From the Microbiology and Tumorbiology Center,
Karolinska Institutet, SE-171 77 Stockholm, Sweden and the
§ Department of Microbiology and Molecular Genetics, Harvard
Medical School, Boston, Massachusetts 021115
Received for publication, February 23, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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BarA is a membrane-associated protein that
belongs to a subclass of tripartite sensors of the two-component signal
transduction system family. In this study, we report that UvrY is the
cognate response regulator for BarA of Escherichia coli.
This conclusion is based upon homologies with analogous two-component
systems and demonstrated by both biochemical and genetic means. We show that the purified BarA protein is able to autophosphorylate when incubated with [ Signal transduction by phosphorylation and dephosphorylation of
cellular proteins plays pivotal roles in the regulation of numerous
cellular processes in both prokaryotes and eukaryotes. In prokaryotes,
these processes are accomplished partly by a family of two-component
signal transducing systems that enable bacteria to adapt to changing
environments (1-3). Typically, such a system comprises a membrane
associated sensor kinase and its cognate response regulator. Signal
reception by the sensor kinase stimulates an ATP-dependent
autophosphorylation at a conserved histidine residue. The
phosphorylated sensor kinase then catalyzes a transphosphorylation of
the cognate response regulator at a conserved aspartate residue, thereby rendering it functional, typically as a transcriptional regulator. Upon cessation or subsidence of signaling, both the sensor
kinase and the response regulator undergo dephosphorylation allowing
signal decay and silencing of the system.
Data derived from genome sequencing projects indicate that an organism
such as Escherichia coli possesses some 30 typical two-component systems (4). The majority of the sensor kinases consist
of an N-terminal cytosolic segment, a canonical pair of transmembrane
segments linked by a periplasmic bridge, and an orthodox transmitter
domain with a conserved histidine residue. However, a few sensors are
more complex; they possess an additional central receiver domain with a
conserved aspartate residue and a C-terminal phosphotransfer domain
with a conserved histidine residue, which is known as the Hpt-domain.
Recent studies have shown that several tripartite kinases catalyze the
phosphorylation of their cognate response regulators via an
ATP The BarA (also known as AirS) sensor kinase (Fig.
1) of E. coli is a member of
the subclass of tripartite sensor kinases. The barA gene was
first identified by its ability, when expressed from a high copy number
plasmid, to phenotypically suppress a deletion mutant of the EnvZ
sensor kinase and to control the OmpR regulator protein. The cross-talk
between BarA and OmpR, however, could not be demonstrated in
vitro, and the cognate response regulator for BarA was not
identified (11). Thus, the suppression phenomenon may be an artifact
resulting from cross-talk of an orphan response regulator in the
absence of its cognate sensor kinase. The barA gene has been
reported to be transcriptionally activated in uropathogenic E. coli strain DS17 upon pyelonephritis-pili attachment to its carbohydrate receptor on eukaryotic host cells and to be required for
activation of the siderophore system. Therefore, it was suggested that
BarA may direct the coordinate regulation of the iron acquisition machinery in uropathogenic E. coli, and play a key role in
colonization during urinary tract infections (12). Recently, BarA was
shown to be involved in the bacterial adaptive responses against
hydrogen peroxide-mediated stress by activating transcription of the
sigma factor RpoS, which in turn controls the expression of KatE, the major catalase of E. coli (13). Here, we present the results of experiments directed to identify the cognate response regulator for
the BarA sensor kinase of E. coli.
