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Journal of Bacteriology, October 2001, p. 5617-5631, Vol. 183, No. 19
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.19.5617-5631.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Global Analysis of the General Stress Response of
Bacillus subtilis
Anja
Petersohn,1,2
Matthias
Brigulla,3
Stefan
Haas,4
Jörg D.
Hoheisel,2
Uwe
Völker,3 and
Michael
Hecker1,*
Institut für Mikrobiologie,
Ernst-Moritz-Arndt-Universität, 17487 Greifswald,1 Laboratorium für
Mikrobiologie, Philipps-Universität, and Max-Planck-Institut
für Terrestrische Mikrobiologie, 35043 Marburg,3 and Abteilung Theoretische
Bioinformatik4 and Abteilung
Funktionelle Genomanalyse,2 Deutsches
Krebsforschungszentrum Heidelberg, 69120 Heidelberg, Germany
Received 16 March 2001/Accepted 9 July 2001
 |
ABSTRACT |
Gene arrays containing all currently known open reading frames of
Bacillus subtilis were used to examine the general
stress response of Bacillus. By proteomics,
transcriptional analysis, transposon mutagenesis, and consensus
promoter-based screening, 75 genes had previously been described as
B-dependent general stress genes. The present gene
array-based analysis confirmed 62 of these already known general stress
genes and detected 63 additional genes subject to control by the stress sigma factor
B. At least 24 of these 125
B-dependent genes seemed to be subject to a second,
B-independent stress induction mechanism. Therefore,
this transcriptional profiling revealed almost four times as many
regulon members as the proteomic approach, but failure of confirmation
of all known members of the
B regulon indicates that
even this approach has not yet elucidated the entire regulon. Most of
the
B-dependent general stress proteins are probably
located in the cytoplasm, but 25 contain at least one membrane-spanning
domain, and at least 6 proteins appear to be secreted. The functions of most of the newly described genes are still unknown. However, their
classification as
B-dependent stress genes argues that
their products most likely perform functions in stress management and
help to provide the nongrowing cell with multiple stress resistance. A
comprehensive screening program analyzing the multiple stress
resistance of mutants with mutations in single stress genes is in
progress. The first results of this program, showing the diminished
salt resistance of yjbC and yjbD mutants
compared to that of the wild type, are presented. Only a few new
B-dependent proteins with already known functions were
found, among them SodA, encoding a superoxide dismutase. In addition to
analysis of the
B-dependent general stress regulon, a
comprehensive list of genes induced by heat, salt, or ethanol stress in
a
B-independent manner is presented. Perhaps the most
interesting of the
B-independent stress phenomena was
the induction of the extracytoplasmic function sigma factor
W and its entire regulon by salt shock.
 |
INTRODUCTION |
Almost 15 years ago we began to
analyze the response of Bacillus subtilis cells to stress
and starvation because these unfavorable conditions are the rule in
natural ecosystems and adaptation to stress and starvation is crucial
for survival in nature. We used the highly sensitive two-dimensional
gel electrophoresis technique to visualize global changes in the gene
expression pattern (24, 25, 38). These studies revealed a
large group of stress proteins that seemed to be induced together by
physical stress such as heat, salt, ethanol, or acid stress, as well as
by glucose, oxygen, or phosphate starvation. This complex induction
profile encouraged us to suggest that these proteins may have a rather
nonspecific, but nevertheless very essential, protective function in
response to stress or starvation, regardless of the specific stress
stimulus. Therefore, the proteins were called nonspecific or general
stress proteins (24, 25, 38).
Subsequently, stress induction of this protein group was shown to be
mediated by the alternative sigma factor
B,
the general stress sigma factor of gram-positive bacteria. W. G. Haldenwang and R. Losick discovered
B more
than 20 years ago (22), but its role and physiological function remained matters of speculation for more than a decade. In the
early 1990s the laboratories of W. G. Haldenwang and C. W. Price independently discovered that the sigB operon was
induced by the same stimuli as the general stress proteins, namely,
either heat, ethanol, or salt stress or entry into the
stationary-growth phase, and that this induction was achieved by
B itself (6, 7, 9, 11). These
findings strongly suggested that the genes encoding the general stress
proteins belong to the
B regulon.
Identification of numerous general stress proteins by N-terminal
sequencing or matrix-assisted laser desorption
ionization-time-of-flight mass spectrometry and subsequent detailed
analysis of gene regulation proved that
B
indeed controls induction of the general stress genes. By use of
transposon mutagenesis C. W. Price and coworkers investigated the
effects of
B on transcription and identified
eight
B-dependent genes (for reviews see
references 26 and 37).
Finally, analysis of the B. subtilis genome for
B-dependent promoters was used to identify
additional members of the
B regulon. This
computer-aided identification of new general stress genes became
feasible because of the highly conserved and distinct consensus
sequence of
B-dependent promoters. Screening
of the potential target genes by oligonucleotide hybridization revealed
more than 20 new genes that are probably under
B control (36). The three
approaches described above and additional genetic and transcriptional
studies have led thus far to the identification of 75
B-dependent general stress genes. The number
of genes identified by each of these approaches is given in Table 3.
