General Stress Transcription Factor ς^B and Sporulation Transcription Factor ς^H Each Contribute to Survival of Bacillus subtilis under Extreme Growth Conditions

Abstract

The general stress response of the bacterium Bacillus subtilis is controlled by the ς^B transcription factor. Here we show that loss of ς^B reduces stationary-phase viability 10-fold in either alkaline or acidic media and reduces cell yield in media containing ethanol. We further show that loss of the developmental transcription factor ς^H also has a marked effect on stationary-phase viability under these conditions and that this effect is independent from the simple loss of sporulation ability.

The general stress response of the bacterium Bacillus subtilis is controlled by ς^B, an alternative ς factor that binds RNA polymerase to direct the transcription of over 50 genes (7, 11, 18, 36). The stresses that activate ς^B include heat, osmotic, and ethanol shocks to exponentially growing cells, as well as the energy limitations imposed on starving cells (2, 6, 11, 13, 35, 36). These stress signals are conveyed to ς^B by a signal transduction network that functions by the newly discovered “partner switching” mechanism, in which key protein interactions are controlled by reversible phosphorylation events (2, 3, 39).

Based on the stresses which lead to ς^B activation, genes expressed under ς^B control are thought to enhance survival when cells persist in a nongrowing state (11, 19, 36). However, early studies found no clear phenotype for ς^B null mutants (8, 16, 21, 23). Consequently, two complementary approaches have been used to determine the physiological role of the ς^B regulon. In the first, two-dimensional gel analysis identified proteins whose synthesis was ς^B dependent (7, 36). A significant discovery of this line of investigation was that ς^B is required for oxidative stress resistance in starved cells (4, 5, 17, 32). In the second approach, a genetic screen identified genes controlled by ς^B, or csb genes, with the view that their predicted products would provide important clues to ς^B function (12, 14). Of the six csb transcription units identified, four encode products that are either known or suspected to interact with the cell envelope (1, 14, 27, 33). Furthermore, one of the six is under the dual control of both ς^B and ς^H, suggesting that in addition to controlling sporulation initiation, ς^H might also regulate a subclass of genes involved in a general stress response (34).

From the results of the csb gene studies, we hypothesized that ς^B might contribute to cell viability under those growth conditions which induce ς^B activity while simultaneously taxing cell envelope function. In support of this notion, we report here that loss of ς^B reduces the viability of stationary-phase cells grown in alkaline or acidic media and also decreases cell yield in Luria broth (LB) containing high concentrations of ethanol. Significantly, loss of ς^H also affects the viability of stationary-phase cells under these same growth conditions.

Strains and growth conditions.

We assayed the growth rate and viability of B. subtilis strains carrying a null mutation(s) in sigB, sigH, or both (Table 1). These strains were inoculated into LB, grown at 37°C to early logarithmic stage, and then diluted 1:100 into one of the three stress media described in the figure legends: alkali, acid, or ethanol stress medium. Cells were grown with vigorous shaking in flasks containing 1/10 volume of medium.

TABLE 1.

B. subtilis strains used in this study

Strain	Genotype	Reference or construction
PB2	trpC2	Wild-type Marburg strain
PB61	spo0A12 trpC2	10
PB153	sigBΔ2::cat trpC2	14
PB384	sigHΔ::cat trpC2	34
PB386	sigBΔ3::spc sigHΔ::cat trpC2	34
PB513	spoIIA::spc trpC2	SM69-1→PB2a
SM69-1	spoIIA::spc	R. Losick (unpublished data)

^aThe arrow indicates transformation from donor to recipient. 

Loss of ς^B function results in diminished survival at alkaline pH.

Maintenance of proton motive force at alkaline pH is thought to be a major energy drain which becomes especially severe during prolonged starvation (38). Bacteria are believed to respond to this stress by partially shifting energy-consuming processes from H⁺- to Na⁺-based energetics or by adopting membrane modifications that may provide an alternative means of maintaining proton motive force (9, 26, 28).

To probe the possible role of the ς^B regulon under these conditions, we tested the growth and survival properties of wild-type and sigB null strains at alkaline pH. In LB medium buffered to pH 9, there was no difference in logarithmic growth rate between the wild type and the sigB mutant. Both strains had a generation time of 50 min in this medium, compared to 20 min in LB medium buffered to pH 7. However, after 5 days in stationary phase at pH 9, we saw a reproducible 10-fold loss of viability in the sigB null mutant (Fig. 1A). This difference was statistically significant by Student’s two-tailed t test, assuming unequal variance (P < 0.01). In contrast, there was no decrease in viability when the sigB null mutant was maintained in medium buffered to pH 7 (Fig. 1B). We conclude that loss of ς^B function has a modest but significant effect on viability during prolonged incubation at alkaline pH.