Bacterial Strains and Plasmids--
To delineate the function of
the E. coli MC4100 BarA protein, we disrupted the gene with
a kanamycin cassette and created a chromosomal null mutant by allelic
gene replacement. The barA gene from MC4100 was amplified by
polymerase chain reaction
(PCR)1 using primers
5'-GCATACGCCAAAATGAGGACAG-3' and 5'-GAAACCAGCGTCATAAAAAGCC-3' (MWG
Biotech) and Pfu polymerase. The 3119-nucleotide
fragment was cloned between the SphI and EcoRV
sites in vector pBR322. A kanamycin resistance cassette was excised
from pUC4K (Amersham Pharmacia Biotech) with BamHI
and ligated between the two BglII sites in the BarA open
reading frame (ORF), creating pBR322BarA::kan. This
construct was digested with EcoRI, blunted with Klenow, and then digested with SphI to release the 3705-nucleotide
barA::kan fragment, which subsequently was
subcloned into pCVD442 (14) using the SmaI and
SphI sites. The resultant construct was conjugated from
strain S17
E. coli strain M15 and plasmids pREP4 and pQE30 were
obtained from Qiagen Ltd. Construction of the pQE30ArcB78-778,
pQE30ArcA, and pQE30CpxR, used for expression of His-tagged derivatives
of ArcB, ArcA, and CpxR, have been described earlier (5, 16, 17). To
create pQE30BarA198-918, primers 5'-CCCGGATCCCATATGCGCGATGTAACCGG-3' and 5'-CCCGGATCCATGCATGCCGATTGCTACTCG-3' were used in the PCR with
chromosomal DNA of strain MC4100 as the template. The PCR product was
digested with BamHI and NsiI and cloned between
the BamHI and PstI sites of pQE30 (Qiagen). To
create pQE30UvrY, primers 5'-CCCGGATCCCATATGATCAACGTTCTACTTGTTGATGACCACG-3' and
5'-CCCGGATCCATGCACGCCTGGCTGGCTGG-3' were used in the PCR with
pCA9505 (15) as the template. The PCR product was digested with
BamHI and NsiI and cloned between BamHI and PstI of pQE30. Oligonucleotides were
purchased from Integrated DNA Technologies Inc. PCR was carried out
using the TacPlus Precision PCR system (Stratagene).
Purification of His6-tagged Proteins--
E. coli M15
cells co-transformed with pREP4 and the appropriate pQE30 derivative
were grown in 1 liter of medium (16 g of tryptone, 10 g of
yeast extract, and 5 g of NaCl/liter) supplemented with 100 µg
ampicillin/ml and 25 µg kanamycin/ml. Expression of the His6-tagged
proteins was induced at mid-exponential phase (A600 nm 0.5-0.6) by the addition of 2 mM isopropyl- Phosphorylation and Transphosphorylation Assays--
Unless
otherwise specified, phosphorylation assays were carried out at room
temperature in the presence of 40 µM
[ Hydrogen Peroxide Sensitivity Assays--
The agar diffusion
assay was performed by dividing an LB-agar plate into three equal
sectors, where ~103 cells of AKP023
(uvrY::cm), AKP014
(barA::kan), and MC4100 (the isogenic wild type
strain) was spread. A filter paper disc impregnated with 0.1 mM H2O2 was placed at the
intersection, the plate was incubated overnight at 37 °C, and the
zone of growth inhibition was measured.
The quantified survival of cultures exposed to hydrogen peroxide was
determined as described previously (13). Briefly, AKP023, AKP014, and
MC4100 were grown in LB, and at mid-exponential phase (A600 nm of 0.2) the three cultures were
divided into two equal portions. One of the portions was challenged
with 1.0 mM hydrogen peroxide, while the other served as a
control. Aliquots of the cultures were removed at 10, 20, 40, and 60 min, diluted appropriately, plated out onto LB-agar plates, and
incubated at 37 °C overnight. The relative survival of the culture
was determined by comparing the viability of the treated cultures to
that of the untreated ones.
Search for Analogous Two-component Systems--
A homology search
approach was undertaken to get insight into the Bar two-component
system. We initially searched for ORFs that were homologous to the
entire amino acid sequence of the BarA sensor kinase in data bases
using the BLASTP 2.0.6 program (National Institutes of Health,
Bethesda, MD). Proteins exhibiting the highest identity to BarA were
the ExpS of Erwinia carotovora (58%) and the GacS (LemA) of
Pseudomonas syringae (37%). ExpA and GacA have been
identified by genetic means as the respective cognate regulators for
these sensors (19, 20). However, supportive biochemical evidence for
these links is lacking. A subsequent search for ORFs homologous to ExpA
and GacA identified a group of typical bacterial response regulator
proteins with high homology (Fig. 2),
including UvrY of E. coli (15), SirA of Salmonella typhimurium (21), and VarA of Vibrio cholerae (22) as
well as highly homologous ORFs in the genomes of Klebsiella
pneumoniae, Yersinia pestis, and Shewanella
putrefaciens. The E. coli uvrY gene derives its
name from a close linkage to the uvrC gene on a bicistronic
transcript (14), a genomic organization shared by all of the above
response regulators. The uvrC gene is known to encode a
subunit of the UvrABC enzyme, which is involved in DNA repair (23), but
mutations in uvrY have no specific effect on this system
(15). Nonetheless, UvrY is the only E. coli protein that
exhibits a considerable homology to ExpA and GacA, suggesting that UvrY
may be the cognate response regulator for BarA.