Many of the general stress genes display basal level transcription from
vegetative
A-dependent promoters. However,
activation of
B activity following metabolic
or environmental stress dramatically increases the transcription of the
general stress genes. As a result of this massive induction of the
regulon, the fraction of total translational capacity utilized for
production of general stress proteins rises from approximately 1% in
growing cells to 20% or even more in starved or stressed bacteria
(8). During exponential growth
B
is kept in an inactive complex by binding to its anti-sigma factor, RsbW (5). Activation of
B
requires the dephosphorylation of an antagonist protein, RsbV, which
then forms a complex with RsbW and releases
B
from its inhibition (1, 17). During exponential growth
RsbV is phosphorylated and inactivated by RsbW (17, 52),
but after the imposition of stress or starvation two specific
PP2C type phosphatases, RsbU and RsbP, can shift the equilibrium
from RsbV~P to RsbV and consequently trigger stress gene activation
(48, 55).
Comparative phenotypic studies of sigB mutants and wild-type
bacteria have meanwhile proven that high-level expression of the
general stress regulon provides stressed or starved cells with
multiple, nonspecific, prospective stress resistance in anticipation of
"future stress" (18, 19, 51). This protective function is particularly important for cells that are no longer able to grow
(51). Therefore, the general stress response might be an essential alternative for all resting Bacillus cells
that do not sporulate efficiently either because the cell density is
too low (21) or because stress conditions (e.g., osmotic
stress, oxygen limitation) do not allow sporulation (28,
39).
Analysis of the precise function of the general stress regulon in
stress management will undoubtedly profit from a comprehensive description of all
B-dependent genes.
Therefore, we decided to use DNA macroarrays for transcriptional
profiling of stress adaptation in B. subtilis to detect the
still missing members of the
B regulon. By
this approach more than 60 new
B-dependent
genes were discovered. The screening was also utilized for the
characterization of
B-independent stress gene
induction. Interestingly, these studies showed salt shock induction of
the regulon of the extracytoplasmic function (ECF) sigma factor
W.
 |
MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The following
B. subtilis strains were used: 168 (trpC2), BSA46
(trpC2 SP
ctc::lacZ), ML6
(trpC2
sigB::
HindIII-EcoRV::cat), BSA272 (trpC2
sigB::
HindIII-EcoRV::cat),
and BSA386 (trpC2 rsbX::spc sup20a
SP
ctc::lacZ; obtained from W. G. Haldenwang). The yjbC (BFA2841) and yjbD
(BFA2842) mutants were constructed by inserting the nonreplicative
plasmid pMUTIN4, carrying fragments of the yjbC and
yjbD structural genes, respectively, lacking the ribosome binding site and the first N-terminal codons, into the corresponding genes via a Campbell type single-crossover event (41).
Strains were grown with vigorous agitation at 37°C in a synthetic
medium with 0.2% (wt/vol) glucose as the carbon source (strains 168, ML6, BFA2841, and BFA2842) (44) or in Luria-Bertani medium
(strains BSA46, BSA272, and BSA386). Ethanol or osmotic stress was
imposed by adding ethanol or NaCl to exponentially growing cells to a final concentration of 4% (vol/vol or wt/vol, respectively). For heat
stress, the temperature was shifted from 37 to 48°C.
Survival of growth-preventing salt stress was examined by transferring
exponentially growing cultures into minimal medium containing an
initial NaCl concentration of 4% (wt/vol). After a preadaptation
period of 30 min, this was raised to a final NaCl concentration of 10%
(wt/vol).
Cell lysis and RNA isolation.
RNA was isolated either
according to the acid phenol method of Majumdar et al.
(34), with the modifications previously described (49), or after mechanical disruption of the cells as
described by Hauser et al. (23). In the latter case
sedimented cells were resuspended in 200 µl of growth medium and
immediately frozen in a small Teflon vessel of a grinding mill (B. Braun Biotec Int., Melsungen, Germany) in liquid
nitrogen. After addition of a tungsten carbide bead, the frozen drops
were mechanically broken for 2 min at top speed. The frozen powder was
instantly taken up in guanidine thiocyanate buffer (4 M guanidine
thiocyanate, 25 mM sodium acetate [pH 5.2], 0.5% [wt/vol]
N-lauroylsarcosinate) and was extracted three times with 1 volume of acid phenol-chloroform-isoamyl alcohol solution (25:24:1,
vol/vol/vol), and twice with chloroform-isoamyl alcohol (24:1,
vol/vol). After ethanol precipitation and washing with 70% ethanol,
the RNA pellet was dried and dissolved in diethyl pyrocarbonate-treated
distilled water.
Preparation of labeled cDNA, array hybridization, and DNA
macroarray regeneration.
