Journal of Bacteriology, October 2002, p . 5275-5281, Vol . 184, No . 19

Cold Shock Response in Sporulating Bacillus subtilis and Its Effect on Spore Heat Resistance

Sara Movahedi and William Waites^*

Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, United Kingdom

Received 25 February 2002/ Accepted 26 June 2002

Bacteria respond to an abrupt decrease in the temperature with the induction of a cold shock response characterized by a coordinate expression of specific proteins classified as cold shock proteins (5, 24) . Cold shock proteins have been found in a broad variety of bacteria (6) and show a high level of sequence identity across species, suggesting a conserved function in bacteria . These proteins protect the cell structures, such as DNA, the membrane, or ribosomes, against dysfunction induced by the low temperature and maintain the efficiency of the metabolism at an optimum level (9) .

The cold shock response in B . subtilis has been investigated at various temperatures at the protein level using two-dimensional (2-D) gel electrophoresis (3, 13, 23) . The sudden exposure of B . subtilis to low temperatures resulted in the induction of as many as 53 total proteins when this organism was shifted to several lower temperatures, with 24 of the proteins being common to all temperatures while others were specific to a particular shock temperature . It has been shown that in B . subtilis, cold shock proteins are essential for protein synthesis at low as well as optimal temperatures and also during the stationary phase (3) .

Previous investigations into the cold shock response of B . subtilis have concentrated on the response of vegetative cells . This is the first study in which the response of sporulating cells to cold shock has been investigated and correlated to the heat resistance of the spores formed subsequently . Since spores that are deficient in SASP have been shown to be more sensitive to a variety of adverse stimuli and have decreased longevity compared to wild-type spores (7), we compared the response of a wild-type and a SASP-deficient strain of B . subtilis .

In this report, we show that cold shock pretreatment of sporulating B . subtilis cells increased the heat resistance of the spores formed from these cells to heat kill at 85 and 90°C, whereas the same pretreatment resulted in spores that were less heat resistant than controls to heat kill at 95 and 100°C . The protein profile of the cold-shocked sporulating cells was studied using 2-D gel electrophoresis, and the level of cold shock proteins induced was related to the subsequent heat resistance of the spores . In addition the response of sporulating B . subtilis cells to ethanol and puromycin stress was investigated . Ethanol and puromycin have been shown to mimic the heat shock response (17, 20) and are a useful tool to investigate the mechanism of induction of heat resistance and heat stress protein induction . Sporulating B . subtilis cells exposed to ethanol or puromycin produced spores which were more heat resistant than controls, and both stresses induced the expression of heat specific shock proteins . However, the rate of induction of these proteins was much lower than that in response to heat stress, and puromycin did not induce class II heat stress proteins .

All strains were grown at 30°C in 2x SG medium (12) supplemented with chloramphenicol (3 µg/ml) for PS 346 and PS 361 and supplemented with the appropriate antibiotic as described by Graumann et al . (3) for the cold shock mutants .

Growth was monitored by determination of the optical density at 600 nm . Sporulation was induced by nutrient exhaustion in 2x SG sporulation medium . Spores were harvested at 24 to 48 h of growth and purified by repeated centrifugation and washing with water purified by reverse osmosis (14) . The spores in all preparations used were refractile and free (>95%) of sporulating cells, cell debris, and germinated spores, as determined with a phase-contrast microscope, and were stored at 4°C in water purified by reverse osmosis .

Stress induction. For protein analysis, samples were taken during sporulation at various times after imposition of stress . The remaining part of the culture was incubated at 30°C until sporulation was completed and spores were released . Different stress conditions were imposed according to the following procedures: heat shock, transfer of the culture from 30°C to 48°C for 30 min; cold shock, transfer of the culture from 30 to 10°C for 30 min; ethanol shock, transfer of the culture to 2x SG medium containing 4% (vol/vol) ethanol for 60 min; puromycin shock, transfer of the culture to 2x SG medium containing puromycin (20 µg/ml) for 60 min; glucose starvation, transfer of the culture to 2x SG medium containing 0.05% (wt/vol) glucose for 60 min .

