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J Bacteriol, July 1998, p. 3730-3733, Vol. 180, No. 14
Department of Food Science and Technology,
University of California, Davis, California 95616
Received 25 November 1997/Accepted 12 May 1998
The general stress response of the bacterium Bacillus
subtilis is controlled by the The general stress response of the
bacterium Bacillus subtilis is controlled by
Based on the stresses which lead to From the results of the csb gene studies, we hypothesized
that 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.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
General Stress Transcription Factor
B and
Sporulation Transcription Factor H Each Contribute to
Survival of Bacillus subtilis under Extreme Growth
Conditions
ABSTRACT
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Abstract
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References
B transcription
factor. Here we show that loss of 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 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.
TEXT
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Abstract
Text
References
B, an alternative factor that binds RNA polymerase
to direct the transcription of over 50 genes (7, 11, 18,
36). The stresses that activate 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 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).
B activation, genes
expressed under 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
B null mutants (8, 16, 21, 23). Consequently,
two complementary approaches have been used to determine the
physiological role of the B regulon. In the first,
two-dimensional gel analysis identified proteins whose synthesis was
B dependent (7, 36). A significant discovery
of this line of investigation was that 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
B, or csb genes, with the view that their
predicted products would provide important clues to 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 B and H, suggesting that in addition
to controlling sporulation initiation, H might also
regulate a subclass of genes involved in a general stress response
(34).
B might contribute to cell viability under those
growth conditions which induce B activity while
simultaneously taxing cell envelope function. In support of this
notion, we report here that loss of 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 H also
affects the viability of stationary-phase cells under these same growth
conditions.
TABLE 1.
B. subtilis strains used in this study
Loss of 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).
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 B function has a modest but significant effect
on viability during prolonged incubation at alkaline pH.
|
H is also involved in a
stress response distinct from sporulation (34), we found
that loss of 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. F is a forespore-specific
transcription factor which acts immediately downstream from
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 H function and not due
to a general loss of sporulation ability.
One function of 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
H-dependent stationary-phase promoter, spo0A
also has a 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, 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 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, B and 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.
|
Loss of B function results in decreased cell yield
in ethanol-containing media.
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, B and
H contribute independently to the ability of B. subtilis to grow in ethanol-containing LB medium.
|
Conclusions.
Because a number of csb gene products
appear to be associated with the cell envelope, we hypothesized that
loss of B function would become evident under
environmental conditions that challenge envelope function. The results
presented here indicate that 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).
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
A-like promoter and a 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 A and B promoters, both
of which are osmoregulated (37). These other studies,
together with our results, implicate 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, H also
contributes to stress resistance in nonsporulating cells. From the
cumulative effects of H and 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 H and B
regulons could be under dual H and 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, H and 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 H and B would then
reflect the relative importance of these independent gene sets to
survival of a given stress.
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ACKNOWLEDGMENTS |
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We thank R. Losick for providing strain SM69-1 bearing the sigF null allele and A. L. Sonenshein for helpful comments on the manuscript.
This research was supported by Public Health Service grant GM42077 from the National Institute of General Medical Sciences.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Food Science and Technology, University of California, Davis, CA 95616. Phone: (530) 752-1596. Fax: (530) 752-4759. E-mail: cwprice{at}ucdavis.edu.
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), in sigH (PB384;
), or in both sigB and sigH (PB386; 
) were
grown to stationary phase in LB medium lacking salt (
) and
spoIIA (sigF) null mutant PB513 (
), which
lacks 
) to that of wild-type strain PB2 (
