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The gram-positive bacterium Bacillus subtilis undergoes the process of sporulation when nutrients become exhausted, and the resulting spores are more resistant than are the growing cells to a variety of environmental insults including heat, UV and gamma radiation, and a number of toxic chemicals (8, 34). Wet heat resistance is probably the most dramatic resistance property of dormant spores, as spores are resistant to about 40°C-higher temperatures than are vegetative cells (8). Spore wet heat resistance is due to a number of factors including dehydration of the spore protoplast or core (8), mineralization of the spore core (8), saturation of spore DNA with /-type small acid-soluble proteins (33, 34), and thermal adaptation, as spores of a single species formed at higher temperatures are more wet heat resistant than are spores formed at lower temperatures (8, 39).

Although many factors contribute to spore wet heat resistance, the identity of the target for wet heat killing of spores is not known. However, two different types of studies have indicated that spore DNA is not the killing target and suggested that some spore protein might be the target (2, 7, 30). If spore killing by wet heat is indeed through protein damage, then it is possible that repair or removal of a damaged protein might be important in spore wet heat resistance. Proteins that can repair or remove denatured proteins in vivo are often members of the heat shock regulon, which is important in the survival of many different bacteria after a heat shock (9, 17). Since sporulation at elevated temperatures results in spores with increased heat resistance and heat shock protein synthesis is increased at elevated temperatures (12, 38), then spores prepared at higher temperatures may also have increased levels of heat shock proteins which may in turn contribute to their increased heat resistance. In order to investigate whether proteins of the heat shock regulon play any role in wet heat resistance of B. subtilis spores, we have examined (i) the effect of mutations in known heat shock genes on spore wet heat resistance, (ii) the effect of mild heat shock at various times during sporulation on spore wet heat resistance, and (iii) the expression of heat shock genes during germination of spore populations which had been killed ~50% by wet heat treatment.

Effects of mutations in heat shock genes on spore wet heat resistance. The heat shock genes of B. subtilis are grouped into at least three classes based on the precise mechanism for the regulation of their expression (11); we examined the effects of mutations in representatives from each of the three classes. Mutations in class I genes included a polar mutation in dnaK, a polar mutation in hrcA, and a nonpolar mutation in hrcA. The mutated class II gene was sigB (14) encoding the RNA polymerase sigma factor, ^B, which directs transcription of other class II genes; consequently, a mutation in sigB abolishes transcription of all class II genes (1, 3, 4, 38). Mutations in class III genes included a mutation in lonA (24, 26) and a nonpolar mutation in ctsR (6); ctsR is a negative regulator of the clpP, clpC, and clpE operons (5, 6, 15), so a mutation in ctsR results in the overexpression of those operons. We had hoped to also study strains with a mutation in clpC, but such strains sporulated extremely poorly, as noted previously (22).

View larger version (64K): [in this window] [in a new window]   FIG. 1.   SDS-polyacrylamide gel electrophoresis analysis of extracts from wild-type (PS832) and nonpolar hrcA mutant (PS3032) B. subtilis spores. Spores were decoated, washed, and lysed as described in the text; 25 µg of protein was run on an SDS-10% polyacrylamide gel; and the gel was stained with Coomassie blue. Lane L contains molecular mass markers whose kilodaltons are given on the left side of the gel, lane 1 is the hrcA spore extract, and lane 2 is wild-type spore extract. The bands labeled a and b are DnaK and GroEL, respectively.

