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.     

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.     

Inhibition by potassium ion of the pregerminative response to L-alanine of unactivated spores of Bacillus cereus T.

The effect of potassium ion on L-alanine-inosine-induced germination of unactivated spores of Bacillus cereus T was studied. Unactivated spores germinated in 0.1 M sodium phosphate buffer (NaPB), but not 0.1 M potassium phosphate buffer (KPB), at pH 8.0 and at 30 C. Inhibition of germination was also observed on incubation of unactivated spores in NaPB containing potassium chloride. Previously it was demonstrated that germination of unactivated spores involves at least two steps, one induced by L-alanine, and the other by inosine. Potassium ion seems to inhibit the response of the spores to inosine, because: (1) Spores that had been preincubated with L-alanine in NaPB or KPB, germinated in NaPB but not KPB in the presence of inosine. (2) During germination in NaPB, incorporation of L-[14C]alanine showed bimodal kinetics with a rapid first phase and a second continuous phase, but in KPB the second phase of incorporation did not occur. The events occurring before germination of unactivated spores are discussed with reference to the initiation of germination.     

Functional relationships between L- and D-alanine, inosine and NH4Cl during germination of spores of Bacillus cereus T

The results of a physiological study of the interaction between NH4Cl, inosine, and the stereoisomers of alanine during germination of spores of Bacillus cereus T are presented. Detailed kinetics for the germination of unheated spores in moderate concentrations of L-alanine (in the absence of auto-inhibition due to alanine racemase) are established, as is the specificity of the stimulatory effect of NH4Cl in relation to other salts, amines, and germinants. The results suggest that NH4Cl and inosine affect an early step in germination closely related to the function of an L-alanine receptor.     

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.     

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.     

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.     

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.     

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.     

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.     

Mechanism and Factors Influencing the Veronal Inhibition of Bacterial Spore Germination

The inhibitory effect of sodium 5,5-diethyl barbiturate (Veronal) on the L-alanine-induced initiation of germination of Bacillus subtilis spores was examined. Veronal reversibly inhibited the initiation of germination by a noncompetitive mechanism. The inhibition was time-independent and it took place whether L-alanine was or was not allowed to permeate the spore before the addition of the inhibitor. The concentration of the inhibitor and the pH of the initiation system were important factors determining the effectiveness of Veronal as an inhibitor. The magnitude of the inhibition increased linearly with decreasing pH at constant concentration and with increasing concentration at constant pH. These results suggest that the inhibition involves a permeability phenomenon related to the access of drug to the active sites in the spore and that the entry of Veronal into the spores is regulated by the concentration of undissociated molecule. At the physiologically important pH of 7.4, initiation with alanine in phosphate buffer at high spore densities (about 10(9) spores per ml) was 50% inhibited by 4 mM Veronal, and 8mM Veronal inhibited initiation completely. L-Alanine initiation in tris(hydroxymethyl)amino-methane-hydrochloride buffer was completely inhibited by 5 mM Veronal. The inhibition could be partially reversed by the combined addition of D-fructose, D-glucose, and K(+). Possible reasons for the failure of otherwise inhibitory concentrations of Veronal to inhibit completely the L-alanine-induced initiation when a combination of fructose, glucose, and K(+) was present and a suggested relationship to two functional roles of L-alanine in the initiation of germination are discussed.     

Inhibition of Bacillus subtilis spore germination by various hydrophobic compounds: demonstration of hydrophobic character of the L-alanine receptor site.

L-Alanine-initiated germination of Bacillus subtilis spores was inhibited by various kinds of hydrophobic compounds. Good correlation of inhibitory effect with hydrophobicity of the compound was demonstrated by using regression analysis in which the hydrophobic character was expressed by the partition coefficient in an octyl alcohol-water system. The correlation coefficient for 20 alcohols was 0.959, and that for 19 miscellaneous compounds was 0.906. Regression lines of the alcohols and other hydrophobic compounds were almost identical, showing that hydrophobic interaction played an important role in inhibition. Diphenylamine was one of the most effective inhibitors examined. n-Octyl, n-nonyl, and n-decyl alcohols were the most effective alcohols. The mode of inhibition by diphenylamine and n-octyl alcohol was a "mixed type" (competitive plus noncompetitive type) with respect to L-alanine; that by D-alanine was competitive inhibition. Sites for diphenylamine, n-octyl alcohol, and D-alanine may have overlapped. Inhibition was reversible by washing; heat resistance, stainability, and germination rate of the washed spores remained unaltered. Thus, we confirmed that the inhibition may occur before the initial trigger reaction of germination and that it may be due to the interaction between a hydrophobic compound and a hydrophobic region closely associated with the L-alanine receptor site on the spore.     