Prior to cDNA synthesis, the quality of
the RNA was routinely verified by standard Northern blot analysis with
digoxigenin-labeled antisense RNA probes specific for known general
stress genes. For cDNA synthesis, 2 µg of total RNA was mixed with 4 µl of a commercially available primer mix (Sigma-Genosys Ltd.) and 3 µl of 10× hybridization buffer (100 mM Tris [pH 7.9], 10 mM EDTA, 2.5 M KCl) in a total volume of 30 µl. The primer mix consisted of
4,107 specific oligonucleotide primers complementary to the 3' ends of
all B. subtilis mRNAs (Sigma-Genosys Ltd.). The sample was
heated to 95°C for 10 min and subsequently cooled to 42°C for
primer annealing. Reverse transcription was performed in a total volume
of 60 µl with SuperScript II reverse transcriptase and
[
-33P]dCTP in the appropriate
buffer for 1 h (Life Technologies, GmbH, Karlsruhe,
Germany). After addition of 2 µl of 1% sodium dodecyl sulfate (SDS),
2 µl of 0.5 M EDTA (pH 8.0), and 6 µl of 3 M NaOH, the remaining
RNA was hydrolyzed by incubation at 65°C for 30 min and at room
temperature for 15 min. Prior to ethanol precipitation, the cDNA
solution was neutralized with 20 µl of 1 M Tris (pH 8.0) and 6 µl
of 2 N HCl. After a wash with 70% ethanol the pellet was carefully
dried and resolved in 100 µl of distilled water. Labeling efficiency
was determined with a liquid scintillation counter. This study was
performed with Panorama B. subtilis gene arrays from
Sigma-Genosys Ltd., which carry duplicate spots of PCR products
representing 4,107 currently known B. subtilis genes. cDNA
denaturation, probe hybridization, and washing of filters were
performed as described by Hauser et al. (23).
The arrays were exposed for 2 and 4 days to storage phosphor screens
(Molecular Dynamics, Sunnyvale, Calif.) and scanned with a Storm
840/860 PhosphorImager (Molecular Dynamics) at a resolution of 50 µm
and a color depth of 16 bits.
Bound cDNA was stripped off the membranes by a short (1-min) washing
step with 250 ml of boiling buffer (5 mM sodium phosphate [pH 7.5],
0.1% SDS), incubation in 250 ml of fresh buffer at 95°C for 20 min,
and two additional wash steps with fresh boiling buffer.
Data analysis.
Hybridization signals were quantified with
ArrayVision software (Imaging Research Inc.) after direct import of the
PhosphorImager files. After subtraction of the background, which was
defined as the median of signals surrounding the entire spot
fields, the overall spot normalization function of ArrayVision
was used to calculate the normalized intensity values of individual
spots, thus facilitating the comparison of results from different
hybridizations and filters. Briefly, this procedure involved two steps:
(i) calculation of the intensity of an average spot by dividing the sum
of the intensities of all PCR product specific signals on the
array by the total number of spots and (ii) dividing the intensity of
the individual spot by the intensity of this average spot.
For each growth condition mRNA was prepared from two independent
cultivations and then used for independent cDNA synthesis and DNA array
hybridizations. For exponentially growing bacteria and ethanol
treatment, three entirely independent replicates were processed. In
total, 32 array hybridizations were performed. For each gene the
average of the normalized intensity values from all the replicate
experiments was calculated. To avoid extreme intensity ratios for genes
close to or below the detection limit, the average normalized intensity
for these low values was arbitrarily set to a value
corresponding to a signal-to-noise ratio of 2. These average values
were then used to calculate expression ratios for the following
comparisons: (i) stressed (ethanol, salt, or heat shock applied for 10 min) versus exponentially growing wild-type strains, (ii) stressed
(ethanol, salt, or heat shock applied for 10 min) versus exponentially
growing sigB mutant cells, (iii) stressed wild-type cells
versus stressed sigB mutant cells (both treated with
ethanol, salt, or heat shock for 10 min), and (iv) the rsbX
sup20a hyperexpression mutant versus the sigB mutant 60 min after ethanol addition. rsbX mutants lack an essential negative regulator of the
B regulatory
cascade, fail to restrict the
B response, and
therefore display artificially high and extended
B activity (50). In the
suppressor mutant (rsbX sup20a) artificially high
B activity is compatible with growth (W. G. Haldenwang, unpublished data).
Experiments involving ethanol, salt, or heat stress (10 min) were
performed with the isogenic B. subtilis strain pair (168 and ML6). In order to substantiate the results and to minimize the number of false positives, experiments involving treatment with
ethanol for 10 min were also performed with an independent strain pair,
BSA46 and BSA272, and Panorama B. subtilis gene arrays (Sigma-Genosys Ltd.) from a different batch. Due to the different strain pair and the different array batches, hybridizations involving this strain pair did not produce exactly the same induction ratios but
confirmed the candidates identified in this study with the strain pair
168 and ML6.
The ratios of the expression levels obtained from the averaged
normalized intensities of all replicate experiments were imported into
GeneSpring 3.2.12 software (Silicon Genetics, Redwood City, Calif.) and used to find additional
B-dependent and
B-independent stress genes. Seventy-five genes
have previously been assigned to the
B
regulon. Twelve of these (clpC, ctsR,
opuE, sms, trxA, yacH, yacI, yacK, ytxG, ytxH,
ytxJ, and yvyD) exhibit an additional stress
induction mechanism, but the remaining 63 (aldY,
bmr, bmrR, bmrU, bofC,
clpP, csbA, csbB, csbD,
csbX, ctc, dps, gsiB,
gspA, gtaB, katB, katX,
nadE, rsbV, rsbW, rsbX,
sigB, yacL, ycdF, ydaD, ydaE, ydaG, ydaP, ydaS,
ydaT, ydbD, ydbP, ydhK,
yfhK, yfkM, yflA, yflT,
yhdF, yhdN, yhxD, yjbC,
yjgB, yjgC, ykgA, ykzA,
yocK, yotK, yoxA, yoxC,
ypuB, yqhA, yqhQ, yqxL,
yrvD, ysnF, ytkL, yugU,
yvrE, yxaB, yxbG, yxcC,
yxkO, and yycD) should display clear
B-dependent induction in response to all three
stresses. These genes were also used to obtain a consensus sequence of
B-dependent promoters.
