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Journal of Bacteriology, November 2001, p. 6422-6428, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6422-6428.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Catalytic Function of an 
/
Hydrolase Is Required for Energy
Stress Activation of the 
B Transcription Factor in
Bacillus subtilis
Margaret S.
Brody,
Kamni
Vijay,
and
Chester
W.
Price*
Department of Food Science and Technology,
University of California, Davis, California 95616
Received 11 June 2001/Accepted 10 August 2001
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ABSTRACT |
The general stress response of Bacillus subtilis is
controlled by the 
B transcription factor, which is
activated in response to diverse energy and environmental stresses.
These two classes of stress are transmitted by separate signaling
pathways which converge on the direct regulators of B,
the RsbV anti-anti- factor and the RsbW anti- factor. The energy
signaling branch involves the RsbP phosphatase, which dephosphorylates RsbV in order to trigger the general stress response. The
rsbP structural gene lies downstream from
rsbQ in a two-gene operon. Here we identify the RsbQ
protein as a required positive regulator inferred to act in concert
with the RsbP phosphatase. RsbQ bound RsbP in the yeast two-hybrid
system, and a large in-frame deletion in rsbQ had the
same phenotype as a null allele of rsbP
an inability to
activate B in response to energy stress. Genetic
complementation studies indicated that this phenotype was not due to a
polar effect of the rsbQ alteration on
rsbP. The predicted rsbQ product is a
hydrolase or acyltransferase of the 
/
fold superfamily, members
of which catalyze a wide variety of reactions. Notably, substitutions
in the presumed catalytic triad of RsbQ also abolished the energy stress response but had no detectable effect on RsbQ structure, synthesis, or stability. We conclude that the catalytic activity of
RsbQ is an essential constituent of the energy stress signaling pathway.
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INTRODUCTION |
In Bacillus subtilis and
related bacteria, a general stress response controlled by the
B transcription factor confers multiple stress
resistance on nongrowing cells (reviewed in references 17
and 26). The activity of B is
governed by a signal transduction pathway with two distinct branches.
One branch is specific for energy stresses, such as carbon, phosphorus,
or oxygen limitation, and the other is specific for environmental
stresses, such as acid, ethanol, heat, or salt stress (20, 41,
43, 44). According to the model shown in Fig.
1, each branch terminates with a
differentially regulated serine phosphatase: RsbU in the environmental
signaling branch and RsbP in the energy signaling branch. When
activated by its particular class of stress, either RsbU or RsbP
engages the common regulators RsbV and RsbW. RsbV and RsbW together
control B activity via a partner-switching
mechanism in which alternate protein-protein interactions are governed
by the phosphorylation state of RsbV (3, 8, 13, 41, 42,
44).
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FIG. 1.
Model of the signal transduction network that activates
the general stress transcription factor B. (A) Two
signaling pathways converge on RsbV-P, the antagonist form found in
unstressed cells. The energy stress branch terminates with the RsbP
phosphatase (Energy PP2C), which contains a PAS domain in its
amino-terminal region (41). In contrast the environmental
stress branch terminates with the RsbU phosphatase (Environmental
PP2C), which is activated by RsbT in response to upstream signaling
elements (2, 20, 42-44). When triggered by stress, either
the RsbP or the RsbU phosphatase removes the serine phosphate from
RsbV-P. Dephosphorylated RsbV then binds the RsbW anti- factor,
forcing it to release B, which can then activate its
target genes (3, 8, 13). We show here that RsbQ (formerly
called YvfQ) is an essential component of the energy stress branch and
propose that it acts in concert with the RsbP phosphatase. (B) Genetic
organization of the rsbQ-rsbP operon. The
rsbQ and rsbP reading frames are denoted
by open rectangles above the kilobase scale. The regions indicated
within rsbP code for the PAS domain (PAS) and the PP2C
phosphatase domain (shaded). Promoter activity (right-angled arrow) has
been proximately located between 0 and 0.7 kb, and a potential
terminator sequence (stem-loop) lies downstream from
rsbP. Energy signaling does not require increased
transcription from this promoter, suggesting that signaling is
accomplished by a posttranslational mechanism (41).