FIG. 1.

Survival of B. subtilis strains under alkaline conditions. The data points shown in all three panels are averages of four independent experiments; differences noted are significant according to a one-factor analysis of variance (P < 0.001). The significance of key pairwise comparisons determined by Student’s t test (unequal variance) is discussed in the text. (A) Strains bearing a null mutation(s) in sigB (PB153; ▵), in sigH (PB384; ◊), or in both sigB and sigH (PB386; ○) were grown to stationary phase in LB medium lacking salt (11) buffered to pH 9.0 with 100 mM 3-(1,1-dimethyl-2-hydroxyethyl amino)-2-hydroxypropanesulfonic acid. The measured pH of the medium was 8.5 at the end of 5 days of incubation. Cell growth was monitored with a Klett-Summerson colorimeter (red filter), and cell viability was determined by serial dilution and plating on tryptose blood agar (Difco Laboratories). Controls included wild-type strain PB2 (▪) and spoIIA (sigF) null mutant PB513 (□), which lacks ς^F activity. (B) The same experiment conducted with phosphate-buffered LB medium, pH 7.0. The symbols are the same as in panel A. (C) Comparison of the alkaline (pH 9.0) viability of strains bearing null mutations in sigH (PB384; ◊) and spo0A (PB61; •) to that of wild-type strain PB2 (▪).

Consistent with the notion that ς^H is also involved in a stress response distinct from sporulation (34), we found that loss of ς^H function was extremely deleterious to survival under prolonged alkaline incubation, with a loss of viability exceeding 10⁶ after 5 days at pH 9 (Fig. 1A). In contrast, no such decrease was observed following prolonged incubation at neutral pH (Fig. 1B). To address the possibility that this loss of viability was primarily a consequence of the impaired sporulation ability of the sigH null mutant, we also tested the viability of a sigF null mutant. ς^F is a forespore-specific transcription factor which acts immediately downstream from ς^H in the developmental cascade (29). Notably, a sigF null mutant was indistinguishable from the wild type during prolonged alkaline incubation (Fig. 1A). We concluded that the diminished viability of the sigH null mutant was specifically due to the loss of ς^H function and not due to a general loss of sporulation ability.

One function of ς^H is to direct the stationary-phase transcription of spo0A, which encodes a response regulator with multiple roles in growing and nongrowing cells (15, 20, 31). Because loss of spo0A function is known to pleiotropically affect stationary-phase viability, we tested whether the decreased alkaline viability observed in a sigH null mutant could be entirely attributed to decreased spo0A function. We first determined that survival of a spo0A null mutant was indistinguishable from that of wild-type cells at pH 7 (data not shown). In contrast, the spo0A null mutation dramatically affected viability under prolonged incubation at pH 9 and the sigH null mutation reproducibly caused a further 15-fold loss of viability (Fig. 1C). This additional 15-fold loss was statistically significant (P < 0.05). Therefore, most, but not all, of the viability loss in the sigH null mutant was due to the loss of spo0A function. Moreover, the comparison shown in Fig. 1C likely overestimated the extent to which loss of spo0A function contributes to loss of alkaline viability in a sigH null mutant. In addition to its ς^H-dependent stationary-phase promoter, spo0A also has a ς^A-like vegetative promoter to provide maintenance levels of Spo0A (15, 20). Therefore, in comparison to the spo0A null mutant, the sigH null mutant would be expected to retain at least some spo0A function. We conclude that, in addition to its role in spo0A transcription, ς^H must control at least one additional gene that significantly contributes to survival under alkaline conditions. And, because the effects of the sigB and sigH null mutations were roughly additive (Fig. 1A), we infer that these two transcription factors control largely independent stress response systems.

Loss of ς^B function results in diminished survival at acidic pH.

We next assayed the growth and survival of wild-type and mutant strains during acid stress. In LB medium buffered to pH 5, there was no difference in logarithmic growth rate between the wild type and the sigB mutant. Both strains had a generation time of 60 to 65 min in this medium, compared to 20 min in LB buffered to pH 7. However, during prolonged incubation at pH 5, we saw a reproducible 10-fold loss of viability in the sigB and sigH null single mutants and a 100-fold loss in the sigB-sigH double mutant (Fig. 2). These differences were statistically significant (P < 0.03). Because the effects of the sigB and sigH mutations were cumulative, ς^B and ς^H contribute independently to stationary-phase survival at acidic pH. And, because a sigF null mutant (Fig. 2) and a spo0A null mutant (data not shown) were similar to the wild type in viability, the diminished acid survival of the sigH mutant was not simply due to an inability to sporulate or an inability to transcribe spo0A during stationary phase.

FIG. 2.