The DNA sequences preceding genes with potential
B-dependent stress induction were subsequently
inspected for the occurrence of the characteristic
35 and
10 boxes
recognized by the RNA holoenzyme carrying
B
with the MotivFinder program (Decodon GmbH, Greifswald, Germany).
World Wide Web access.
The complete data sets for all the
growth conditions investigated are available online
(http:///www.uni-marburg.de/mpi/voelker/functional -genomics).
 |
RESULTS |
Identification of
B-dependent general stress
genes.
DNA macroarrays that contain all currently known genomic
open reading frames of B. subtilis were used to record the
comparative transcriptional profiles of exponentially growing cells and
cultures exposed to mild ethanol, heat, or salt stress. Bacteria were
exposed to the stresses for 10 min in order to achieve maximal
transcriptional induction.
Prior to quantification of the array data, the reproducibility of the
array experiments was estimated by comparing the normalized spot
intensities in scatter diagrams (Fig. 1).
Array data from hybridizations of independent samples from the same
cultivation condition always yielded high Pearson correlation
coefficients (see Fig. 1A for an example; r = 0.992).
As expected, Pearson correlation coefficients calculated for comparison
of untreated and stressed samples of the wild type or for comparison of
ethanol-stressed samples from a wild-type strain and from the
corresponding sigB mutant were lower (r = 0.8995 and r = 0.9266, respectively), and the scatter
diagrams displayed genes induced and genes repressed by ethanol stress
(Fig. 1B and C).

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FIG. 1.
Scatter diagrams of normalized spot intensities. (A)
Spot intensities of two array hybridizations with two different
unstressed samples from the wild-type strain 168 (wt co1 versus wt
co2). (B) Spot intensities of array hybridizations from a nonstressed
probe of strain 168 (wt co) and an ethanol-stressed sample of the same
strain (wt EtOH). (C) Comparison of spot intensities of filters
hybridized with probes of wild-type strain 168 (wt EtOH) and its
isogenic sigB mutant ML6 ( sigB EtOH),
both treated with ethanol. For the presentation, spot intensities of
the 4,107 genes have been normalized and the duplicate spots on the
filter have been averaged as described in Materials and Methods. r,
Pearson correlation coefficient.
|
|
In this study a gene was considered to require
B for stress induction when it complied with
all of the following three criteria. (i) Expression of the gene had to
be induced more than twofold by at least two of the three stresses in
the wild type. (ii) The ratio of induction had to be at least 2 in
three of the four mutant comparisons employed, i.e., wild type versus
sigB mutant after heat, salt, or ethanol stress and
expression in the rsbX sup20a mutant versus expression in
the sigB mutant 60 min after exposure to ethanol stress.
(iii) A
B-dependent promoter had to be located
in front of the gene or the transcriptional unit to which the gene
belonged. These criteria clearly differentiated between specific and
general
B-dependent stress genes.
Table 1 lists the 101 genes that met
these selection criteria. Fifty-one genes belong to the
group of 75
B-dependent genes already
described in the literature, and 50 genes correspond to potential new
members of the
B regulon.
However, it was apparent that we preferentially missed
B-dependent genes subject to an additional
B-independent stress induction mechanism,
because those genes would not always display much stronger induction in
the wild type than in the sigB mutant. Therefore, we applied
a modified two-step discrimination protocol to the same set of data to
hunt for this class of genes. Table 2
displays the results of this search, which required twofold stress
induction by at least two of the three stress factors in the wild type
and the sigB mutant. Potential candidates passing this first
test were subsequently screened for the presence of the conserved
B-dependent promoter. This search strategy
identified 24 additional genes, 11 of which had previously been
described as general stress genes. The latter group includes
yvyD, which remained inducible by ethanol stress in the
sigB mutant at the
H-dependent
promoter (16), as well as the clpC operon and
clpP, both of which remained stress inducible in a
sigB mutant at a
A-dependent
promoter after inactivation of the CtsR repressor by stress (20,
32). trxA also belongs to this group, but the mechanism for stress induction in the sigB mutant has not
been clarified yet (40). Assigning the other 13 genes to
the
B regulon is more complicated because all
of them still displayed stress induction in a sigB
mutant (Table 2). In the case of the presumed yceCDEFGH
operon this additional stress induction mechanism seems to involve the
ECF sigma factor
W (see below). Detailed
transcriptional analysis will be necessary to confirm
B dependency for each single gene listed in
Table 2.
In total this DNA array analysis revealed 125
B-dependent genes, 24 of which seem to be
subject to a second,
B-independent stress
induction. We confirmed 62 of the 75
B-dependent genes known from the literature.