Assessment of spore heat resistance. Spore wet heat resistance was assessed by incubating purified spores in water in thin-wall capillary tubes (catalog no . C-6148; Sigma) at temperatures of 85 to 100°C and plating serial dilutions of unheated and heated spores on 2x SG plates (1.5% [wt/vol] agar) at 30°C for 24 h . Spores were heated at 60°C for 10 min before inoculation on 2x SG plates to kill any remaining vegetative cells . Spore heat resistance was expressed as D values . The D value is the time needed at temperature t for a 10-fold reduction in spore viability . D values were calculated as the negative reciprocal of the slope of the regression line plotted with the values of the straight portion of thermal death curves (log survivors versus heating time) . At least 12 different time points were used for each determination, and each heat resistance determination was carried out in quadruplicate with three repeats per experiment .

Preparation of protein samples. Samples were taken at intervals after heat shock, and crude cell extracts were prepared as previously described (18) . Cells were harvested by centrifugation (4°C, 18 000 x g, 10 min), washed three times in 0.1 M Tris-HCl-1 mM EDTA (pH 7.5), and the pellet was resuspended in 1 ml of disruption buffer (10 mM Tris-HCl [pH 7.5], 5 mM MgCl₂, 2 mM phenylmethylsulfonyl fluoride) . Cells were disrupted using a mini-bead beater and 0.1-mm-diameter zirconia beads (both from Biospec Products) for up to 10 1-min disruptions on ice, and the cell debris was removed by centrifugation (4°C, 42 000 x g, 10 min) .

2-D gel electrophoresis. The proteins present in the sporulating B . subtilis cells were resolved on 2-D gels using the products and protocols of Amersham Pharmacia Biotech . (Uppsala, Sweden) . Proteins were resolved by isoelectric focusing on a precast Immobiline DryStrip with a linear pH gradient (pH 3 to 10 or pH 4 to 7) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% acrylamide gels for the second dimension . For analytical gels 100 µg of crude cell extract was applied to gels and proteins were stained with a Pharmacia Biotech silver stain kit . For preparative 2-D protein gel electrophoresis, 500 µg of the crude protein extract was separated and proteins were visualized using Coomassie blue R-350 (Phast Gel BlueR; Amersham Pharmacia Biotech) .

Computer-aided analysis of 2-D gels. Images of the gels were captured using a Sharp JX-330 flat-bed scanner, and image analysis of the protein profiles was performed using Amersham Pharmacia Biotech ImageMaster 2-D Elite software . The relative amount of each protein spot was calculated and expressed by the software as the percentage spot volume and represented the intensity of each individual spot compared to the intensity of the whole gel .

Western blotting and N-terminal amino acid sequencing. For the analysis of the N-terminal protein sequences, Coomassie-stained protein spots were excised from the preparative 2-D gels, pooled and concentrated according to the protocol of Rider et al . (21), and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore) by semidry, discontinuous horizontal electroblotting for 1 h at 0.8 mA/cm² . The proteins were stained with Coomassie blue R-350 and sequenced on an Applied Biosystems A473a protein sequencer .

 
Following cold shock, protein extracts of sporulating cells of mutant (SASP^-) and wild-type (SASP⁺) strains were prepared and studied using 2-D gel electrophoresis . A total of 13 proteins were either overexpressed or induced as a result of cold shock (Fig . 1; Table 2) . Identities of eight of these proteins were confirmed: CspB, CspC, CspD, ribosomal protein S6, peptidylprolyl isomerase (PPiB), FbaA, elongation factor Ts (EF-Ts), and IlvC . These proteins have previously been shown to be cold shock but not heat shock proteins in vegetative cells of B . subtilis (4, 5) .