Survival of B. subtilis strains under acidic conditions. The data points shown are averages of four independent experiments; differences noted are significant according to a one-factor analysis of variance (P < 0.001). Strains bearing a null mutation(s) in sigB (PB153; ▵), in sigH (PB384; ◊), or in both sigB and sigH (PB386; ○) were grown to stationary phase in LB medium buffered to pH 5.0 with 200 mM 2-(N-morpholino)ethanesulfonic acid. The measured pH of the medium was 5.5 at the end of 5 days of incubation. Cell growth and viability were monitored as described in the Fig. 1 legend. Controls included wild-type strain PB2 (▪) and spoIIA (sigF) null mutant PB513 (□).

Loss of ς^B function results in decreased cell yield in ethanol-containing media.

ς^B activity is strongly induced by ethanol shock (11, 36), and ethanol is known to have multiple effects on membrane function (reviewed in reference 22). Consequently, we tested the influence of various concentrations of ethanol on the growth of sigB and sigH null mutants. Our initial experiments found that growth of B. subtilis cells in LB medium led to rapid alkalification whether or not ethanol was present (data not shown). To eliminate the influence of alkalification, we therefore performed experiments with phosphate-buffered LB medium. At ethanol concentrations of up to 6% (wt/vol), there was no difference in growth rate between the wild-type and mutant strains; in greater-than-9% ethanol, neither the wild type nor the mutants could grow (data not shown), but with ethanol concentrations of 7, 8, and 9%, there was a clear difference in cell yield between the wild-type and mutant strains. Figure 3 shows the results of a typical growth experiment with 8% ethanol, in which sigB and sigH single mutants showed a fourfold-lower cell yield than either the wild-type or sigF control strain and the sigB-sigH double mutant manifested a 10-fold-lower cell yield. Because the effects of the sigB and sigH null mutations were approximately additive, ς^B and ς^H contribute independently to the ability of B. subtilis to grow in ethanol-containing LB medium.

FIG. 3.

Growth and yield of B. subtilis strains in LB medium containing 8% ethanol. The A₄₂₀ of strains bearing a null mutation(s) in sigB (PB153; ▵), in sigH (PB384; ◊), or in both sigB and sigH (PB386; ○) was monitored by placing samples into 0.9% (vol/vol) formaldehyde and measuring A₄₂₀ in a Varian DMS100 spectrophotometer, essentially as described by Neidhardt et al. (30). Over the range shown, A₄₂₀ was found to be directly correlated with viable cell counts, with 1 U of A₄₂₀ equal to 10⁸ CFU/ml. Growth medium was phosphate-buffered LB lacking salt (11) and supplemented with 8% (wt/vol) ethanol. Controls included wild-type strain PB2 (▪) and spoIIA (sigF) null mutant PB513 (□).

Conclusions.

Because a number of csb gene products appear to be associated with the cell envelope, we hypothesized that loss of ς^B function would become evident under environmental conditions that challenge envelope function. The results presented here indicate that ς^B significantly contributes to the survival of B. subtilis cells during prolonged starvation at extremes of pH and also contributes to the ability to grow in medium containing high ethanol concentrations. Although the molecular basis for these observations is not known, our findings do extend the phenotype of a sigB null mutant beyond the previously described sensitivity to oxidative stress (4, 5, 17, 32).

Other studies have suggested additional roles for the ς^B regulon in preserving cell viability but have not associated these roles with a clear phenotype for a sigB null mutant. In one study, the ClpC operon was shown to be under the dual control of a ς^A-like promoter and a ς^B-dependent promoter (24). In addition to the ClpC protein, which is a pleiotropic regulator of stress responses, the ClpC operon also encodes products involved in DNA repair (25). In another study, expression of the OpuE transporter, which is required for proline-dependent osmoprotection, was also found to be under the control of dual ς^A and ς^B promoters, both of which are osmoregulated (37). These other studies, together with our results, implicate ς^B in the control of genes which are important for resistance to oxidative stress, for repair of DNA damage, for osmoprotection, for growth in ethanol, and for viability at extreme pH.

One unexpected finding is that in addition to its well-established role in controlling the sporulation process, ς^H also contributes to stress resistance in nonsporulating cells. From the cumulative effects of ς^H and ς^B under the three stress conditions tested, it is clear that these transcription factors independently contribute to stress response. Two different scenarios are consistent with this result. First, all alkali, acid, and ethanol stress genes in the ς^H and ς^B regulons could be under dual ς^H and ς^B control. In this view, the cumulative effect of these two transcription factors would primarily reflect their relative contribution to the expression of the same subset of stress genes. Alternatively, ς^H and ς^B could regulate largely independent sets of stress genes, only some of which are under the control of both transcription factors. In this latter case, the cumulative effect of ς^H and ς^B would then reflect the relative importance of these independent gene sets to survival of a given stress.