Most notably, all the
B-dependent genes
identified by the proteomics approach were confirmed by this array
analysis (Table 3). This observation is
not surprising, because the proteomics approach should have mainly
detected genes displaying strong expression as well as clear
transcriptional induction. Also, most of the genes identified by
transcriptional studies (27, 37) or by the
transposon-based approach of Price and coworkers (10, 12)
were confirmed (Table 3). However, only 14 of 24 genes newly described
as
B dependent by a promoter consensus search
(36) were validated by DNA macroarrays. The reason for the
surprisingly low validation rate of this group is not yet known. The
list of genes already described in the literature as
B dependent but not confirmed by this DNA
macroarray analysis includes aldY, csbA,
csbB, opuE, ydbP, ydhK,
yotK, yoxA, ypuB, yqhA,
yqhQ, yrvD, and ytkL. We cannot
exclude the possibility that a few of these genes constitute false
positives that were described in earlier studies. However, we suspect
that in some cases the apparent lack of detection by this DNA array
approach might also be an artifact due to differences in the amount or
quality of the PCR products on the membrane or in the primers utilized
for the cDNA synthesis. One member of the latter group is certainly
opuE, whose
B dependency has been
unambiguously demonstrated (43).
Induction of
B-dependent genes ranged from
twofold to several hundredfold. Such extreme induction ratios might be
explained by the fact that
B, which is almost
inactive during exponential growth, exclusively controls some
B-dependent genes. Frequently, general stress
genes contain additional promoters, in most cases
A-dependent promoters, that allow significant
basal expression level during growth. Accordingly, the stress induction
ratios of genes in the latter group are lower than those for the former.
When the data were analyzed, it was apparent that salt and ethanol
triggered much stronger induction of the
B
regulon than heat stress, although heat stress was effective in
inducing heat-specific stress proteins (see below). The reason for this
difference is not clear but might be related to the influence of the
stresses on growth and consequently their stringency. Both ethanol and
salt reduced the growth rate slightly at the concentration used (final
concentration, 4%), whereas a temperature shift from 37 to 48°C
still stimulated growth.
Sometimes not all genes of an operon met the stringent criteria
applied. If the missing genes were flanked by genes displaying a clear
induction pattern (e.g., yfhL) or if the operon structure had been experimentally proven (e.g., the bmrU bmr
bmrR operon), the genes were added to the table, since the
failure of detection was most likely caused by the limitations of the
array analysis described above. Applying stringent criteria to the
searches will certainly minimize the detection of false-positive
candidates, but at the risk of producing false negatives. Besides the
genes listed in Tables 1 and 2, we uncovered a group of genes with either a less conserved
B-dependent promoter
or a stress induction pattern just failing to fulfill the requirements
outlined above. These 38 genes (aldY, aroI,
mtrA, purK, rbfA, spoIIQ,
yabK, yacO, yazC, ycsE,
ydcF, yddS, yeaC, yerD,
yfjB, yfkC, yfkT, yfmG,
yfmK, ygxA, yhdE, ykrS, ykrT, ykyB, ykzC, yrrU,
ysdB, ytzB, ytzE, yumB,
yusD, yusS, yutK, yvaM,
ywdJ, ywdL, ywlB, and ywmF)
are currently the subject of detailed Northern blot analysis to clarify
their potential
B dependence.
Locations and functions of new general stress proteins.
The
proteomic approach almost exclusively identified general stress
proteins that were localized in the cytoplasm. The transposon mutagenesis as well as the promoter search- and oligonucleotide screening-based approaches have already indicated that the synthesis of
membrane proteins is also subject to
B
control, leading to the assumption that
B also
contributes to the maintenance of the integrity of the cell envelope
during stress (19, 36). Inspection of the
B-dependent gene products described in this
study for membrane-spanning helices (MSH) revealed
that 25 of them contain at least one
potential MSH (Table 4). Furthermore, at least six of the
general stress proteins seemed to contain signal sequences indicating
an extracellular location. Four of those proteins are potential
lipoproteins and are most likely attached to the outside of the
cytoplasmic membrane (Table 5).
The functions of most of these newly described
B-dependent genes are still unknown. Probably
the proteins encoded by those genes are involved, like the proteins
already known, in the development of nonspecific multiple stress
resistance in starving cells or in growing cells subject to harsh
stress. In order to define the kind of stress resistance in which the
individual genes are involved, a comprehensive screening program
analyzing the stress resistance of mutants with mutations in single
stress genes is in progress (41). This screening has
already revealed a number of stress genes that have dramatic effects on
resistance to specific stress factors. Figure
2 displays the sensitivities of two
selected mutants to growth-preventing salt stress (see reference
51). The newly described yjbCD operon, which is
at least in part under
B control (see Table
2), therefore codes for proteins that are somehow involved in salt
resistance.

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FIG. 2.
Survival of B. subtilis strains during
growth-preventing salt stress. The wild-type strain 168 (solid squares)
and its isogenic mutants with mutations in sigB (ML6)
(open squares), yjbC (BFA2841) (solid triangles), and
yjbD (BFA2842) (solid circles) were grown in a synthetic
medium and exposed to salt stress. Survival was determined by plating
appropriate dilutions on Luria-Bertani agar plates. Cultures were
pretreated with a mild salt stress of 4% NaCl for 30 min at time zero
before the sodium chloride concentration was raised to 10% (wt/vol).
