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.     

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.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

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.     

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.     

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.     

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.     

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.     

Revival of Bacillus subtilis spores from biocide-induced injury in the germination process

Spores of Bacillus subtilis NCTC 8236 were treated with glutaraldehyde, Lugol's iodine, polyvinylpyrrolidone-iodine (PVP-I), sodium hypochlorite or sodium dichloroisocyanurate (NaDCC). After exposure survivors were enumerated on nutrient agar containing potential revival agents (subtilisin, lysozyme, calcium dipicolinate, calcium lactate). Of these, only calcium lactate had any significant enhancing effect and then only with iodine-treated spores. Calcium lactate (9 mmol 1⁻¹) in nutrient broth enhanced the rate and extent of germination of iodine-treated spores but not of spores previously subjected to glutaraldehyde, hypochlorite or NaDCC.     

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.     

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 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+.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.     

Germination properties of a spore coat-defective mutant of Bacillus subtilis.

The presence of the gerE36 mutation in strains of Bacillus subtilis 168 resulted in poor germination of their spores in a range of germinants, as measured by the fall in absorbance of spore suspensions. Although resistant to heat and organic solvents, spores were sensitive to lysozyme; electron microscopy revealed that their coat structure was incomplete. These spores responded to germinants by losing heat resistance and changing from phase bright to phase gray. The release of dipicolinic acid and the fall in absorbance of spore suspensions reached only 75 and 50% of wild-type levels, respectively, but followed the same time course as the loss of heat resistance. Although the germination response was incomplete, the concentration of L-alanine required to elicit it was the same for the mutant as for the wild type. The properties of mutant spores suggest that an intact spore coat is not required for the initial interaction between germinant and spore, but that the coat layers may contain molecules important in later stages of germination. In transduction with phage SPP1, the gerE36 mutation mapped between citF and ilvB and was 90% cotransduced with citF2. The gerE mutation identifies the location of a gene important for the progress of late stages of spore formation.     

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.     

Inhibition of germinant binding by bacterial spores in acidic environments.

Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination. Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites. C. botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5. Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7. Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate. The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM). Nonspecific uptake of L-[3H]alanine by C. botulinum spores was not inhibited at pH 4.5. Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site. This may represent a general mechanism for inhibition of spore germination in acidic environments.     

Germination properties as marker events characterizing later stages of Bacillus subtilis spore formation

At various stages during spore formation sporangia were shocked by cold treatment or with toluene, and the germination requirements of the prespores were examined. Up to 5 h after induction of sporulation (t5) germination was spontaneous; i.e., it occurred without any added germinants. After t5, during stages V and VI, the capacity for spontaneous germination diminished progressively, and the spores acquired a need for externally added germinants. At t6 this need was satisfied by either L-alanine or a mixture of KCl, glucose, and fructose. By t8, the latter response had disappeared. The spores germinated only with L-alanine, and the response was much slower. Experiments with chloramphenicol showed that the germination properties of the spores appearing between t6 and t8 were the expression of events in protein synthesis that had occurred before t5. Although the germination requirements developed at about the same time as heat resistance, they could be dissociated from heat resistance in wild-type and mutant cells. The germination properties of the developing spores are additional marker events characterizing the later stages of sporulation, as follows: (i) spontaneous germination (up to the end of stage IV); (ii) germination requirements that are satisfied by KCl-glucose-fructose or L-alanine (stage V); and (iii) slow germination response with L-alanine only (stage VI).