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 proteins in membrane vesicles of Bacillus subtilis

Fractionation of B. subtilis cells after heat shock, from 37 degrees C to 54 degrees C, shows an increase in synthesis of proteins localized in cell membranes and a decrease in synthesis of proteins localized in cytosol. There is no such effect of heat shock at temperature of 45 degrees C. Autoradiograms of electrophoretically separated proteins, labelled during heat shock at 54 degrees C, reveal 26 heat-shock proteins (hsps) in membrane vesicles and 11 hsps in cytosol, five of which are common to both fractions. Heat shock at 45 degrees C induces 18 hsps localized in membrane vesicles and 13 hsps localized in cytosol, six of which are common to both fractions. Results are interpreted as showing a relevant role of membrane proteins in cell response to shock at high temperature, pointing to two steps of defense against heat stress.     

Translocation and induction of alpha B crystallin by heat shock in rat glioma (GA-1) cells.

Response to heat shock of alpha B crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of alpha B crystallin at growing phase. When the growing cells were heated at 45 degrees C, concentrations of alpha B crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of alpha B crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45 degrees C for 15 min), the concentration of alpha B crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that alpha B crystallin is a small heat shock protein.     

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.     

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.     

Heat shock affects cell cycling in the neural plate of cultured rat embryos: a flow cytometric study

The effects of heat shock on cell cycling in the mammalian neuroectoderm were determined by applying heat shocks to cultured rat embryos at the neural plate stage, as part of a study on the teratogenic effects of heat shock on neural development. The heat shocks had been characterized previously (Walsh et al.: Teratology 36:181-191, 1987) with respect to their effects on the gross morphological development of the rat embryos. The effects on cell cycling were observed in DNA histograms of neural plate cells recorded in a flow cytometer after staining with DAPI. The mild heat shock (42 degrees C for 10 min) arrested cells at entry to S phase. The teratogenic heat shock (43 degrees C for 7.5 min) arrested cells at entry to S phase also but for a longer time and inhibited cycling through S phase. After each arrest, a synchronized peak of cells later entered S phase and progressed through the cycle. The induced-thermotolerance heat shock, which was the mild heat shock followed after an interval by the teratogenic heat shock, showed that pre-treatment with the mild heat shock reduced the magnitude of the response to the teratogenic heat shock. The cell-cycle inhibitor ICRF 159 showed the effects on cycling rates of the heat-shock treatments. The arrest of cells at entry to S phase by heat shock may function to prevent cells entering DNA synthesis under non-optimal conditions. We report estimates of proportions of non-proliferative cells in the neural plate of the rat embryos.     

Thermal adaptation in CHO cells at 40 degrees C: the influence of growth conditions and the role of heat shock proteins

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.     

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.     

Effects of thermoradiation on bacteria.

A 60Co source was used to determine the effects of thermoradiation on Achromobacter aquamarinus, Staphylococcus aureus, and vegetative and spore cells of Bacillus subtilis var. globigii. The rate of inactivation of these cultures, except vegetative-cell populations of B. subtilis, was exponential and in direct proportion to temperature. The D10 (dose that inactivates 90% of the microbial population) value for A. aquamarinus was 8.0 Krad at 25 degrees C and 4.9 Krad at 35 degrees C. For S. aureus, D10 was 9.8 and 5.3 Krad at 35 and 45 degrees C, respectively. Vegetative cells of B. subtilis demonstrated a rapid initial inactivation followed by a steady but decreased exponential rate. The D10 at 25 degrees C was 10.3 Krad, but at 35 and 45 degrees C this value was 6.2 and 3.8 Krad, respectively. Between 0 and 95 Krad, survival curves for B. subtilis spores at 75 degrees C showed slight inactivation, increasing in rat at and above 85 degrees C. The D10 values for spores at 85 and 90 degrees C were 129 and 92 Krad, respectively. Significant synergism between heat and irradiation was noted at 35 degrees C for A. aquamarinus and 45 degrees C for S. aureus. The presence of 0.1 mM cysteine in suspending media afforded protection to both cultures at these critical temperatures. On the other hand, cysteine sensitized B. subtilis spores at radiation doses greater than 100 Krad. The combined effect of heat and irradiation was more destructive to bacteria than either method alone.     

Initiation of Bacillus spore germination by hydrostatic pressure: effect of temperature.