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The means by which energy and environmental stress signals enter their
respective branches of the pathway are unknown, and it is clear that
additional signaling components remain to be identified (1, 33,
41, 45). Here we focus on the energy signaling branch. The
amino-terminal half of the RsbP phosphatase contains a Per-ARNT-Sim
(PAS) domain (41), similar to those found in a wide
variety of proteins involved in sensing fluctuations in redox, light,
or oxygen (38). In some proteins, such PAS domains
function by binding a ligand or a chromophore and in others by
controlling protein-protein interactions with other proteins. As part
of a search for new elements of the energy signaling branch, we report
here that RsbP interacts with RsbQ, a positive regulator essential for
energy stress activation of B. Moreover, our
genetic analysis indicates that the predicted catalytic activity of
RsbQ is required for energy stress signaling in B. subtilis.
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MATERIALS AND METHODS |
Bacterial strains and genetic methods.
Escherichia
coli DH5 (Gibco BRL, Gaithersburg, Md.) was host for all
plasmid constructions, while E. coli
BL21(DE3)pLysS (Novagen, Madison, Wis.) was the expression host for
protein purification. Standard recombinant DNA methods were as
described by Sambrook et al. (32). B. subtilis strains used are shown in Table
1. B. subtilis PB2
and its derivatives were made competent by standard methods
(12). We used the four-primer method of site-directed mutagenesis (18) to make three separate alterations within
the cloned rsbQ gene. To make the large, in-frame deletion
of rsbQ (rsbQ
2), we removed 546 bp from within
the 807-bp coding region, deleting triplets 38 to 219. To make the
missense mutations, triplet 96 was changed from serine (TCC) to alanine
(GCC) to yield rsbQS96A and triplet 247 was changed from
histidine (CAT) to alanine (GCA) to yield rsbQH247A. Each
PCR-mutagenized product was cloned into the pCP115 integration vector
(27), yielding pMB56 (rsbQ2), pMB58
(rsbQH247A), and pMB59 (rsbQS96A). We then
introduced the deletion or missense mutations into the homologous copy
of rsbQ on the B. subtilis chromosome
by means of a two-step allele replacement method (35).
Substitution of the rsbQ2 allele was confirmed by PCR
amplification. Substitution of the missense mutations was confirmed by
chromosomal sequencing.
-Galactosidase accumulation assays.
All cultures were
grown in a shaking water bath at 37°C. For energy stress experiments,
cells were either grown into stationary phase in buffered Luria broth
(LB) lacking salt (9) or subjected to carbon and
energy starvation in a minimal salt medium (5) containing
0.05% glucose. For environmental stress experiments, cells were grown
to early logarithmic phase in buffered LB lacking salt. At this point,
either NaCl (0.3 M final concentration) or ethanol (4% [vol/vol]
final concentration) was added to one of two parallel cultures. For all
experiments, samples were collected at the times indicated and treated
as described by Miller (22). Cells were washed with Z
buffer and permeabilized using sodium dodecyl sulfate and chloroform.
Protein concentrations were determined using the Bio-Rad Protein Assay
reagent (Bio-Rad, Richmond, Calif.). Activity was defined as
A420 × 1,000 per minute per
milligram of protein.
Yeast two-hybrid analysis.
We used the Matchmaker Two-Hybrid
System (Clontech, Palo Alto, Calif.) to detect possible interactions
among RsbP, RsbQ, RsbT, and RsbU. The rsbQ and
rsbP reading frames were fused to either the GAL4 DNA
binding domain in plasmid pGBT9 or the GAL4 activation domain in
plasmid pGAD424, as previously described for rsbT and rsbU (44). These constructs were then paired in
yeast SFY526 cells by selecting double transformants on minimal plates.
