Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.     

Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment

The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

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.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.     

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-dependent activation of spore-lytic enzyme precursor by germinated spores of Clostridium perfringens

A precursor of the spore-lytic enzyme of Clostridium perfringens was extracted with alkali from dormant spores of the organism. The enzyme precursor was activated by incubating it with germinated spores which had been treated with alkali. The activation was greatly enhanced by the addition of 3-phosphoglycerate, suggesting that the conversion of precursor to active enzyme depends on endogenous energy-producing metabolism during germination.     

Protein Synthesis During Fungal Spore Germination II. Aminoacyl-soluble Ribonucleic Acid Synthetase Activities During Germination of Botryodiplodia theobromae Spores

The specific activities of 13 aminoacyl-soluble ribonucleic acid (sRNA) synthetases were measured at various time intervals during the germination of Botryodiplodia theobromae conidiospores. The enzyme activities were low or absent in ungerminated spores, and they increased rapidly as germination proceeded. When extracts of the ungerminated spores were prepared with mortar and pestle, very little or no enzyme activity was detected. When the extracts were prepared with a mechanical homogenizer, however, they exhibited some enzyme activity, although less than did the extracts from germinated spores. Enzyme activities from germinated spores were approximately the same, regardless of the method of preparation. The enzyme fraction from ungerminated spores prepared with a mechanical homogenizer could also stimulate incorporation of phenylalanine into polyphenylalanine in the presence of ribosomes, polyuridylic acid, and sRNA, although the activity was approximately only 15 to 20% that of a similar enzyme fraction from germinated spores. It is concluded that ungerminated spores of B. theobromae contain active aminoacyl-sRNA synthetases and transfer enzymes, although the activities are low when compared to germinated spores.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

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.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

Biochemical Studies of Bacterial Sporulation and Germination XVII. Sulfhydryl and Disulfide Levels in Dormancy and Germination

A fourfold increase in sulfhydryl content upon germination of Bacillus megaterium spores was observed by the standard fluorescein mercuric acetate assay as reported by others. However, assay of ruptured dormant spores or the use of N-ethylmaleimide and a denaturing agent on intact spores showed a constant sulfhydryl level in dormancy and in germination. The apparent increase in sulfhydryl groups observed on germination was shown to be due to inaccessibility of most sulfhydryl groups in the dormant spore to sulfhydryl reagents. The disulfide content of dormant spores showed no change on germination, nor was any evidence found for production of low-molecular-weight sulfhydryl or disulfide compounds during germination.     

Glutamic Acid Decarboxylase in Spores of Bacillus megaterium and Its Possible Involvement in Spore Germination

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase (GAD). Although dormant spores showed no GAD activity, spores given sonic treatment and heat-activated spores had high activities when assayed for this enzyme. Several parameters of GAD in heat-activated spores were examined. The effects of KCN, NaN(3), 2,4-dinitrophenol, and KF on GAD activity were examined. Only KCN was an effective inhibitor of GAD activity in heated spores and was also shown to be the only effective inhibitor of GAD activity in vegetative bacteria. Similar patterns of inhibition were obtained with GAD activity and with spore germination, KCN being the only effective inhibitor of both, although at different concentrations. Spore GAD activity in heat-activated spores showed a loss with storage at 4 C; on the other hand, storage at 25 C was not accompanied by a loss, but, to the contrary, showed an increase in GAD activity of about 30%. A comparison of GAD activity at different times during germination, growth, and sporulation showed it to be highest in freshly germinated spores. Although vegetative cells contained GAD activity, the level in log-phase cells was approximately one-half the level obtained with freshly germinated spores. Heat-activated mutant spores with a requirement of gamma-aminobutyric acid for germination gave no GAD activity. GAD activity appeared in mutant spores after germination and increased to levels comparable to parent spores after 9 min of germination.     

Cyclic AMP,phosphodiesterase, and spore activation inPhycomyces blakesleeanus

A soluble cyclic AMP phosphodiesterase was demonstrated in crude extracts ofPhycomyces spores. During an activating heat treatment of the spores the cyclic AMP phosphodiesterase activity was reduced to some 15% of its value in dormant spores. During early germination the activity slowly increased. No difference was found in the behavior of the enzyme from dormant and activated spores during gel filtration and anion exchange chromatography or in its sensitivity toward heat denaturation. After the spores were heated at different temperatures there was a coincidence between germination induction and cyclic AMP phosphodiesterase inactivation. 3-Isobutyl-1-methylxanthine induced an increase in both cyclic AMP concentration and trehalase activity in the spores and led to complete germination of the spores.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

Role of uricase in the triggering of germination of Bacillus fastidiosus spores.

The likelihood that uric acid was the only compound capable of triggering germination of Bacillus fastidiosus spores was reinforced by the finding that ureidoglycollic acid, urea, NH4Cl, 2,8-dihydroxypurine and a combination of L-alanine and O-carbamoyl-D-serine were ineffective as germinants. Uric acid-triggered germination of B. fastidiosus was prevented by a range of inhibitors that also inhibited uricase activity in dormant spore extracts. O2 uptake during germination started immediately after addition of uric acid, possibly as a consequence of the oxidation of uric acid by the enzyme uricase. Germination showed a dependence on uric acid concentration, with a relatively high Km (4-5 mM). During the first 10 min of germination of heat-activated spores there was no detectable change in the number of spore-cortex reducing groups, indicating that selective cortex hydrolysis is not involved in the trigger mechanism of germination of B. fastidiosus. On the basis of the results, a model is proposed in which re-initiation of uricase activity is the mechanism by which B. fastidiosus spores are triggered to emerge from the dormant state.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene.

The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCI-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S. Miyata, R. Moriyama, N. Miyahara, and S. Makino, Microbiology 141:2643-2650, 1995). The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca2+, Mg2+, or Mn2+) for its activity. The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg2+. The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls. However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam in its cortical peptidoglycan. This indicates that the enzyme recognizes the delta-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination. Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore. A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined. The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358. The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form. It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme. A possible mechanism for cortex degradation in C. perfringens S40 spores is discussed.