Germination of Unactivated Spores of Bacillus cereus T

Heat-activated spores of Bacillus cereus T germinate rapidly in the presence of l-alanine alone or inosine alone. In contrast, unactivated spores can not germinate in the presence of either germinant alone but rapidly in the presence of both germinants. The highest level of cooperative action of l-alanine and inosine on the germination was observed when they were present in a ratio 1 :1. Preincubations of unactivated spores with l-alanine or inosine had opposite effects on the subsequent germination in the presence of both germinants: preincubation with l-alanine stimulated the initiation of subsequent germination, while preincubation with inosine inhibited it. These results suggest that germination of unactivated spores initiated by l-alanine and inosine includes two steps, the first initiated by l-alanine and the second prompted by inosine. The effect of preincubation of unactivated spores with l-alanine was not diminished by washings. The pH dependence of the preincubation of unactivated spores was not so marked as that of the subsequent germination in the presence of inosine.     

Oxidative Activation of Bacillus cereus Spores

A study was made of the activation of Bacillus cereus strain T spores by using the oxidizing agent sodium perborate. The degree of activation was measured with constant germination conditions by using L-alanine, inosine, adenosine, and L-alanine plus adenosine as germination stimulants. The germinal response following the various treatments was compared with the responses obtained with heat activation. It was concluded that the optimal time for activation with 30 mM sodium perborate at room temperature was about 4 hr. If the exposure time was greatly extended, the spores would germinate spontaneously. When the perborate treatment followed heat activation, the germinal response to L-alanine was stimulated, to inosine retarded and without apparent effect for adenosine or L-alanine plus adenosine. Results of experiments designed to demonstrate deactivation by slow oxidation showed that spores activated with sodium perborate were not deactivated by slow oxidation, whereas those activated by heat were. A deactivation study using mercaptoethanol as the deactivation agent showed that both methods of activation could be deactivated after a 24-hr exposure, but this deactivation was reversible by extending the exposure to mercaptoethanol. The results of heat-sensitivity studies revealed that about 70% of the sodium perborate-activated spores were heat sensitive after 60 min in a germination menstruum of L-alanine plus adenosine, whereas similarly treated heat-activated and nonactivated spores were about 99.99% heat sensitive, respectively.     

Ultrastructural and Chemical Changes in Spores of Bacillus cereus after Action of Disinfectants

Spores of Bacillus cereus subjected to 5% NaOH at 50° and 0.25% NaOCl at 20° undergo a 99% loss in viability in 11 and 19.5 min, respectively. Kinetic studies show that the release of Ca²⁺ and dipicolinic acid occur at different rates and that extensive loss of both ribonucleic and deoxyribonucleic acids occur under these conditions of disinfection. Electron micrographic studies reveal a progressive degradation of the spore integuments. Both chemical and ultrastructural observations made in this study lead to the conclusion that the primary lethal effect of either disinfectant is the modification of outer spore coats leading to a disruption of normal permeability barriers.     

Role of Calcium in Biphasic Germination of Bacillus cereus T Spores Sensitized to Lysozyme

Biphasic germination induced by inosine in the presence of Ca²⁺ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10. The first phase of the germination was stimulated by Ca²⁺ in the concentration-dependent manner, showing the optimal concentration at 0.5-1.0 mM. The second phase appeared to be insensitive to the cation. The optimal temperatures for the first and the second phase were 25 C and 40 C, respectively; the optimal pHs for the two phases were 7-9 and around 7.5, respectively. Heat resistance and dipicolinic acid of the SDS-DTT-treated spores were lost mostly during the first phase. A Ca²⁺-specific chelator, glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca²⁺, while it had no inhibitory effect on the second phase. In contrast, the divalent cations examined, except Mg²⁺ and Sr²⁺, affected not only the first phase but also the second phase. The order of inhibitory effect on the first phase was Hg²⁺ > Zn²⁺ > Ba²⁺, Co²⁺, Cu²⁺ > Mn²⁺; on the second phase, it was Hg²⁺ > Cu²⁺ > Zn²⁺ > Co²⁺ > Mn²⁺ > Ba²⁺.     

Cytochrome Pigments in Spores of Bacillus cereus T

Absorption spectra of dormant spores of Bacillus cereus T suspended in glycerol showed peaks characteristic of cytochrome pigments.     