Effect of different factors on the germination of spores in S and P- variants of Bacillus brevis]

The effect of temperature, duration of heating and the presence of L-alanine and L-glutamine in the medium on the spore germination was studied with the S and P- variants of Bacillus brevis which did not contain gramicidin S and with the R and P+ varants obtained on a defined medium with beta-phenyl-beta-alanine, an inhibitor of the biosynthesis of gramicidin S. The experiments were carried out according to the scheme of complete factor experiment. Germination of the spores was found upon their incubation in a defined medium with L-alanine within two hours after their preliminary heating at 80 degrees C during 45 minutes (S variant), at 60 degrees C during 45 minutes (R variant+trace amounts of gramicidin S), at 80 degrees C during 15 minutes (P+ variant/trace amounts of gramicidin S). Germination of the spores of the P- variant was best upon heating to 60 degrees C during 45 minutes. Gramicidin S is presumed to inhibit, to a certain extent, germination of the spores of its producing culture.     

Germination-initiation and inhibitory activities of L- and D-alanine analogues for Bacillus subtilis spores. Modification of methyl group of L- and D-alanine

The ability of 33 compounds of L-alanine analogues over a wide range of concentrations to initiate germination of Bacillus subtilis spores was determined, and the inhibitory activity against L-alanine-initiated germination was determined for the above compounds and 22 of their D- and DL-isomers. Nineteen L-isomers were able to initiate the germination. The maximum germination rate and the apparent binding affinity of the germinant were obtained from concentration-germination response curves. Not only D-isomers but also L-isomers of many alanine analogues showed inhibitory action on L-alanine-initiated germination. The apparent binding affinity of an inhibitor was calculated by Schild's method. D-Alanine, D-serine, glycine, D-2-amino-n-butyric acid, D-cysteine, D-norvaline, and D-threonine were competitive inhibitors for the L-alanine action. Analysis of the relation between the structure of the side chain of L- and D-alanine analogues and their apparent affinity suggested that there are separate binding portions, which differ in size and electrostatic nature, for germination and for inhibition on the receptor. Certain L-alanine analogues had a dualistic property of initiating germination at low concentrations and inhibitory activity at higher concentrations, i.e., autoinhibition. The autoinhibitory phenomenon might be explained by the above postulation of the presence of separate binding portions for germination and for inhibition.     

Inhibitory Effect of 8-(N,N-Diethylamino)octyl-3, 4, 5-Trimethoxybenzoate Hydrochloride (TMB-8) on Germination of Bacillus cereus T Spores

Effect of 8-(N,N-diethylamino)octyl-3, 4, 5 - trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L -alanine and inosine was investigated. TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores. The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L -alanine. Addition of Ca²⁺ reversed the inhibitory effect of TMB-8 in a dose-related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.     

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.     

Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores

In this work, we have used spores of Bacillus subtilis that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0. 5%), and a pasteurization step (90 degrees C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the L-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate on L-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.     

Differing L-alanine germination requirements of hypochlorite-treated Clostridium botulinum spores from two crops.

Similar populations of hypochlorite-treated spores were enumerated from two crops of Clostridium botulinum 12885A produced by the same procedure; however, germination required different L-alanine concentrations. Lactate permitted the germination of spores from both crops with suboptimal L-alanine concentrations. The data suggest that the spores differ slightly in chemical or structural composition.     

Differing L-alanine germination requirements of hypochlorite-treated Clostridium botulinum spores from two crops

Similar populations of hypochlorite-treated spores were enumerated from two crops of Clostridium botulinum 12885A produced by the same procedure; however, germination required different L-alanine concentrations. Lactate permitted the germination of spores from both crops with suboptimal L-alanine concentrations. The data suggest that the spores differ slightly in chemical or structural composition.     

Proposed role of lactate in germination of hypochlorite-treated Clostridium botulinum spores

Clostridium botulinum 12885A spores treated with hypochlorite required added DL-calcium lactate for L-alanine germination. Lactate was the active component of calcium lactate. Equimolar concentrations of L-malate, but not of DL-propionate, could replace lactate, suggesting that the alpha-hydroxy acid structure is important. Neither lactate nor malate was an effective germinant for buffer-treated or hypochlorite-treated spores. If the L-alanine concentration was increased 100-fold (to 450 mM), the lactate germination requirement was overcome. The data suggest that the L-alanine germination sites were modified by hypochlorite so that a higher concentration of alanine was required for activity. Lactate appeared to be an activator of modified or non-hypochlorite-modified L-alanine germination sites.