Transcriptional activation of the lacZ reporter gene in
these transformants was determined qualitatively using a colony lift
filter assay according to the manufacturer's protocol.
Overexpression of rsbQ and rsbP
products in B. subtilis.
We used PCR
to amplify the individual rsbQ and rsbP reading
frames along with their predicted ribosomal binding sites, placing them
under control of the
isopropyl--D-thiogalactopyranoside (IPTG)-inducible Pspac promoter in the multicopy expression vector pDG148 (36). Plasmid pMB64, carrying rsbQ, was
transformed into B. subtilis PB198 (wild-type)
and PB567 (rsbP1::spc). Plasmid pKV2, carrying rsbP, was transformed into PB198 and PB605
(rsbQ2). These strains also carried a
B-dependent ctc-lacZ fusion in
single copy at the amyE locus (10) to permit
measurement of B activity. Expression from the
plasmid was induced by adding IPTG (1 mM final concentration) to
cultures in the early logarithmic phase of growth.
Construction of overexpression clones and purification of
His-tagged proteins from E. coli.
To
purify the wild-type, S96A, and H247A forms of RsbQ, we fused the
appropriate coding region to a hexahistidine tag in the pET15b
expression vector (Novagen). Hexahistidine-tagged proteins were
purified from E. coli BL21(DE3)pLysS extracts on
nickel-nitrilotriacetic acid metal affinity columns (Qiagen, Valencia,
Calif.) according to the manufacturer's protocol.
Trypsin proteolysis.
Purified wild-type and mutant RsbQ
proteins were subjected to limited trypsin proteolysis as follows.
Trypsin (L-1-tosylamido-2-phenyl ethyl chloromethyl ketone
[TPCK]-treated type XIII from bovine pancreas; Sigma, St.
Louis, Mo.) was used to digest a 500-fold-molar excess of the target
protein in a buffer containing 25 mM Tris (pH 8.0), 50 mM NaCl, and 1 mM CaCl2. Digestions were performed at 25°C and
were stopped at 2-min intervals by adding phenylmethylsulfonyl fluoride
(1 mM final concentration), removing the mixtures to ice, and then
freezing them at 
80°C. Samples were electrophoresed on sodium
dodecyl sulfate (SDS)-15% polyacrylamide gels and were visualized by
Coomassie blue staining.
Detection of RsbQ by Western blotting.
Polyclonal antibody
was raised by injecting rabbits with the purified, His-tagged RsbQ. To
test antibody specificity, extracts of PB2 and PB604
(rsbQ2) were prepared from early-stationary-phase cells.
For determining RsbQ levels in vivo, strains PB2, PB633 (rsbQS96A), and PB631 (rsbQH247A) were grown in
buffered LB lacking salt, and samples were taken at the indicated times
during the logarithmic and stationary growth phases. Cells were
harvested by centrifugation, washed in 1 ml of sonication buffer (10 mM Tris, pH 7.4, and 5 mM MgCl2), and resuspended in
a minimal volume (50 to 100 µl) of the same buffer containing 10 µg
of lysozyme. Following a 10-min incubation at 37°C, cells were broken
by sonication and centrifuged to remove cell debris from the extracts.
Approximately 100 µg of total cell protein per lane (as determined by
Bio-Rad Protein Assay) was separated on an SDS-12% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Blots were blocked and washed in phosphate-buffered saline-Tween (PBST) (10 mM Na2HPO4, pH
7.2; 137 mM NaCl; 2.7 mM KCl; 3% [vol/vol] Tween 20) containing 5%
(wt/vol) nonfat dry milk (BLOTTO) and were then exposed to the rabbit
polyclonal anti-RsbQ antiserum for 1 h at
25oC. Following four washes with BLOTTO and PBST,
blots were incubated at 25°C for 1 h with the secondary
antibody, a goat anti-rabbit immunoglobulin G peroxidase conjugate
(Sigma). Washes were repeated as before, with the addition of a final
wash in PBS. Bound antibody was detected using the ECL Plus Western
blotting detection kit (Amersham Pharmacia Biotech, Piscataway, N.J.)
according to the manufacturer's instructions.
