Germination of unactivated spores of Bacillus cereus T. Effect of preincubation with L-alanine or inosine on the subsequent germination.

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.     

Stimulation of germination of unactivated Bacillus cereus spores by ammonia

Inclusion of ammonia in germinant mixtures containing L-alanine and inosine stimulated germination of unactivated Bacillus cereus spores at rates equal to those obtained using heat-activated spores without ammonia. D-Alanine had little effect on germination of heat-activated spores, but severely inhibited germination of unactivated spores in the presence of ammonia. Ammonia did not replace the requirement for either L-alanine or inosine: all three compounds were required for rapid germination. Kinetic analysis suggested that the functions of ammonia and L-alanine were more closely related than the functions of ammonia and inosine. With rate-saturating concentrations of L-alanine and inosine, germination rates showed saturation kinetics for ammonia with a Km for NH4Cl of 5 mM. Comparisons of the effects of salts, amines and pH on germination rates suggested that NH4OH rather than NH+4 was the rate-limiting form of ammonia. In comparisons of various strains of B. cereus, stimulation of germination by ammonia occurred in all cases, although spores of most soil isolates germinated more rapidly than B. cereus T spores in the absence of ammonia.     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

Inhibition by ammonium ion of germination of unactivated spores of Bacillus cereus T induced by l-alanine and inosine.

Studies were carried out on the inhibitory effect of NH4+ on germination of spores of Bacillus cereus T induced by L-alanine and inosine. Kinetic analysis showed that NH4+ inhibited the germination competitively. Its inhibitory effect was greater when the unactivated spores had been preincubated with L-alanine. NH4+ did not inhibit the response of unactivated spores to L-alanine during preincubation. These results suggest that L-alanine sensitizes the spores to the inhibitory effect of NH4+.     

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.     

GerN, an antiporter homologue important in germination of Bacillus cereus endospores

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Mechanism of Ca2+ and dipicolinic acid requirement for L-alanine induced germination of Bacillus cereus BIS-59 spores

Spores prepared from different sporulating media containing varying amounts of Ca and dipicolinic acid (DPA), exhibited differential responses to germination in L-alanine (0.25 M). Ca-spores with moderately high Ca and DPA contents could be triggered to germination by L-alanine, whereas P-spores with low contents of Ca and DPA could not be germinated by L-alanine unless Ca2+ or DPA was exogenously added. The initiation of L-alanine induced germination by P-spores in the presence of 45CaCl2 was associated with a marked uptake of 45Ca2+. Experiments involving stepwise extraction of 45Ca from prelabelled spores indicated that a part of the spore calcium may be involved in L-alanine induced germination. Both Ca2+ and DPA seemed to have a stimulatory effect on the incorporation of 14C-L-alanine.     

Correlation between spore structure and spore properties in Bacillus megaterium

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.     

Involvement of the spore coat in germination of Bacillus cereus T spores.

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Involvement of the spore coat in germination of Bacillus cereus T spores

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Uptake of l-[14C]-alanine by glutaraldehyde-treated and untreated spores of Bacillus subtilis

The effect of glutaraldehyde on the uptake of L-alanine, and subsequent germination, in spores of Bacillus subtilis NCTC 8236 was examined. Germination was induced by single amino acids, D-glucose and phosphate buffer at 37 degrees C. L-alanine was the best germinant of all amino acids tested. Pretreatment of spores with low concentrations of acid and alkaline glutaraldehyde inhibited subsequent germination, complete inhibition being observed at concentrations of 0.1% (w/v). This concentration also prevented the loss of heat resistance of spores placed in germination medium and exposed to 75 degrees C. Radioactive studies indicated that maximum uptake of L-alanine occurred after ca 30 min at 37 degrees C. Only 1.2% of available L-alanine was taken up during germination. Pretreatment of spores with glutaraldehyde did not interfere with L-alanine uptake at aldehyde concentrations up to 0.5% (w/v). However, this was significantly reduced at a glutaraldehyde concentration of 1.0% (w/v). Minimal differences were observed between acid and alkaline forms of the aldehyde. The results are discussed in terms of the mode of action of glutaraldehyde.     

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.     

A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies

Bacillus anthracis spore germination is usually detected in vitro by alterations in spore refractility, heat resistance, and stainability. We developed a more quantitative, sensitive, and semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry. The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing a fluorescent nucleic acid-binding dye which stained germinated B. anthracis but not ungerminated (UG) spores. Spore germination in the presence of several germinants was characterized. Although L-alanine and inosine alone stimulated rapid germination in this assay, a medium containing optimal concentrations of L-alanine, adenosine, and casamino acids gave low background fluorescence, stimulated germination completely, and at a reasonable rate. Suspensions of heat-activated, UG spores of B. anthracis strain Ames were preincubated with antibodies (Abs) against whole spores to assess their effect on germination. Analyses of the germination data obtained revealed significant differences between spores pretreated with these Abs and those treated with non-immune sera or IgG. Germination inhibitory activity (GIA) was detected for several polyclonal rabbit anti-spore Ab preparations. These included anti-Ames strain spore antisera, IgG purified from the latter, and spore affinity-purified Abs from antisera elicited against four strains of B. anthracis. Abs elicited against UG as well as completely germinated Ames spores inhibited germination. Abs were ranked according to their GIA, and those specific for UG spores usually exhibited greater GIA. Direct binding to spores of these Abs was detected by an ELISA with whole un-germinated Ames spores. Although specific binding to spores by the anti-spore Abs was shown, their titers did not correlate with their GIA levels. Current efforts are focused on identifying the spore antigens recognized by the anti-spore Abs, characterizing the role of these targeted antigens in disease pathogenesis, and evaluating the ability of specific anti-spore Abs to protect against infection with B. anthracis.     

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.     

Factors Affecting the Rate of Heat-induced Spore Germination in Dictyostelium discoideum

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12, germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Cultures of the slime molds were grown in association with Escherichia coli B/r as the host bacterium; spores taken from plates of synthetic medium had a higher final germination value than spores from complex medium containing peptone and yeast extract. Young spores germinated more rapidly than older spores. Optimal germination occurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphate allowed the most rapid germination. After heat shocking, spores were diluted into fresh oxygenated buffer to provide enough oxygen for completion of germination. Germination occurred most rapidly between incubation temperatures of 22 and 25 C.     

L-alanine and inosine enhancement of glucose triggering in Bacillus megaterium spores

Both rate and extent of germination of Bacillus megaterium 14581 (ATCC) spores are considerably augmented when L-alanine and inosine are added to the glucose commonly used as triggering agent for this strain. This enhancement does not arise from heterogeneity in germination requirements of the dormant spore, but is rather a consequence of the combined action of glucose and either or both of the added reagents on a sizeable fraction of spores unable to germinate in glucose alone. Nearly half of the spores that eventually germinate in the mixture of germinants used are either triggered by glucose or are sensitized by it to subsequent triggering by L-alanine and inosine in the first 10 s of imbibition. For a good number of these spores, then, triggering consists of a sequence of separable events.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

Involvement of calcium in germination of coat-modified spores of Bacillus cereus T.

The effect of calcium on germination of coat-modified Bacillus cereus T spores was investigated. Coat-modified spores produced either by chemical extraction (SDS-DTT-treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS-DTT-treated spores could germinate slowly in the presence of L-alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat-modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat-modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat-modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which L-alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS-DTT-treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.     

Germinability of coat-lacking spores of Bacillus megaterium

Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus megaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germinability must not exist in the coat.