Heat activation of Streptomyces viridochromogenes spores.

The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.     

Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores

Here we describe the functional relationship between YabG and transglutaminase (Tgl), enzymes that modify the spore coat proteins of Bacillus subtilis. In wild-type spores at 37 degrees C, Tgl mediates the crosslinking of GerQ into higher molecular mass forms; however, some GerQ multimers are found in tgl mutant spores, indicating that Tgl is not essential. Immunoblotting showed that spores isolated from a yabG mutant after sporulation at 37 degrees C contain only very low levels of GerQ multimers. Heat treatment for 20 min at 60 degrees C, which maximally activates the enzymatic activity of Tgl, caused crosslinking of GerQ in isolated yabG spores but not in tgl/yabG double-mutant spores. In addition, the germination frequency of the tgl/yabG spores in the presence of l-alanine with or without heat activation at 60 degrees C was lower than that of wild-type spores. These findings suggest that Tgl cooperates with YabG to mediate the temperature-dependent modification of the coat proteins, a process associated with spore germination in B. subtilis.     

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.     

A two-dimensional protein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation

The heat resistance of spores of Bacillus subtilis formed at 30 degrees C was enhanced by pretreatment at 48 degrees C for 30 min, 60 min into sporulation, for all four strains examined. High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance. The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles. The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation. The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.     

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.     

Cold shock response in sporulating Bacillus subtilis and its effect on spore heat resistance

Cold shock and ethanol and puromycin stress responses in sporulating Bacillus subtilis cells have been investigated. We show that a total of 13 proteins are strongly induced after a short cold shock treatment of sporulating cells. The cold shock pretreatment affected the heat resistance of the spores formed subsequently, with spores heat killed at 85 or 90 degrees C being more heat resistant than the control spores while they were more heat sensitive than controls that were heat treated at 95 or 100 degrees C. However, B. subtilis spores with mutations in the main cold shock proteins, CspB, -C, and -D, did not display decreased heat resistance compared to controls, indicating that these proteins are not directly responsible for the increased heat resistance of the spores. The disappearance of the stress proteins later in sporulation suggests that they cannot be involved in repairing heat damage during spore germination and outgrowth but must alter spore structure in a way which increases or decreases heat resistance. Since heat, ethanol, and puromycin stress produce similar proteins and similar changes in spore heat resistance while cold shock is different in both respects, these alterations appear to be very specific.     

Characterization of a Bacillus cereus protease mutant defective in an early stage of spore germination.

Temperature-sensitive sporulation mutants of Bacillus cereus were screened for intracellular protease activity that was more heat labile than that of the parental strain. One mutant grew as well as the wild type at 30 and 37 degrees C but sporulated poorly at 37 degrees C in an enriched or minimal medium. These spores germinated very slowly in response to alanine plus adenosine or calcium dipicolinate. During germination, spores produced by the mutant rapidly became heat sensitive, but released dipicolonic acid and mucopeptide fragments more slowly than the wild type and decreased only partially in density while remaining phase white (semirefractile). In freeze-etch electron micrographs, the mature spores were deficient in the outer cross-patched coat layer. During germination, the spore coat changes associated with wild-type germination occurred very slowly in this mutant. Although the original mutant was also a pyrimidine auxotroph, reversion to prototrophy did not alter any of the phenotypic properties discussed. Selection of revertants that germinated rapidly or sporulated well at 37 degrees C, however, resulted in restoratin of all wild-type properties (exclusive of the pyrimidine requirement) including heat-stable protease activity. The reversion frequency was consistent with an initial point mutation, indicating that a protease alteration resulted in production of spores defective in a very early stage of germination.     

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.     

Heat-shock responses in two leguminous plants: a comparative study

Relative growth rates, basal and acclimated thermotolerance, membrane damage, fluorescence emission, and relative levels of free and conjugated ubiquitin and HSP70 were compared after 2 h of treatment at different temperatures between Prosopis chilensis and Glycine max (soybean), cv. McCall, to evaluate if the thermotolerance of these two plants was related to levels of accumulation of heat shock proteins. Seedlings of P. chilensis germinated at 25 degrees C and at 35 degrees C and grown at temperatures above germination temperature showed higher relative growth than soybean seedlings treated under the same conditions. The lethal temperature of both species was 50 degrees C after germination at 25 degrees C. However, they were able to grow at 50 degrees C after germination at 35 degrees C. Membrane damage determinations in leaves showed that P. chilensis has an LT(50) 6 degrees C higher than that of soybean. There were no differences in the quantum yield of photosynthesis (F(v)/F(m)), between both plants when the temperatures were raised. P. chilensis showed higher relative levels of free ubiquitin, conjugated ubiquitin and HSP70 than soybean seedlings when the temperatures were raised. Time-course studies of accumulation of these proteins performed at 40 degrees C showed that the relative accumulation rates of ubiquitin, conjugated ubiquitin and HSP70 were higher in P. chilensis than in soybean. In both plants, free ubiquitin decreased during the first 5 min and increased after 30 min of heat shock, conjugated ubiquitin increased after 30 min and HSP70 began to increase dramatically after 20 min of heat shock. From these data it is concluded that P. chilensis is more tolerant to acute heat stress than soybean.     

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.     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.     

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.     

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

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

A comprehensive analysis of heat shock protein synthesis in human peripheral lymphocytes: the effect of penicillin/streptomycin.

A reliable experimental procedure is described for the simultaneous characterisation of a comprehensive range of heat shock proteins (hsps) in human peripheral lymphocytes. In this system, a mild heat shock from 37 to 42 degrees C for 1 h induced the synthesis of hsps 105, 90, 70, 60, 57, 47, 40, 27 and 16. Densitometric analyses of 35[S]-methionine labelled protein gels indicated that levels of these hsps peaked at 3 to 4 h, following post-heat shock recovery at 37 degrees C. The presence of penicillin and streptomycin in the cell culture medium, appeared to have little effect on the kinetics of hsp synthesis. The present method can be used for relatively small blood samples and its relative ease of application and reproducibility make it appropriate for screening the expression of hsps in human lymphocytes from a range of individuals.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.