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RESULTS |
rsbQ is required to activate B in
response to energy stress.
As shown in Fig. 1B, rsbQ is
located in an operon together with rsbP, the structural gene
for the serine phosphatase which activates B
under conditions of energy stress. Although the promoter for the
rsbQP operon has not been located precisely, fusion analysis found that energy signaling does not require increased operon transcription, leading to the notion that signaling is largely accomplished via a posttranslational mechanism (41).
Moreover, because a similar arrangement of rsbQ and
rsbP homologues is also found in Streptomyces
coelicolor, we inferred that both gene products might affect a
common cellular process (41). To determine whether RsbQ is
involved in B regulation, we made a large
in-frame deletion within the rsbQ coding region and
substituted it for the chromosomal copy via a two-step allele
replacement procedure. The resulting null mutant also contained a
single-copy transcriptional fusion between the B-dependent ctc promoter and an
E. coli lacZ reporter gene to provide an assay
for B activity.
As shown in Fig.
2, the strain bearing
the
rsbQ2 null allele was unable to induce expression of
the reporter fusion in two
different energy stress assays: entry into
stationary phase in
buffered LB (Fig
2A) and glucose limitation in a
minimal medium
(Fig.
2B). In contrast, the
rsbQ2 null
mutant retained the ability
to respond to signals of environmental
stress. Logarithmically
growing cells were challenged by the addition
of either 0.3 M
salt (Fig.
2C) or 4% ethanol (Fig.
2D). Response to
ethanol stress
was similar in the mutant and wild-type strains, whereas
response
to salt stress was decreased about threefold by the
rsbQ2 null
allele. Although it appears that
rsbQ plays a role in response
to salt stress, perhaps due to
an effect on the levels of
B in unstressed
cells,
rsbQ function is clearly not required for
the
environmental stress response as it is for the energy stress
response.
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FIG. 2.
RsbQ function is required for energy stress signaling.
Effect of the rsbQ2 allele on -galactosidase
accumulation from a B-dependent transcriptional fusion
in response to entry into stationary phase in buffered LB medium (A),
carbon and energy starvation in a minimal medium containing 0.05%
glucose (B), addition of 0.3 M NaCl to logarithmically growing cells in
buffered LB (C), or addition of 4% ethanol (EtOH) to logarithmically
growing cells in buffered LB (D). Time zero indicates when the stress
was imposed on wild-type PB198 ( ) and on the PB605 mutant bearing
the rsbQ2 allele ( ).
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The phenotype elicited by the
rsbQ2 null allele was
essentially the same as that previously reported for the
rsbP1::
spc null allele
(
41). This raised the possibility that the in-frame
deletion in
rsbQ was polar on
rsbP gene
expression. To address
this concern, we performed a complementation
experiment in the
rsbQ null background.
B activation in response to stationary-phase
stress was restored
by insertion of a single copy of
rsbQ+ at the
thr locus, in
trans to the
rsbQ2 allele at the
rsbQ locus
(data not shown). We therefore conclude that
rsbQ encodes a positive
regulator which plays an essential
role in transmitting energy
stress signals to
B.
Activation of B by RsbP requires RsbQ function in
vivo.
In the parallel environmental signaling pathway (Fig. 1),
the RsbU phosphatase is activated by direct protein-protein interaction with RsbT, the product of the gene immediately upstream from
rsbU. RsbT therefore functions as a regulatory subunit of
the RsbU environmental signaling phosphatase (21, 44). To
probe whether a similar relationship exists between RsbQ and the RsbP
energy-signaling phosphatase, we first asked if the two proteins could
interact to activate the yeast two-hybrid system. As shown in Table
2, RsbQ and RsbP together activated
transcription of the yeast reporter fusion. Moreover, this interaction
was specific in that there was no detectable activation when RsbQ was
paired with RsbU or when RsbP was paired with RsbT.