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Identification of
B-independent stress genes.
The DNA array data had thus far been used only for the identification
of genes strictly requiring
B for stress
induction or possessing a
B-dependent stress
induction component. However, the same array data also provide a
comprehensive picture of genes inducible by ethanol, salt, or heat
stress independently of
B. Table
6 lists genes displaying
significant induction by only one stress or a combination of two or all
three stresses. In those cases stress induction is very likely
B independent, because similar or even
stronger induction was observed in the sigB mutant and the
corresponding transcriptional units seemed to lack
B-dependent promoters. Seven genes
(murG, sacC, yugJ, yutG,
ywaC, ywnF, and ywoA) seemed to lack a
potential
B promoter and displayed at least
twofold induction in the wild type and the sigB mutant under
all stress conditions tested. For most of these genes, the precise
biochemical functions of the products have not yet been determined.
In addition to
B-independent stress genes
induced by all three stresses, there are proteins induced at least
2.5-fold by heat alone or heat plus ethanol (Table 6). Because ethanol
may induce cellular signals similar to those induced by heat stress,
genes induced by ethanol and heat stress might actually prove to encode specific heat stress proteins. The well-known members of the HrcA regulon belong to this group. Induction of the HrcA regulon by ethanol
was more pronounced in the sigB mutant, most likely because
B-dependent stress gene induction did not
compete for the limiting RNA polymerase core enzyme in this mutant.
The group of genes preferentially induced at least threefold by ethanol
stress alone is somewhat surprising, since it was not recognized in the
proteomic studies. For most of those genes neither an induction
mechanism nor their functions in adaptation to ethanol can be inferred
from the currently available data. Induction of the ureAB
operon is most likely accomplished via
H,
which has already been shown to be involved in stress induction of the
ytxGHJ operon and the yvyD gene (16,
47).
Activation of the ECF sigma factor
W following salt
shock.
Investigation of the genes specifically induced after
imposition of salt stress revealed 64 genes that were at least
threefold induced in the wild type and the sigB mutant or
that belonged to an operon fulfilling this criterion (Table 6). In
accordance with expectations, screening for genes specifically induced
by salt stress revealed four (opuA, opuB,
opuC, and proHJ) of the already known
osmoregulated operons of B. subtilis (13).
Interestingly, the gbsAB operon, encoding proteins for the
conversion of choline to the osmoprotectant glycine betaine
(13), also displayed salt shock induction. Quite
surprisingly, the gene coding for the ECF sigma factor
W was clearly induced following salt shock.
Frequently, the genes of ECF sigma factors, sigW included,
are subject to autoregulation (29). Consequently, the list
of salt-induced genes included 23 genes previously described as
W dependent (30). Screening of
the regulatory regions of the remaining salt-induced genes revealed
that 11 of them either possessed a putative
W
promoter or belonged to a potential
W-dependent transcriptional unit. Further
extending this analysis, we inspected (i) the salt induction pattern of
other previously described members of the
W
regulon (30) for their response to salt shock and (ii)
genes displaying stress induction via
B and at
least one other mechanism (Table 2) for putative
W promoters. This approach suggested 14 more
genes that seemed to belong to a
W-dependent
transcriptional unit displaying salt shock induction. An overview of
the induction of the
W regulon by salt shock
is presented in Table 7. Examination of the data indicates that many of these potential
W-dependent genes were also
although to a
lesser extent
induced by ethanol (Tables 6 and 7), an effect that was
more pronounced in the sigB mutant than in the wild type
strain.
Twenty-three of the salt-induced proteins contain at least one putative
MSH, and five seem to be exported or attached to the membrane as
lipoproteins. These proteins are involved either in the acquisition of
compatible solutes (the Opu-class of proteins) (13) or in
the compensation of constraints imposed by salt stress on the membrane
or the cell wall.
Because other stress stimuli such as oxidative, alkaline, or acid
stress were not considered in this study, care should be taken in the
classification of genes as stress specific from these data alone. AhpC
and AhpF, for instance, should be considered oxidative stress
proteins, because both are particularly induced by peroxide (3,
14). In this case induction by salt stress most likely reflects
a secondary oxidative stress.
 |
DISCUSSION |
The
B-dependent general stress regulon is
one of the largest regulons of B. subtilis. The discovery
and functional characterization of almost all
B-dependent genes will be necessary for a
comprehensive understanding of the physiological role of this huge
regulon. Therefore, the DNA array technique was used to detect the
candidates not yet found by proteomics, transcriptional analysis,
consensus promoter-based transcriptional screening, or transposon
mutagenesis (2, 8, 10, 12, 26, 36, 37). The DNA array
induction pattern of the previously published
B-dependent genes (see Materials and Methods
for a comprehensive list) was utilized to formulate the following
criteria for identifying the remaining members of the regulon: (i)
induction in the wild type by at least two of the three stresses
analyzed (heat shock, salt stress, and ethanol stress), (ii)
B dependency of stress induction, that is,
absence in the sigB mutant and/or presence for a prolonged
time in an RsbX
suppressor mutant that
displayed prolonged and increased
B activity
following stress, and (iii) presence of a putative
B-dependent promoter in front of the gene or
operon. This approach is validated by the fact that it detected 51 of
the 64 genes already known to be strictly
B
dependent. In addition to this large group, 50 new genes, all subject
to the control of a putative
B-dependent
promoter, were identified. In order to also facilitate the recognition
of genes with an additional
B-independent
stress induction component, target genes displaying stress induction in
the wild type and the sigB mutant were screened for the
presence of the typical
B promoter structure
in the regulatory region. This adjustment of the data analysis revealed
11 already known
B-dependent genes for which
complex regulation had been described previously as well as 13 new candidates.