 
The same proteins were expressed in both wild-type (SASP⁺) and mutant (SASP^-) strains, but the level of expression of the stress proteins in the mutant strain was less marked than that in the wild-type strain (data not shown) . In both strains, cold shock proteins were detected within 15 min of cold shock and the biochemical response was maintained for at least 60 min before declining to preshock levels 2 h after cold shock . The level of cold shock proteins in spores was also the same as in those observed in unshocked sporulating cells (results not shown) . The maximal increase in induction of cold shock proteins was apparent 30 to 60 min after cold shock, as was previously found for heat shock proteins (18) . In order to confirm the function of these proteins as cold-specific stress proteins, the level of expression of these proteins in cold-stressed cells was compared to that of sporulating cells exposed to heat shock or glucose starvation . The 13 proteins listed in Table 2 are the only ones induced or overexpressed specifically in response to cold shock and not heat shock or glucose starvation (data not shown) . Only two proteins (cold shock proteins 8 and 9) are induced specifically in response to cold shock; the rest are present in control cells, but their level increases dramatically after cold shock (Table 2) . The majority of the proteins are low-molecular-mass proteins (7.2 to 38 kDa) and have acidic pIs (pH 4.2 to 5.8) compared with the heat-specific stress proteins isolated previously (18), which are spread over a wider range of molecular masses and pIs (11 to 87 kDa and pH 4.5 to 6.8) . The low-molecular-mass proteins may well be spore coat proteins, but we have not verified this .

In order to investigate whether cold shock proteins induced as a result of cold stress play any role in the heat resistance of B . subtilis spores, the effect of mutations in known cold shock genes on spore heat resistance was examined . As D values in Table 3 show, there is no significant difference between the heat resistance of the wild-type strain and the mutants in the major cold shock proteins CspB, CspC, and CspD or the double mutant in CspC and CspD . Strain 64BC is unable to sporulate, whereas 64BD produces a reduced number of CFU under sporulation conditions (Weber and Marahiel, personal communication) . However, under our laboratory conditions both the double mutant strains 64BD and 64BC were unable to sporulate . All the strains displayed increased heat resistance of spores after cold shock pretreatment at 85 and 90°C and reduced heat resistance at 95 and 100°C, as found previously with the SASP⁺ and SASP^- strains .

 
Determination of heat resistance response of B . subtilis to ethanol and puromycin stress. The effect of puromycin and ethanol stress on subsequent heat resistance of spores of B . subtilis was investigated . One hour after the start of stationary phase, sporulating cultures of wild-type (SASP⁺) and mutant (SASP^-) strains of B . subtilis were exposed to ethanol or puromycin stress as described in Materials and Methods, and the resistance of the spores formed was compared to those of untreated spores . D values at 85, 90, 95, and 100°C were obtained for both wild-type and mutant cells (Table 4) .

 
Both ethanol and puromycin stress induced heat resistance in spores of both strains, as evidenced by the increase in D values in response to heat kill . Exposure of sporulating cells of B . subtilis to 4% ethanol for 60 min, 60 min into sporulation, increased resistance to heat at 100°C by 1.8-fold for the SASP^- mutant and by 1.6-fold for the wild-type spores . Puromycin treatment increased resistance to heat at 100°C by 1.5-fold for the SASP^- mutant and by 1.3-fold for the wild-type spores . This is in comparison with three-and twofold increases in D, respectively, when the cells were subjected to heat pretreatment .

No specific stress proteins were detected after ethanol or puromycin pretreatment of B . subtilis cells in either SASP⁺ or SASP^- cells . Instead the pattern of protein induction was very similar to that obtained after heat shock . The 11 heat-specific stress proteins described previously (18) were all induced in sporulating B . subtilis cells after ethanol or puromycin shock, but the pattern of induction was different compared to that obtained after heat stress . The induction of the major heat specific stress protein GroEL is shown in Figure 2 . Similar results were obtained for other heat-specific stress proteins . After heat shock the level of stress proteins increased to reach a maximum at 30 to 60 min after stress and thereafter decreased, whereas there was a steady increase in the level of heat shock proteins after ethanol or puromycin shock, which peaked at 120 min after stress and diminished to prestress levels 180 min after application of stress . Unlike ethanol and heat, puromycin did not induce class II stress response proteins such as Ctc or RsbW (results not shown) .

 
This is the first study in which spore heat resistance after cold shock treatment of sporulating B . subtilis cells has been investigated . In a previous study we showed that heat pretreatment of sporulating B . subtilis cells is followed by increased heat resistance in the spores formed subsequently (18) . Similar results were obtained here after treatment with ethanol or puromycin . With cold shock pretreatment, there is a similar increase in the heat resistance of spores at 85 and 90°C, but interestingly, cold shock pretreatment decreases heat resistance at higher temperatures compared to untreated controls for all the strains investigated . The reason for this difference in response is as yet unknown .