We next used overexpression experiments to further characterize the
relationship between RsbQ and RsbP. We moved each gene
into a multicopy
plasmid that placed its expression under control
of the
LacI-repressible, IPTG-inducible Pspac promoter. pMB64
(bearing the
rsbQ coding region) was introduced into the wild-type
strain
and also into a strain harboring the
rsbP1::
spc null allele,
whereas pKV2
(bearing the
rsbP coding region) was introduced into
the
wild-type strain and also into a strain harboring the
rsbQ2 null allele. To permit measurement of
B activity, these four strains each contained
a single-copy
ctc-lacZ reporter
fusion.
As shown in Fig.
3, overexpression of
rsbQ during early logarithmic growth did not immediately
induce expression of the reporter
fusion in wild-type cells. Induction
occurred only when cells
encountered the energy stress of stationary
phase, and the magnitude
of this induction was reduced compared to the
wild-type control
in which
rsbQ was not overexpressed.
Moreover, overexpression
of
rsbQ could not compensate for
the loss of
rsbP function in
the strain bearing the
rsbP1::
spc null allele. The
phenotype
manifested in this latter strain was the same as that of the
rsbP null mutant without overexpression of
rsbQ:
ctc-lacZ was not induced
by energy stress. The failure of
rsbQ overexpression to induce
B
activity in logarithmically growing cells stands in marked contrast
to
the results obtained by Yang et al. (
44) in their study of
the environmental stress pathway. These authors found a strong,
rsbU-dependent induction of
B
activity immediately upon overexpression of the
rsbT
regulator.
From the experiments shown in Fig.
3, we conclude that RsbQ
does
not act as a regulatory subunit for its cognate phosphatase, as
does RsbT.
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FIG. 3.
Overexpression of either RsbP or RsbQ cannot compensate
for the loss of the other. At time zero (arrow) IPTG was added to
logarithmically growing cells to induce overexpression of either
rsbP (carried in pKV2) or rsbQ (carried
in pMB64). Samples were periodically removed for assay of
-galactosidase activity both before and after cells had entered
stationary phase in buffered LB medium (indicated by the dashed line at
60 min). , wild-type PB198;  , wild-type PB606 with
rsbQ overexpressed;  , wild-type PB607 with
rsbP overexpressed;  , PB719 mutant bearing the
rsbP1::spc allele with
rsbQ overexpressed; , PB716 mutant bearing the
rsbQ2 allele with rsbP
overexpressed.
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We then performed the reciprocal experiment to determine the dependence
of
rsbP upon
rsbQ. As shown in Fig.
3,
overexpression
of
rsbP during early logarithmic growth
caused a rapid and significant
increase in
B
activity in wild-type cells. This activation occurred well before
the
energy stress of stationary phase, whereupon a further increase
in
B activity could be seen. However, this result
required the presence
of least one copy of
rsbQ. In the
absence of
rsbQ function, overexpression
of
rsbP
had no apparent effect. The phenotype manifested in this
experiment was
therefore the same as that of the
rsbQ2 null mutant
without overexpression of
rsbP:
ctc-lacZ was not
induced by energy
stress. We conclude that overexpression of either
rsbQ or
rsbP is not sufficient to compensate for
the loss of the other
regulator.
Serine 96 and histidine 247 of RsbQ are important for energy stress
signaling.
In order to suggest the in vivo function of RsbQ, we
first considered the Cluster of Orthologous Groups of proteins (COG) to
which it belonged. The COG database (http://www.ncbi.nlm.nih.gov/COG) is a phylogenetic classification of proteins encoded by 44 complete microbial genomes representing 30 lineages, and it allows detection of
both close and distant relationships (37). RsbQ (formerly called YvfQ) is a member of COG 0596predicted hydrolases or
acyltransferases (/ hydrolase superfamily). Examples of
the / hydrolase superfamily are widely distributed and manifest
unusually diverse catalytic activities, functioning variously as
proteases, lipases, peroxidases, epoxide hydrolases, dehalogenases, and
esterases (23, 25). While their primary amino acid
sequences are not highly conserved, members of the / hydrolase
superfamily share a common three-dimensional core structure containing
the catalytic domain. Within this domain is a catalytic triad in an
invariant order: nucleophilic residue/acidic residue/histidine residue
(23, 25). The residues comprising this triad can be
broadly separated in the primary sequence but are brought together in
the tertiary structure.