Suspensions of Bacillus cereus T, B. subtilis, and B. pumilus spores in water or potassium phosphate buffer were germinated by hydrostatic pressures of between 325 and 975 atm. Kinetics of germination at temperatures within the range of 25 to 44 degrees C were determined, and thermodynamic parameters were calculated. The optimum temperature for germination was dependent on pressure, species, suspending medium, and storage time after heat activation. Germination rates increased significantly with small increments of pressure, as indicated by high negative deltaV values of -230 +/- 5 cm3/mol for buffered B. subtilis (500 to 700 atm) and B. pumilus (500 atm) spores and -254 +/- 18 cm3/mol for aqueous B. subtilis (400 to 550 atm) spores at 40 degrees C and -612 +/- 41 cm3/mol for B. cereus (500 to 700 atm) spores at 25 degrees C. The ranges of thermodynamic constants calculated at 40 degrees C for buffered B. pumilus and B. subtilis spores at 500 and 600 atm and for aqueous B. subtilis spores at 500 atm were: Ea = 181,000 to 267,000 J/mol; deltaH = 178,000 to 264,000 J/mol; deltaG = 94,000 to 98,300 J/mol; deltaS = 264 to 544 J/mol per degree K. These values are consistent with the concept that the transformation of a dormant to a germinating spore induced by hydrostatic pressure involves either hydration or a reduction in the visocosity of the spore core and a conformational change of an enzyme.     

Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability

A bank of pTV32 (Tn 917 lacZ) - generated Bacillus subtilis mutants were examined on milk agar for the ability to produce proteases at 48 degrees C. A single mutant, BUL786, was isolated, which could hydrolyze casein after overnight incubation at 48 degrees C. This mutant secreted protease 10 fold more at 48 degrees C when compared to 37 degrees C, and part of the activity appears to be 48 degrees C-specific. At high temperatures, other strains of B. subtilis, including hyperprotease secretors, were unable to secrete protease to any significant degree. The BUL786 strain is missing the 97K major heat shock protein. Since a number of other proteins also appear to be secreted at 48 degrees C, this mutant may be a hypersecretor of exported proteins at temperatures greater than 45 degrees C.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Effect of heat shock on acetylcholinesterase activity in chick muscle cultures

The effect of heat shock was studied on the acetylcholinesterase activity of chick muscle primary cultures. In cultures transferred from 37 degrees C to 45 degrees C, a sharp drop in activity was followed by rapid spontaneous recovery. The time of onset of recovery resembled the time needed for expression of heat shock proteins. In cultures exposed to heat shock at 45 degrees C and allowed to recover at 37 degrees C, reappearance of acetylcholinesterase activity did not involve de novo protein synthesis since it was not prevented by cycloheximide. Our data raise the possibility of a role for heat shock proteins as molecular chaperones in rescuing heat-denaturing acetylcholinesterase.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

The Effects of Temperature Shock on Transcription and Replication in Rhynchosciara americana (Diptera: Sciaridae)

The chromosomal response to temperature shock in Rhynchosciara americana is similar to that observed in other Diptera. After a 33 degrees C/90 min or a 36 degrees C/30 min shock the reaction for RNA polymerase II (RpII) is enhanced at five loci. The most prominent of these was identified by in situ hybridization as the site of the hsp70 gene. At 33 degrees C, an accumulation of heat shock factor (HSF) and an increase in the level of RpII was observed at some heat shock loci after 5 min and reached a maximum after 15 min at most loci. The pattern of accumulation of HSF and RpII at individual heat shock loci was similar and their increases were generally coordinated among the loci. RpII gradually decreased at sites active prior to shock, the rate of decrease varying with the site. The B2 DNA puff retained RpII for a significant length of time while the histone locus still contained RpII after a shock of 90 min. With a 36 degrees C/30 min shock, the size of the heat shock puffs and the intensities of HSF and RpII peaked at 1-4 h post stress. The level of HSF declined rapidly after 1 h while the level of RpII remained high for an additional 4 h. The reaction of the DNA puffs to heat shock varied. Usually they did not regress completely and retained traces of RpII. BrdU incorporation continued at both amplifying and non-amplifying bands after shock but on average it appeared depressed for about 24 h post stress.     

Heat-induced modulation of lamin B content in two different cell lines

Previous work in our laboratory indicates that the nuclear matrix protein lamin B is a "prompt" heat shock protein, which increases significantly when human U-1 melanoma and HeLa cells are exposed to 45.5 degrees C for 5-40 min. Using Western blotting, we found that the lamin B content in U-1 and HeLa cells increased to a greater extent during post-heat incubation at 37 degrees C than during the heat dose itself. When HeLa cells were heated at 45.5 degrees C for 30 min, and then incubated at 37 degrees C for up to 7 h, lamin B content was increased significantly (1.69-fold maximum increase at 3 h) compared to unincubated heated cells. Also, thermotolerant HeLa cells showed a greater increase (up to 1.72-fold) in lamin B content during subsequent heating compared to nontolerant cells. The increase in lamin B content in thermotolerant cells, or when heated cells were incubated at 37 degrees C, was also observed in U-1 cells. HeLa cells heated in the presence of glycerol (a heat protector) showed a 1.21-1.72-fold increase in lamin B content compared to cells heated for 10-30 min without glycerol. In contrast, lamin B content decreased 1.23-1.85-fold when cells were heated for 10-30 min in the presence of procaine (a heat sensitizer) compared to cells heated without procaine. These data suggest that lamin B may play an important role in the heat shock response, and that modulation of lamin B content by heat sensitizers or protectors may play a role in regulation of heat sensitivity.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)