The protein profile of cold-shocked sporulating B . subtilis cells was investigated using 2-D gel electrophoresis . Compared with unstressed sporulating cells, a total of 13 proteins were either over expressed or induced . This is in contrast to previous investigations in which as many as 53 proteins were induced in vegetative B . subtilis cells in response to cold shock (5) . Eight of these proteins were confirmed to be CspB, CspC, CspD, S6, PPiB, FbaA, EF-Ts, and IIvC, all of which have also been identified as cold shock proteins in vegetative cells of B . subtilis . (4, 5) . The three most prominent cold shock proteins in sporulating cells were found to be CspB, CspC, and CspD as found previously for vegetative cells of B . subtilis (2, 3) .

Similar to our findings, Graumann et al . (2) found that in vegetative cells, the induction of cold-induced proteins decreased 1 h after cold shock and after 2 h general protein synthesis resumed . The physiology of bacteria is severely affected by cold shock (10) . In B . subtilis transferred from 40 to 20°C, an 80-min growth lag is induced as a consequence of inhibition of RNA and DNA and of the protein synthesis (11) . These processes are gradually recovered, with the spores attaining a new metabolic state equilibrium which corresponds to the low temperature .

In order to investigate whether the CspB, CspC, and CspD proteins play any role in the heat resistance of B . subtilis spores, the effect of cold shock pretreatment of sporulating cells of deletion mutants in one or more of these proteins on subsequent heat resistance of spores formed was investigated . None of the deletion mutants showed a decreased heat resistance compared to controls . It is apparent that none of these proteins play a role in the increased heat resistance of cold-shocked B . subtilis spores . Further investigations are in progress to determine whether deletion in the genes for these Csp proteins results in compensation and overexpression of other Csp proteins . Graumann et al . (3) showed that deletion of CspB, CspC, and CspD can result in complementation in vivo . They found that deletion of one or two of the major cold shock proteins led to an increase in the synthesis of the major cold shock proteins both at 37°C and after cold shock, suggesting that cold shock proteins down-regulated the production of members of this protein family . It may be that in our study also the expression of some proteins was upregulated to compensate for the deletion of the major Csp proteins . Hence, we cannot entirely rule out the involvement of cold shock proteins in increased heat resistance .

We have shown that ethanol and puromycin treatment of sporulating B . subtilis cells increases heat resistance in spores formed subsequently . The same stress proteins induced after heat shock were also induced after ethanol and puromycin stress, although no class II general stress proteins were induced after puromycin stress . We have previously shown that a sigB mutant also showed increased heat resistance in response to heat pretreatment, albeit at a lower level than wild-type cells (18) . The observation that puromycin pretreatment of sporulating cells induced a smaller increase in heat resistance in spores formed subsequently may be related to the fact that no class II ^B-dependent general stress proteins were induced .

It has been argued that, since puromycin does not induce class II genes, the signals for the induction of class I and class II genes are different (17) . However, it has recently been proposed (16) that this is because puromycin inhibits the de novo synthesis of ^B, which in turn reduces the induction of ^B-dependent genes .

The loss of stress proteins later in sporulation found here and previously (18) showed that they are unlikely to repair heat damage during germination and outgrowth . This view has been confirmed by Melly and Setlow (15) . It would seem, therefore, that stress proteins act to alter spore structure . The different effects of heat and ethanol stress versus cold stress suggest that these changes are specific, since heat and ethanol produce similar stress proteins and increase heat resistance while cold shock produces different stress proteins and reduces spore resistance to higher temperatures .

Practically, these findings could have important implications for the food industry, particularly in the area of cooked chilled foods (1), which are generally processed with mild heat treatments and rely on refrigeration for preservation . Spores are abundant in the environment and can withstand both mild heat treatments and refrigeration temperatures, and heat shock has now been shown to increase the heat resistance of spores of Bacillus megaterium (22), Clostridium perfringens (8), and Clostridium botulinum (Stringer and Peck, personal communication) as well as B . subtilis .

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