Comparison of the predicted RsbQ sequence to well-characterized
/
hydrolases yielded good candidates for the catalytic triad
residues.
Based on the alignments shown in Fig.
4,
we chose serine
96 as the potential catalytic nucleophile for RsbQ and
histidine
247 as the potential catalytic histidine. In contrast, the
assignment
of aspartate 219 as the acidic residue was less certain due
to
other nearby candidates. We therefore focused our analysis on
serine
96 and histidine 247.
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FIG. 4.
Partial alignment of RsbQ with well-characterized
members of the / hydrolase superfamily. The crystal structures
have been solved and the residues comprising the catalytic triads have
been suggested or established for BpoA2, a bromoperoxidase from
Streptomyces aureofaciens (16); DhlA, a
haloalkane halidohydrolase from Xanthobacter
autotrophicus (14, 28, 29, 39, 40); and EchA, an
epoxide hydrolase from Agrobacterium radiobacter
(24, 31). RsbQ and these other family members are aligned
by the Reverse Position Blast algorithm (4) against the
consensus for the / superfamily found in the Conserved Domain
Database, maintained by the National Library of Medicine
(www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). RsbQ was 23.5%
identical to the / hydrolase fold consensus over a 226-residue
overlap (E = 3 × 10 15). The two
regions surrounding the proposed catalytic triad residues are shown
here. Residues in boldface indicate the positions conserved in at least
three of the four proteins. (A) Residues surrounding the proposed
nucleophile S96 of RsbQ, shown here in larger font together with the
S A substitution. The catalytic nucleophiles in the other proteins
and in the consensus are also identified by the larger font. (B)
Residues surrounding the proposed acidic residue D219 and the proposed
histidine residue H247 of RsbQ, shown here in larger font together with
the HA substitution at H247. The catalytic acidic and histidine
residues in the other proteins and in the consensus are also identified
by the larger font.
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If serine 96 and histidine 247 comprised part of a catalytic triad in
RsbQ and if the enzymatic activity of RsbQ was important
for its
signaling function, we would predict that changing either
residue would
affect
B activation in response to energy
stress. We therefore constructed
two mutant versions of
rsbQ, one in which an alanine replaced
serine 96 (RsbQS96A)
and another in which an alanine replaced
histidine 247 (RsbQH247A).
These mutant versions were substituted
for the wild-type copy of
rsbQ on the chromosome. The resulting
mutant strains also
contained a
ctc-lacZ reporter fusion to measure
B activity. As shown in Fig.
5, replacement of either serine 96
or
histidine 247 with alanine completely abolished induction of
the
reporter fusion in response to energy stress. These results
suggest
that the catalytic activity of RsbQ is required for energy
stress
signaling. However, to strengthen this conclusion, it was
necessary to
test the possibility that the S96A and H247A alterations
might only
indirectly perturb catalytic activity as a result of
their primary
effects on RsbQ structure, synthesis, or stability.
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FIG. 5.
Residues S96 and H247 of RsbQ are required for energy
stress signaling. -Galactosidase accumulation from a
B-dependent transcriptional fusion as cells were grown
to stationary phase in buffered LB medium. , wild-type PB198;  ,
PB634 mutant bearing the rsbQS96A allele; , PB632
mutant bearing the rsbQH247A allele.
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The S96A and H247A alterations do not detectably affect RsbQ
structure or accumulation.