BarA Autophosphorylation and Transphosphorylation of UvrY--
To
test whether UvrY is the cognate response regulator for BarA, we
cloned, overexpressed, and purified UvrY and BarA as His6-tagged proteins. For the purposes of this study, and to facilitate the purification of the sensor protein, we used BarA-(199-918),
deprived of amino acid residues 1-198 that constitute the
transmembrane segments. Previous studies on several sensor kinases
showed that removal of the transmembrane segments does not affect the
processes of autophosphorylation and the subsequent
transphosphorylation of the cognate regulator proteins (24-29).
Purified His6-BarA-(199-918) (hereafter referred to as 'BarA) was
incubated with [
We then addressed the question of whether BarA is able to
transphosphorylate UvrY in vitro. The sensor kinase was
incubated with [ The Transphosphorylation of UvrY by BarA Is Specific--
The
specificity of the UvrY transphosphorylation by 'BarA was then
determined. As expected, UvrY failed to undergo autophosphorylation with [
Finally, we tested if UvrY was able to accept the phosphoryl group from
ArcB, the tripartite sensor kinase of the Arc two-component system of
E. coli. As shown in Fig. 5B, ArcB was an
efficient kinase for its cognate response regulator, ArcA, but failed
to transphosphorylate UvrY at a detectable level. It should also be
noted that, even though ArcB failed to transphosphorylate UvrY, a
reduction in ArcB-P was observed. Thus, it seems reasonable to conclude
that BarA specifically transphosphorylates UvrY and that the two
proteins constitute a new two-component system.
Inactivation of Either BarA or UvrY Leads to Hydrogen Peroxide
Hypersensitivity--
It has previously been reported that mutations
in the barA gene lead to poor growth in iron-limiting media
(12) and to hypersensitivity when challenged with exogenous hydrogen
peroxide (13). Based on our biochemical data, we predicted that a
uvrY mutant strain would exhibit similar phenotypes as the
barA mutant.
The iron limitation effect on the growth of a uvrY and a
barA mutant strain was tested first. AKP023
(uvrY::cm), AKP014
(barA::kan), and MC4100 (wild type) were cultured
in either minimal media or LB supplemented with either the iron
chelators desferrioxamine or
The hydrogen peroxide sensitivity was next assayed by the agar
diffusion method. An LB-agar plate was divided into three equal sectors, where ~103 cells of each strain (AKP023, AKP014,
and MC4100) were spread. A filter paper disc impregnated with 0.1 mM H2O2 was placed at the
intersection, and the plate was incubated overnight at 37 °C. As
seen in Fig. 6A, the
uvrY and the barA mutant strains were more
sensitive to hydrogen peroxide than the wild type strain. The radius of
the zone of growth inhibition of AKP023 and AKP014, respectively, was
1.9 and 1.6 times larger than that of the wild type strain (Fig.
6A). The hydrogen peroxide sensitivity of the barA and uvrY mutant strains was further analyzed
by measuring cell viability during hydrogen peroxide challenge. AKP023,
AKP014, and MC4100 were cultured in LB, and at mid-exponential growth (A600 ~ 0.2) the three cultures were
challenged with 1 mM H2O2, as
described under "Experimental Procedures." Fig. 6B shows
that both uvrY and barA mutant strains were much
more sensitive to hydrogen peroxide than the wild type strain (Fig.