In total we describe 125 genes that belong to the
B regulon in this study. For the new members
of the regulon detected in this study,
B
dependency is highly probable but has to be confirmed by additional transcriptional studies in each case. Northern blot hybridizations have
been conducted and confirmed
B dependency for
ycnH, yjgD, and yqgZ.
However, a few genes described as
B dependent
in earlier studies, such as aldY, csbA,
csbB, opuE, ydbP, ydhK,
yotK, yoxA, ypuB, yqhA,
yqhQ, yrvD, and ytkL, were not
detected by our approach. The majority of these belong to a group of
genes that had been found to be
B dependent by
a consensus promoter-directed slot blot hybridization screening
(36). Of the 24 new candidates identified by this strategy, only 14 could be confirmed in the present investigation. Possible reasons for this failure include (i) the complex control of
genes, which could blur the
B dependency,
especially if it was combined with a weak
B
promoter that showed only a low induction rate, and, alternatively, (ii) false-positive candidates described in earlier studies. We suspect
(iii) that some genes were not confirmed because of artifacts due to
either the quality or quantity (or both) of the PCR product on the
membrane or the quality of the primers utilized for synthesis of the
labeled cDNA. opuE is a clear example of this class, because its
B-dependent stress induction has been
unequivocally demonstrated (43, 53).
Therefore, it should be stressed that the real number of
B-dependent genes might be even higher.
Thirty-eight genes displayed
B dependency but
failed to comply with all the criteria applied in this study. Those
genes had to be listed separately either because they did not display
induction by multiple stresses or because they lacked the
well-conserved
B-dependent promoter, although
they exhibited much stronger induction in the wild type than in the
sigB mutant. Failure to display an obvious
B promoter, for a gene that shows clear
B-dependent induction, might reflect indirect
control, probably via a transcriptional regulator subject to
B-dependent induction. However, this
hypothesis remains to be substantiated by experimental data. Besides
additional
B-dependent stress genes, this list
of 38 genes probably also contains some false-positive candidates.
Detailed transcriptional analysis of each single gene or operon is
currently being performed so that a final decision on their
B dependency can be made.
Although the limitations of the DNA array hybridization at best allow a
semiquantitative comparison of the expression profiles of different
genes, the variations in the expression level of the
B-dependent genes were striking. Most of the
B-dependent genes displaying the strongest
induced signals on the DNA macroarrays have already been found by the
proteomic approach, which should preferentially identify the strongly
expressed genes. Examples of this group are ctc,
gsiB, clpP, ydaD, yflT, and
ykzA. Three other strongly expressed
B-dependent genes have not yet been identified
on two-dimensional gels. This is not surprising, because one of them
encodes a membrane protein (ytxG), and the other two most
likely escaped detection by the proteomic approach because their small
products have a very basic pI (csbD and ywzA).
The reasons for the strong expression of these genes are not
immediately apparent because most of their promoters do not show what
we currently believe to be perfect
35 (GTTTAA) and
10 (GGGWAW)
boxes. In the case of gsiB the strong ribosome binding site,
leading to high stability of the mRNA, seems to be an additional factor
contributing to a high expression rate (31). For the other
genes the factors determining strong expression still need to be elucidated.
The complete description of all members of a regulon is only a
prerequisite for a full understanding of its physiological role.
Detailed biochemical and physiological studies must now follow to
obtain substantially new information on the physiological role of the
B regulon. Previous studies showed that
B-dependent stress proteins provide the
starved or stressed cell with oxidative, pH, salt, and heat stress
resistance (3, 18, 19, 51). So far only Dps has been shown
to be required for oxidative stress resistance (4), and
the
B-dependent proteins essential for salt,
heat, and acid resistance are not known. Because the clpC
operon or the clpP gene remains heat inducible in a
sigB mutant, a limiting amount of ClpC or ClpP should not be
the main reason for the impaired heat stress resistance of a
sigB mutant (for reviews see references 26 and 27). The newly identified
B-dependent genes do not immediately help to
answer this question, because most of them encode proteins of thus far
undefined function. However, many membrane proteins belong to this
group, indicating an essential role in the maintenance of cell envelope
or transport capacity, as already discussed by C. W. Price
(19, 37). Experimental evidence for this suggestion has
been provided by studies by E. Bremer's group, who showed that some
genes encoding proteins involved in the uptake of compatible solutes
are at least partly under
B control (43,
53). A few of the new
B-dependent genes
seem to encode proteins with interesting functions. YfhF, a probable
cell division inhibitor, might prevent division under conditions of
severe stress, giving cells time to recover. Some of the products might
be involved in detoxification, such as the products of the
yceCDEFGH operon, which seems to encode toxic anion
resistance proteins, or that of yqgZ, which encodes a
potential arsenate reductase. Other proteins seem to perform functions
in maintaining the redox balance of the cell, including the products of
yxnA, ycnH, and yvaA, encoding a
glucose-1-dehydrogenase, a potential succinate semialdehyde
dehydrogenase, and a hypothetical oxidoreductase, respectively.