Effect of heat shock during sporulation on spore wet heat resistance. As noted above, it is known that, when cultures of the same strain are sporulated at different temperatures, the spores from cultures sporulated at the higher temperature are more wet heat resistant than are those sporulated at lower temperatures (8, 39). It has also been reported previously that when Bacillus megaterium or B. subtilis cultures at 27 or 30°C were shifted to 45 or 48°C for 30 min at 1 to 2 h into sporulation, the resultant spores were more heat resistant than were those from cultures which had not been subjected to a temperature shift or had been shifted earlier or later in sporulation (21, 28), and we obtained similar results. Cultures were sporulated at 30°C by the resuspension method (36) to ensure the maximum synchrony of the sporulation process, shifted to 45°C for 30 min at various times during sporulation, and returned to 30°C for the remainder of sporulation, and the heat resistance of the resulting spores was measured (Fig. 2). The same experiment was also performed during sporulation in 2× SG medium (23) (data not shown). In both cases, spores from cultures that were shifted to 45°C at various times in sporulation were more heat resistant than were those from cultures not shifted at all, with cultures shifted 2 h into sporulation consistently giving spores with slightly more heat resistance (Fig. 2 and data not shown). We also used Western blot analysis to examine the level of DnaK and GroEL in spores from cultures sporulated at 30°C in 2× SG medium and either shifted to 45°C for 30 min or not shifted. Cleaned spores (OD₆₀₀ of ~75) were lyophilized and dry ruptured for 8 min with glass beads as the abrasive, and the dry powder was suspended in 500 µl of cold 25 mM Tris-HCl (pH 7.4)-5 mM EDTA-0.1 mM phenylmethylsulfonyl fluoride. After incubation for 30 min at 4°C, the extract was centrifuged, an aliquot of the supernatant fluid was run on an SDS-polyacrylamide gel, proteins were transferred to nitrocellulose-based paper, and DnaK and GroEL were detected as described above. There was no (<15%) difference in the levels of either GroEL or DnaK in the more heat-resistant spores from the appropriately heat-shocked culture compared to the spores from the non-heat-shocked culture (data not shown). These results agree with those of a recent study using two-dimensional gel electrophoresis (21) which found a transient, but no permanent, increase in the levels of a number of heat shock proteins in cells from sporulating cultures that had been heat shocked. These latter results thus strongly suggest that increased spore levels of heat shock proteins are not responsible for the increased heat resistance of spores from appropriately heat-shocked cultures. To prove this point conclusively, we performed the same temperature shift during sporulation of sigB and dnaK strains. Again, spores from cultures of these mutant strains that were shifted to 45°C at ~2 h into sporulation exhibited increased wet heat resistance compared to that of spores from unshifted cultures (data not shown), strongly indicating that heat shock proteins are not involved in the elevated wet heat resistance of spores from cultures subjected to a heat shock. We also analyzed the core wet density (37) of spores from cultures which had or had not been subjected to a 30-min shift from 30 to 45°C at the second hour of sporulation. Since these values were both 1.355 ± 0.01 g/ml, there was no difference in the core wet density and thus the core water content of these spores, indicating that this is not the cause of the increased heat resistance of spores from appropriately heat-shocked sporulating cultures (8).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Heat resistance of spores from wild-type B. subtilis cultures shifted from 30 to 45°C for 30 min during sporulation. Sporulation of strain PS832 was in resuspension medium with the time of initiation of sporulation defined as the time of resuspension. Spores were cleaned and wet heat resistance at 90°C was measured as described in the text. Similar results were obtained in experiments performed twice, as well as during sporulation in nutrient exhaustion (2× SG) medium. The symbols used are as follows: , unshifted culture; , culture shifted at the initiation of sporulation; , culture shifted at the second hour of sporulation; , culture shifted at the fourth hour of sporulation. Error bars have been omitted for clarity, but the spores produced in the culture that was shifted at the second hour of sporulation had D90 values (see Table 3) that were from 28 to 110% (±10%) greater in multiple experiments than values for spores from unshifted cultures or from cultures shifted earlier or later in sporulation.

Expression of heat shock genes during germination of heat-treated spores. The results given above strongly suggest that heat shock proteins play no significant role in spore wet heat resistance and suggest that repair or removal of heat-damaged proteins during spore germination and outgrowth is not important to spore survival after heat treatment. Heat shock of growing cells is known to induce the synthesis of a number of heat shock proteins in a variety of species (17). If removal or renaturation of damaged proteins is not important in spore heat resistance, then wet heat treatment of dormant spores would not result in the production of heat shock proteins during subsequent spore germination. In order to test this prediction, we examined the expression of a number of heat shock genes during germination of wet heat-treated spores. The genes examined were groEL, dnaK, ctc, lonA, and clpC, and their expression was monitored by measuring -galactosidase synthesis from transcriptional fusions in which the promoter of the gene in question was fused to the promotorless bgaB gene encoding the thermostable -galactosidase from Bacillus stearothermophilus; these bgaB fusions were inserted into the amyE locus on the B. subtilis chromosome (20). If any of these gene products are important in repairing or removing a heat-damaged protein, we would expect to see an increase in -galactosidase synthesis from the bgaB fusion during germination of heat-treated spores compared to synthesis in germinating unheated spores (31). Cleaned spores carrying the various bgaB fusions were prepared at 37°C in 2× SG medium (23) and treated with wet heat to give ~50% killing. Both heated and unheated spores (OD₆₀₀ of 20) were germinated at 37°C in 25 ml of Spizizen's minimal medium (35) plus 0.1% Casamino Acids and also containing 5 µCi of [³H]leucine in order to measure total protein synthesis (31). Samples were taken throughout germination and outgrowth (~3 h), -galactosidase activity and total protein synthesis were determined, and the -galactosidase specific activity was calculated relative to total protein synthesized (31), since there is essentially no -galactosidase from any of these bgaB fusions in spores (data not shown). The -galactosidase specific activity would be higher in the culture from heated spores if expression of the heat shock gene in question had been induced during germination and outgrowth by prior spore heat treatment (31). However, upon analysis of the expression of all five heat shock genes, we found less than a 25% difference in the -galactosidase specific activities in germinating-outgrowing cultures from heated versus unheated spores (data not shown). In contrast, in vegetatively growing cells, heat shock results in a 4- to 25-fold induction of expression of these same genes (11, 24, 38). From these data, we conclude that the heat shock genes that we tested are not induced by prior heat treatment of spores.

Conclusions. The findings in this work allow three major conclusions. First, as reported by two other groups (13, 21, 28), a heat shock at an early time in sporulation results in an increase in wet heat resistance of the resultant spores. This effect does not appear to involve the heat shock response, as there was no elevation in the level of heat shock proteins in the spores with elevated wet heat resistance, as also found in a recent study (21), and we also found that mutations in several heat shock genes did not abolish this phenomenon. While the specific reason for the effect of a heat shock at the second hour of sporulation is not clear, it may be simply the result of a minor, albeit global, alteration in transcription at a key time in sporulation which results in production of spores with slightly altered properties, including slightly increased wet heat resistance. Indeed, recent work has indicated that global alterations in transcription during sporulation can significantly alter spore properties (29).