6B). It is worth noting that the uvrY mutant
showed a more severe survival defect than the barA mutant
strain, in accordance with the agar diffusion assay. Thus, both the
BarA and the UvrY gene products are needed for full protection
against hydrogen peroxide-mediated stress, consistent with the
conclusion that the two proteins belong to the same two-component
system.
The experiments presented in this study identify the UvrY protein
as the cognate response regulator for the BarA sensor kinase in
E. coli, based on the following biochemical findings. First, purified BarA protein autophosphorylates when incubated with
[ These results have a bearing on a whole family of analogous
two-component systems, including the ExpS/ExpA of E. carotovora and the GacS/GacA of Pseudomonas
sp. (Table I), as the links between the sensor and regulator components
in these cases have only been defined genetically. Other members of
this group of highly conserved proteins are the response regulators
SirA of S. typhimurium and VarA of V. cholerae
(Fig. 2). All response regulators of this family share not only
considerable sequence homology (Fig. 2) but also genomic organization;
first, the regulator genes are not linked with the sensor gene on the
chromosome, and second, the regulator genes are directly followed by
the uvrC ORF on a bicistronic transcript such that the two
ORFs overlap for a short stretch. UvrC encodes a subunit of the UvrABC
enzyme complex, which is involved in DNA repair (23). However,
mutations in expA and uvrY have no specific
effect on UV-induced DNA repair (15, 19). Nonetheless, most of those
regulators, with the exception of UvrY the function of which has not
yet been discovered, are known to be involved in the virulent life
style of the respective bacteria (Table
I). ExpA and GacA of E. carotovora and Pseudomonas sp., respectively,
are key regulators for secreted proteases, toxins, and other
extracellular compounds that are required for the pathogenic nature of
these bacteria. The relationship between the regulators is also
underlined by the fact that an expA mutation in E. carotovora can be complemented with the E. coli uvrY
gene (19). Interestingly, it was recently reported that GacA regulates some of the genes under its control at the translational level; this
was demonstrated by drastic changes in GacA-dependent
expression upon changes of the ribosomal binding site of these
mRNAs (32). The involvement of GacA in the process of translation,
however, is most likely to be exerted indirectly by controlling the
expression of a transcriptional regulator. The varA gene
product of V. cholerae regulates transcription of the major
subunit of the toxin-coregulated pilus and the production of cholera
toxin. Mutations in varA lead to a lack of autoagglutination
and a decreased virulence in an infant mouse model (22). The
SirA gene product of S. typhimurium, which shows a
96% identity to UvrY of E. coli, regulates the expression of hilA, which in turn controls virulence genes on
pathogenicity islands (Table I). It is noteworthy that a S. typhimurium sirA mutant has been shown to be avirulent in a bovine
intestinal infection model (33). The recently released
Salmonella genome sequence revealed a close homologue
to the E. coli barA gene. Intriguingly, a recent report also
demonstrated that a mutation in the BarA homologue of
Salmonella decreased expression of the
hilA-controlled invF-lacZ and
prgH-lacZ fusions, thus supporting a conclusion that
Salmonella BarA and SirA proteins are involved in the same signal transduction pathway (34).
-32P]ATP but not with
[
-32P]ATP or [
-32P]GTP.
Phosphorylated BarA, in turn, acts as an efficient phosphoryl group
donor to UvrY but not to the non-cognate response regulators ArcA,
PhoB, or CpxR. The specificity of the transphosphorylation reaction is
further supported by the fact that UvrY can receive the phosphoryl
group from BarA-P but not from the non-cognate tripartite sensor ArcB-P
or ATP. In addition, genetic evidence that BarA and UvrY mediate the
same signal transduction pathway is provided by the finding that both
uvrY and barA mutant strains exhibit the same
hydrogen peroxide hypersensitive phenotype. These results provide the
first biochemical evidence as well as genetic support for a link
between BarA and UvrY, suggesting that the two proteins constitute a
new two-component system for gene regulation in Escherichia
coli.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His
Asp
His
Asp phosphorelay (5-7) as well as the
dephosphorylation of the phospho-response regulator by a reverse
Asp
His
Asp
Pi phosphorelay (8). In addition, a SixA
(signal inhibitory factor X) protein was suggested to be able to
specifically catalyze the dephosphorylation of the histidine-P of the
phosphotransfer domain of ArcB (9). Furthermore, intracellular
metabolic intermediates have been reported to enhance the rate of
autophosphorylation of the ArcB sensor kinase (10). This complex mode
of action is believed to provide multiple levels of control that play
important roles in the "fine tuning" of such a system.
