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.     

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.     

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.     

Trypsinlike enzymes from dormant and germinated spores of Bacillus cereus T and their possible involvement in germination.

Trypsin-like enzymes were studied in dormant, activated, and germinated spores of Bacillus cereus T. Dormant spores contained two heat-labile enzyme activities. One was extractable with 2 M KCl and hydrolyzed azo-albumin. The second, a trypsinlike activity, was not extractable with 2 M KCl and hydrolyzed benzoyl-L-arginine-p-nitroanilide. Because of their heat instability, these two enzyme activities are probably not involved in the germination of heat-activated spores. Upon germination of heat-treated spores, a trypsinlike protease which was not detected in intact dormant spores was activated or exposed. This enzyme, when measured in intact germinated spores, hydrolyzed benzoyl-DL-arginine-p-nitroanilide but not azo-albumin and was inhibited in situ by sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid and Hg2+. There was a correlation between the inhibition of germination and enzymatic activity by sulfhydryl-blocking reagents. The enzyme was also inhibited by leupeptin, tosyl-L-lysine chromoethyl ketone, and tosyl-L-arginine methyl ester. Good correlation existed between the inhibition of germination and enzymatic activity by these agents. Electron micrographs showed that in the presence of trypsin inhibitors, the spores did not lose their cortex. The protein extracts of the inhibited spores formed a somewhat different electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the protein extracts of dormant or germinated spores.     

Isolation and characterization of two distinct fractions from the inner membrane of dormant Bacillus megaterium spores

Two distinct membrane bands were obtained after sucrose velocity gradient centrifugation of crude inner membranes from dormant Bacillus megaterium spores disrupted under conditions which minimized endogenous enzyme action. These two inner membrane fractions (termed LD and HD) contained similar amounts of total and individual phospholipid species. However, LD and HD differed significantly in phospholipid/protein ratios (4.3 and 0.47 mg/mg, respectively), equilibrium densities (1.12 and 1.18 g/cm3), NADH oxidase specific activity (less than 0.01 and 0.13 mumol/min X mg), and content of specific proteins. In contrast, crude membranes prepared in identical fashion from germinated spores gave only a single inner membrane band (termed G) on sucrose velocity gradients. G had a phospholipid/protein ratio of 0.98 mg/mg, an equilibrium density of 1.16 g/cm3, and an NADH oxidase specific activity of 2.1 mumol/min X mg. Essentially all of the proteins present in LD or HD or both were found in G, consistent with the latter membrane being derived from a mixture of LD and HD. No evidence was found suggesting that there is significant degradation of dormant spore inner membrane protein upon spore germination.     

Role of menaquinone in inactivation and activation of the Bacillus cereus forespore respiratory system.

The respiratory systems of the Bacillus cereus mother cell, forespore, and dormant and germinated spore were studied. The results indicated that the electron transfer capacity during sporulation, dormancy, and germination is related to the menaquinone levels in the membrane. During the maturation stages of sporulation (stages III to VI), forespore NADH oxidase activity underwent inactivation concomitant with a sevenfold decrease in the content of menaquinone and without major changes in the content of cytochromes and segment transfer activities. During the same period, NADH oxidase and menaquinone levels in the mother cell compartment steadily decreased to about 50% at the end of stage VI. Dormant spore membranes contained high levels of NADH dehydrogenase and cytochromes, but in the presence of NADH, they exhibited very low levels of O2 uptake and cytochrome reduction. Addition of menadione to dormant spore membranes restored NADH-dependent respiration and cytochrome reduction. During early germination, NADH-dependent respiration and cytochrome reduction were restored simultaneously with a fourfold increase in the menaquinone content; during germination, no significant changes in cytochrome levels or segment electron transfer activities of the respiratory system took place.     

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)     

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.     

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.     

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.     

Mutual relationship between antibiotics and resting spores of Bacillus subtilis: morphological changes and macromolecular synthesis after germination of spores treated with cyclic polypeptide and aminoglycoside antibiotics

Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin. Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetable cells, some mesosomes and DNA fibrils. On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B- and gramicidin S-treated resting spores. With gramicidin S-treated germinated spores a few vacuoles were formed in the cytoplasm. Both polymyxin B- and gramicidin S-treated germinated spores showed little or no synthesis of DNA, RNA, and protein. The vegetative cells derived from streptomycin-treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early. Their DNA and RNA synthesis was normal, whereas protein synthesis was low. In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin-treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled D-alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal. The outgrowing cells from gentamicin-treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled D-alanine. Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited. These results coincide with the growth curves of individual antibiotic-treated resting spores.     

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.     

Studies of pyridine nucleotide oxidizing enzymes from human neutrophils

An NADH cytochrome c reductase has been identified in plasma membrane fractions from neutrophils in addition to the superoxide producing NADPH oxidase which has been extensively studied by other investigators. Activation of neutrophils resulted in increased enzyme activities but to different degrees; the NADH cytochrome c reductase increased 2 fold in specific activity and the NADPH oxidase 30 fold. Treatment of the plasma membrane fraction with sonication and differential centrifugation yielded a particulate fraction (R2) with a 2 fold increase in specific activities of both enzymes and concentrations of cytochrome b and FAD. The cytochrome b in the preparation was not reduced under anaerobic conditions by either NADH or NADPH. Treatment of preparations of R2 with deoxycholate or potassium thiocyanate separated the two enzymes yielding particulate preparations with only NADPH oxidase or NADH cytochrome c reductase activity, respectively.     

Increasing Activity of Germinating Bacillus subtilis Spores to Incorporate Thymidine Triphosphate into Deoxyribonucleic Acid After Detergent Treatment

The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.     

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.     

The role of osmotic pressure in the germination of Nosema algerae spores

Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.     

Conversion of Intrasporal Trehalose into Reducing Sugars During Germination of Nosema algerae (Protista: Microspora) Spores: A Quantitative Study

ABSTRACT. Carbohydrates were extracted from dormant, stimulated and germinated spores of Nosema algerae . Concentrations of total sugars were measured by the Anthrone test. Non-reducing sugars were quantified by NaOH hydrolysis followed by the Anthrone reaction, and reducing sugars by the Nelson's test. Glucose was measured by the o -toluidine test and a glucose oxidase assay. The concentrations of trehalose in the cytoplasm of the dormant, ungerminated spore was estimated to be in excess of 1.0 M. Trehalose decreased by 70% during the five-minute course of germination. All of the lost trehalose was converted to reducing sugar of which 70–78% was glucose. The osmotic potential increase caused by catabolism of trehalose appears to be sufficient for germination.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Effects of alpha- and beta-D-glucose on germination of spores of Bacillus megaterium QM B1551.

The effects of D-glucose anomers on the germination of dormant spores of Bacillus megaterium QM B1551 were studied, alpha-D-Glucose (1 mM) slightly initiated the germination of the dormant spores during 10 min incubation at 37 degrees C, while about 60% of the dormant spores became germinated with beta-D-glucose (1 mM) in the same conditions. From the above observations and the finding that only a trace amount of alpha- or beta-D-glucose may bind with the dormant spores, it is speculated that the beta-D-glucose-stereospecific receptor site for the germination exists on the surface of the dormant spores of the bacillus.