To address the possibility that the
S96A and H247A alterations might affect RsbQ structure, we used limited
trypsin proteolysis to compare the conformation of mutant and wild-type
RsbQ proteins. His-tagged proteins were overexpressed in E. coli and purified as described in Materials and Methods. As
shown in Fig. 6, the mutant proteins were
not substantially altered in their trypsin susceptibility relative to
the wild type. This implies that the native conformation of RsbQ was
not materially disrupted by the S96A or H247A substitutions.
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FIG. 6.
The S96A and H247A substitutions do not affect trypsin
susceptibility of RsbQ. His-tagged versions of wild-type RsbQ (W), the
S96A substitution (S), and the H247A substitution (H) were purified by
metal affinity chromatography and subjected to limited trypsin
digestion. Samples were removed at 2-min intervals and digestion was
terminated by addition of phenylmethylsulfonyl fluoride. Protein
fragments were separated by SDS-polyacrylamide gel electrophoresis and
stained with Coomassie blue. The mobilities of the marker protein are
given on the left, labeled with their masses (in kilodaltons).
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To determine whether the S96A and H247A substitutions affected
synthesis or stability of the mutant proteins, we used Western
blotting
experiments to compare steady-state, in vivo levels of
mutant and
wild-type RsbQ. Polyclonal antibody against RsbQ was
raised by
injecting rabbits with the His-tagged protein purified
from
E. coli; the specificity of this antibody is
shown in Fig.
7A. When tested against a
whole-cell extract of wild-type cells
that had been subjected to
SDS-polyacrylamide gel electrophoresis,
the anti-RsbQ antibody detected
three proteins with distinctly
different mobilities. One of these
proteins had the mobility expected
for RsbQ and was missing from an
extract of the
rsbQ2 null mutant.
Figure
7B shows that
the signals from this protein were the same
in the wild type and the
two substitution mutants at multiple
points in the growth curve. We
infer from these results that the
alterations in the mutant proteins
had no significant effect on
synthesis or stability of RsbQ in vivo.
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FIG. 7.
The S96A and H247A substitutions do not affect
steady-state levels of RsbQ in vivo. Polyclonal antibody raised against
purified RsbQ was used to probe Western blots of whole-cell extracts
separated by SDS-polyacrylamide gel electrophoresis. (A) The anti-RsbQ
antibody detected three clear signals in the wild-type PB2 extract (W).
One of these signals was absent in the extract from the PB604 mutant
bearing the rsbQ2 allele (Q); this signal had the
mobility expected for RsbQ (29.9 kDa). Marker protein masses (in
kilodaltons) are shown on the left. (B) The wild-type PB2 (W), the
PB633 mutant bearing the rsbQS96A allele (S), and the
PB631 mutant bearing the rsbQH247A allele (H) were grown
in buffered LB medium. The time before or after entry into stationary
phase at which each group of three samples was taken is indicated in
minutes at the top. At each time point, the anti-RsbQ antibody detected
a signal of similar strength in all three strains. This signal
increased in magnitude (relative to total cell protein) as cells
entered stationary phase. Only that portion of the gel containing the
RsbQ signal is shown; mobilities of the 29.3- and 34.7-kDa marker
proteins are given on the left.
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From the sum of these analyses, we conclude that the loss of energy
stress signaling caused by the RsbQS96A and RsbQH247A
substitutions
most likely resulted from altered catalysis and
not from a disruption
of protein structure, synthesis, or stability.
We therefore propose
that RsbQ possesses an enzymatic activity
that plays an essential role
in the energy stress signaling pathway
which activates the general
stress
response.
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DISCUSSION |
Bacteria use a variety of mechanisms to sense and respond to
impending energy stress. These include control of individual enzyme
activities by energy charge (6) as well as control of regulatory proteins by reaction centers which most frequently sense
oxygen tension or electron flux (7, 38). Some of these reaction centers are provided by PAS domains, and in the
best-characterized examples the PAS domain binds a chromophore specific
for the parameter sensed, such as heme in the oxygen sensors FixL and
Dos (11, 15) and flavin adenine dinucleotide in the redox
sensors NifL and Aer (30, 34). In eukaryotic signaling
pathways, PAS domains are also known to mediate ligand binding or
protein-protein interactions (38), but these aspects of
PAS function have not yet been demonstrated in prokaryotic systems.