Ultrastructural Changes During Germination of Dictyostelium discoideum Spores

Spores of Dictyostelium discoideum undergo significant changes in fine structure during germination. The mitochondria progressively become less dense and lose their peripherally attached ribosomes, and the tubuli become more pronounced as germination proceeds. During this period, the three-layered spore wall breaks down in two stages: first, the outer and middle layers are ruptured as a unit, and, second, the inner wall is breached. Crystals and dark (lipid) bodies disappear shortly before or during emergence of the myxamoebae. Autophagic vacuoles are found in dormant spores and throughout the entire germination process. The addition of cycloheximide to germinating spores inhibited the loss of the crystals and dark (lipid) bodies. In addition, the drug inhibited the breakdown of the inner wall layer. Cycloheximide did not prevent the formation of the water expulsion vesicle or the apparent function of the autophagic vacuoles.     

Ultrastructural Analysis During Germination and Outgrowth of Bacillus subtilis Spores

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.     

Activation Energy for Glucose-Induced Germination of Bacillus megaterium Spores

The maximum germination rate of Bacillus megaterium QM B1551 spores in glucose increased, and the lag before its attainment decreased, with increasing germination temperature. The activation energy for germination (μ = approximately 20 kcal/mole), based on rate or on lag, was consistent with an enzymatic mechanism.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.     

Ultrastructural Changes Associated with Germination and Outgrowth of an Appendage-Bearing Clostridial Spore

The sequence of events occurring during the germination and outgrowth of appendage-bearing spores of Clostridium bifermentans was studied by phase-contrast and electron microscopy. The mature spore was characterized ultrastructurally as having the normal spore components as well as long tubular appendages which orginated from the surface of the spore coat. Spores were incompletely enclosed by a distinctly laminated exosporium which possessed hairlike projections on its outermost layer. During germination, structural changes were observed in the core, core wall, cortex, and spore coat layers. Cortical material was extruded from the spore during outgrowth, which usually occurred from the pole opposite the appendages. The subunits comprising the structure of the appendages and the morphology of the mature appendages were observed. No discernible changes could be observed in the spore appendages during germination and outgrowth.     

Germination of Spores of Bacillus cereus in Milk and Milk Dialysates: Effect of Heat Treatment

S ummary . The germination of spores of Bacillus cereus was studied in milk and in media consisting of the low M.W. fraction of milk. Dialysates, centrifugates, filtrates and acid whey supported germination to an extent similar to that in the milk from which they were derived. HTST (72° for 15 sec) pasteurized milk or derived media supported appreciable germination whereas raw milk or media derived from it supported little or none. Whey produced by the action of rennet was an exception in that it was equally stimulatory for germination whether derived from raw or pasteurized milk. Heat treatments for 15 see using temperatures between 65–75° rendered the milk most suitable as a germination medium but temperatures > 80° were necessary for spore activation. Of the 2 effects, activation was the more important; at treatment temperatures > 80° germination was increased despite the less favourable medium which resulted. The extent of germination in pasteurized milk varied with different isolates and could be related to their source, those from pasteurized milk germinating the most readily. The practical implications of these findings are discussed together with the preliminary work to examine the nature of the germination factor(s) produced during HTST pasteurization.     

Effects of Temperature on Activation, Germination, and Outgrowth of Bacillus megaterium Spores

The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Chemical Composition of Exosporium from Spores of Bacillus cereus

Homogeneous fragments of exosporium were extricated in centigram amounts from dormant spores of Bacillus cereus and analyzed for intrinsic constituents. The membrane proved to be chemically complex but not unique, consisting mainly of protein (52%), amino and neutral polysaccharides (20%), lipids (18%), and ash (4%). Seventeen common amino acids were identified by chromatography, and were present in usual proportions except for low levels of cystine-cysteine, methionine, tyrosine, and histidine. Glucosamine was the only amino sugar, and glucose and rhamnose were the principal neutral sugars. The lipid fraction contained 5.5% cardiolipin as the only phospholipid, 12.5% neutral lipids, and at least 19 fatty acids, among which normal C(16) and C(18) ones predominated. Calcium and phosphorus occurred in the ash. Small amounts of teichoic, ribonucleic, and dipicolinic acids were believed to represent contamination.     

Factors Influencing Germination of Bacillus subtilis Spores via Activation of Nutrient Receptors by High Pressure

Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl₂.