synthesis of the 48-kDa outermost layer protein and induction of two initial enzymes for galactosamine-6-phosphate polymer synthesis, evidence that these biochemical events are regulated in the cascade system during morphogenesis of the outermost layer.     

Levels of Small Molecules and Enzymes in the Mother Cell Compartment and the Forespore of Sporulating Bacillus megaterium

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.     

Occurrence of galactosamine-6-phosphate as a constituent of Bacillus megaterium spore coat

Galactosamine-6-phosphate was identified as a component of the coat of the Bacillus megaterium QM B1551 spore. It was one of the main constituents of the outermost layer of the spore coat, but it was absent from the other integuments including the cortex. These findings suggest that galactosamine-6-phosphate comprises the phosphorus-containing skeleton structure of the spore coat.     

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.     

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.     

Morphogenesis of the membrane-bound electron-transport system in sporulating Bacillus megaterium KM.

The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM?HAVE BEEN COMPARED WIth those of similar fractions prepared from exponential-phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a + a3, and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential-phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but DL-glycerol 3-phosphate and L-malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH oxidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five-fold increase in NADH-cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and L-malate oxidases; DL-glycerol 3-phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors.     

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.     

Regulation of phosphoglycerate phosphomutase in developing forespores and dormant and germinated spores of Bacillus megaterium by the level of free manganous ions.

The large depot of phosphoglyceric acid (PGA) which is accumulated within spores of Bacillus megaterium is greater than 99% 3-phosphoglyceric acid (3-PGA). The 3-PGA depot is stable in forespores and dormant spores, but is utilized rapidly during spore germination. When spores were germinated in KBr plus NaF, the PGA depot was not utilized, but 13% of the 3-PGA was converted to 2-PGA. These data suggest phosphoglycerate phosphomutase as the enzyme which is regulated to allow 3-PGA accumulation during sporulation. Young isolated forespores, in which 3-PGA was normally stable, utilized their 3-PGA rapidly when incubated with Mn2+ plus the divalent cation ionophore X-537A; Mn2+ or ionophore alone or Mg2+ or Ca2+ plus ionophore was without effect. Young forespores contained significant amounts of Mn2+. However, forespore Mn2+ exchanged slowly with exogenous Mn2+ and was removed poorly by toluene treatment. This suggests that much of the forespore Mn2+ is tightly bound to some forespore component. Since phosphoglycerate phosphomutase from B. megaterium has an absolute and specific requirement for Mn2+, these data suggest that the activity of this enzyme in vivo may be regulated to a large degree by the level of free Mn2+. Indeed, the activity of this enzyme in forespore or dormant spore extracts was stimulated greater than 25-fold by Mn2+, whereas comparable extracts from cells or germinated spores were stimulated only two- to fourfold.     

Dielectric characterization of forespores isolated from Bacillus megaterium ATCC 19213

Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium.     

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.     

Effect of Lysozyme on Resting Spores of Bacillus Megaterium

Resting spores of Bacillus megaterium ATCC 9885 were found to be markedly affected by lysozyme. Exposure to as little as 1.5 mug of lysozyme per ml caused the spores to lose refractility, the darkened spores to shed their coat structures, and the spore central bodies to lyse. The spores of seven other strains of B. megaterium and seven other Bacillus species were not similarly affected by lysozyme. Proteolytic enzymes such as pronase, trypsin, pepsin, and subtilisin did not induce the change. The action of lysozyme differed in certain important respects from that of common "physiological" germinants. Its action was considered to be direct via its enzymatic attack on exposed sites directly accessible in the resting spores of B. megaterium ATCC 9885.     

Mineralization and heat resistance of bacterial spores.

The heat resistances of the fully demineralized H-form spores of Bacillus megaterium ATCC 19213, B. subtilis var. niger, and B. stearothermophilus ATCC 7953 were compared with those of vegetative cells and native spores to assess the components of resistance due to the mineral-free spore state, presumably mainly from dehydration of the spore core, and to mineralization. Mineralization greatly increased heat resistance at lower killing temperatures but appeared to have much less effect at higher ones.