Here we report that RsbQ is a positive regulator required for energy
stress activation of the B transcription
factor. Moreover, RsbQ interacts in the yeast two-hybrid system with
the RsbP energy-signaling phosphatase, which contains a PAS domain in
its amino-terminal region (41). We presently have no
additional biochemical evidence to corroborate this interaction, nor
have we detected a hypothetical ligand or chromophore which the PAS
domain of RsbP might bind. However, we have shown that the predicted
catalytic activity of RsbQ is important for its signaling role, and on
the basis of our genetic analysis we can propose a model of RsbQ function.
We first considered the possibility that RsbQ might hydrolyze a small
molecule and thereby produce a direct activator of the RsbP
phosphatase. However, simply overexpressing RsbQ in growing cells did
not trigger the energy stress response (Fig. 3), so this model would
also require that RsbQ activity is somehow regulated by energy stress.
Such energy stress regulation was not apparent even when the cells that
were overexpressing RsbQ subsequently entered stationary phase. In
contrast, overexpressing the RsbP phosphatase in growing cells did
immediately induce a mock energy stress response, and this response was
intensified upon subsequent challenge with an authentic energy stress.
We interpret these results to indicate that in unstressed cells the
RsbP phosphatase has a low intrinsic activity which normally sets the
steady-state level of functioning B, a
necessary feature of its autocatalytic induction mechanism (19). Overexpression of RsbP would rapidly increase the
total amount of this intrinsic phosphatase activity, and, therefore, of
functioning B. Moreover, we presume that RsbP
activity in vivo is stimulated by energy stress (41).
Therefore, induction of B activity would occur
upon energy stress in wild-type cells, in which RsbP levels are normal,
and also in cells in which RsbP levels have been artificially elevated.
We infer that the catalytic activity of RsbQ is required for RsbP
phosphatase activityboth for the low intrinsic activity as well as
for the activity stimulated by energy stress (Fig. 3). We therefore
propose that the target of the RsbQ activity is RsbP itself. In this
view, the RsbP phosphatase would be synthesized in an inactive form and
converted to an active form by RsbQ. RsbQ could conceivably modify the
covalent structure of RsbP by means of its presumed hydrolytic or
acyltransferase activity or perhaps modify the hypothetical chromophore
which binds the PAS domain of RsbP.
The relationship of the RsbQ and RsbP energy stress regulators is
clearly different from that of the RsbT and RsbU environmental stress
regulators. RsbT lies directly on the signaling pathway, and it passes
the environmental stress signal to the RsbU phosphatase via a specific
protein-protein interaction (21, 44). In contrast, RsbQ
does not appear to lie directly on the energy signaling pathway but
instead acts catalytically to render the RsbP phosphatase competent to
receive the signal. This difference in the relationship between the
terminal regulators of the energy and environmental branches suggests
that the upstream sections of these two signaling pathways will also
prove to function by distinctly different mechanisms.
| |
ACKNOWLEDGMENTS |
We thank Tania Baker for her helpful discussions and Valley
Stewart for his critical comments on the manuscript.
This research was supported by Public Health Service grant GM42077 from
the National Institute of General Medical Sciences. Kamni Vijay was a
predoctoral trainee supported in part by Public Health Service training
grant GM07377 from the National Institute of General Medical Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Science and Technology, University of California, Davis, One
Shields Ave., Davis, CA 95616-8598. Phone: (530) 752-1596. Fax: (530) 752-4759. E-mail: cwprice{at}ucdavis.edu.
Present address: MJ Research, Inc., South San Francisco, CA 94080.
| |
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Abstract
Introduction