The superoxide dismutase SodA is certainly required for detoxification
of superoxide, whereas the potential intracellular proteinase YraA
might be required to degrade proteins that cannot be repaired. However,
detailed functional analysis will be necessary to ascertain the precise
functions of these proteins in stress management.
One of the interesting findings of this study is the surprisingly large
number of genes with unknown functions that belong to the
B regulon (see Tables 1 and 2). Of the
4,100 B. subtilis genes, about 1,700 code for proteins with
still unknown functions. Elucidation of the functions of all these
proteins is a great challenge for future research. Allocating unknown
proteins to their regulation groups is a useful approach for a
preliminary prediction of their functions. This approach indicates that
almost 100
B-dependent proteins with still
unknown functions are probably involved in the development of multiple
prospective stress resistance in cells entering the stationary-growth
phase or in the development of heat or osmotic stress resistance.
However, detailed phenotypic screening of mutants is necessary to
assign each protein to a single facet of stress resistance as a first
step and to uncover its exact function by detailed biochemical
experiments. Such experiments are in progress in order to gain a more
comprehensive picture of the physiological role of this huge regulon in
stress adaptation. The first results of this screening are presented in
Fig. 2. Obviously, inactivation of yjbC renders B. subtilis almost as sensitive to growth-preventing salt stress as a
sigB null mutant. In the yjbD mutant,
too, stress resistance is significantly diminished from that in the
wild type. It is noteworthy that yjbD, the downstream gene
of an operon (yjbCD) that is at least partially
B dependent, encodes a protein (YjbD) whose
Lactococcus lactis homolog seems to affect degradation of
nonnative proteins and thereby stress tolerance (H. Ingmer, personal
communication). Further studies are in progress to analyze the precise
physiological role of both B. subtilis proteins in more detail.
Despite some restrictions discussed above, this study clearly shows
that the DNA array technique is a very useful approach for defining the
structure and function of already known or still unknown regulons.
Fewer than 30% of the regulon members had been identified thus far by
a proteomics approach, and low-abundance proteins or proteins with
membrane-spanning domains had been missed entirely. Even though the
proteomics approach will still have wide application because the final
and active products of gene expression, as well as information on
posttranslational modification or protein sorting, can be visualized
only by proteomics, it will be replaced by the DNA array technologies
for the purpose of defining regulon structure. The latter approach is
easier to handle and is certainly more comprehensive, but even such a
sophisticated screening will require detailed follow-up experiments to
validate the data, including quantification of the results.
Among
B-independent stress induction
phenomena, the salt shock induction of the
W
regulon was certainly the most interesting.
W
is one of seven ECF type sigma factors of B. subtilis, the
functions of all of which are still not well understood. In general
this class of sigma factors controls uptake or secretion of specific molecules and ions or responses to a variety of stresses
(33).
W in particular has been
implicated in detoxification responses and the production of
antimicrobial compounds (30, 46). Although a
sigW mutant displays altered resistance to cell wall
biosynthesis inhibitors (46), no difference in the zone of
inhibition was observed when a sigW mutant and its
corresponding wild type were exposed to HCl, NaOH, NaCl, EDTA,
dithiothreitol, 2-mercaptoethanol, lysozyme, SDS, hydrogen peroxide,
methyl viologen, metal ions, and various antibiotics (30).
In this study we provide evidence that the
W
regulon is induced by salt shock. This is most likely not an osmotic
but an ionic effect, because none of the known osmoregulated genes of
B. subtilis possesses a
W promoter.
Probably salt shock interferes with the cell envelope or the transport
capacity of the cell and thereby triggers induction of the
W regulon. Sensing of transport processes has
already been implicated in triggering
W
activity (46). Salt shock does not seem to be the only
stress inducing the
W regulon. Recently,
Schumann and coworkers discovered alkaline shock induction of the
W regulon (54). Alkaline shock
could also interfere with the transport capacity of the cell and thus
release
W from its inhibition by its
anti-sigma factor RsiW.
 |
ACKNOWLEDGMENTS |
A. Petersohn and M. Brigulla contributed equally to this work.
We are grateful to W. G. Haldenwang for providing strain BSA386
and to Jeff Errington for construction of the BFA2841 and BFA2842
mutants. We thank N. Hauser, M. Scheideler, and T. Beißbarth for
initial help with the gene array hybridizations and analysis of the data.
This work was supported by grants from the Deutsche
Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the BMBF, the European Union (GLG2-CT-1999-01455) and the Max-Planck-Gesellschaft to J.H., U.V., and M.H.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Ernst-Moritz-Arndt-Universität
Greifswald, Friedrich-Ludwig-Jahn-Str. 15, 17487 Greifswald, Germany.
Phone: 49-3834-864200. Fax: 49-3834-864202. E-mail:
hecker{at}biologie.uni-greifswald.de.
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