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Fig. 1.
Schematic representation of BarA and
UvrY. Upper panel, the BarA sensor kinase. The
N-terminal transmembrane domain was predicted based on a hydrophobicity
plot analysis. The primary transmitter domain is shown with the
conserved His-302 and the catalytic determinants N, D/F, and G. The
G sequence typifies the nucleotide-binding motif. The receiver
domain is shown with the conserved Asp-718, and the phosphotransfer
domain is shown with the conserved His-861. Lower panel,
UvrY is shown with its N-terminal receiver domain containing the
conserved Asp-54 and its C-terminal helix-turn-helix domain (HTH
domain).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pir to MC4100 (nalidixic acid-resistant), and bacteria
were selected with nalidixic acid (20 µg/ml) and kanamycin (50 µg/ml). Co-integrates were further selected on LB plates containing 5% sucrose and kanamycin (100 µg/ml). Resolution products were subsequently selected for growth on plates containing kanamycin and
inability to grow on plates containing ampicillin. Western blot was
used to confirm the absence of the BarA protein. Strain CS4923
(uvrY::cm) (15) was kindly provided by Dr. G
Moolenaar. Both barA and uvrY mutants were
reintroduced into MC4100 using P1-vir lysates to create AKP014 and
AKP023, respectively, and confirmed genotypically by Southern blotting
and PCR. Growth was followed by measuring turbidity at
A590 in Luria broth and M9 minimal medium with
0.2% (w/v) glucose without any antibiotics.
-D-thiogalactopyranoside. Cultures were harvested after 5 h of induction. Protein
purification was performed at 4 °C under nondenaturing conditions,
as described in protocol 5 for native purification of cytoplasmic
proteins in the QIAexpressionist manual (Qiagen). Purification was
based on affinity chromatography using the chelate absorbent
Ni-nitrilotriacetic acid resin that interacts with the 6xHis tag. The
proteins were eluted by imidazole, which was subsequently removed by
dialysis (16). Following dialysis, the proteins were concentrated in Centricon 10 units (Amicon) and stored at
20 °C. The Coomassie Plus protein assay reagent (Pierce) was employed to estimate protein concentrations, using bovine serum albumin as a standard. SDS-PAGE of
the purified proteins revealed that each had the expected molecular weight and that the preparations were essentially homogenous (data not
shown). PhoB, a kind gift of M. Prahalad and C. T. Walsh (Harvard Medical School), was prepared as described (18).
-32P]ATP (specific activity 2 Ci/mmol, PerkinElmer
Life Sciences), 33 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, and 10%
glycerol. The reactions were initiated by the addition of
[
-32P]ATP, terminated by the addition of an equal
volume of 2× SDS sample buffer, and immediately subjected to SDS-PAGE
on 15% gels. In time course experiments, the samples were mixed with
the SDS sample buffer and kept on ice until the last portion was taken. The radioactivity of proteins resolved in the gels was determined qualitatively by autoradiography of the dried gels with X-Omat AR
(Kodak). A PhosphorImager (Molecular Dynamics) was used for quantitative analyses.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
A, sequence alignment of: a,
E. coli UvrY; b, S. typhimurium SirA; c, E. carotovora ExpA;
d, V. cholerae VarA; and e, P. aeruginosa GacA; regulator proteins and the homologous ORFs of:
f, K. pneumoniae contig 1572 (incomplete
sequence); g, Y. pestis contig 669; and
h, S. putrefaciens contig 5557. The receiver
domain, amino acids 1-99, contains the catalytic site
(DD) and the site of phosphorylation
(D). The helix-turn-helix motif suggested to mediate DNA
binding is shown in bold at position 169-187.
Asterisks indicate conserved residues. B,
percentage of identity between the listed regulator proteins.
-32P]ATP, and the time course of the
reaction was followed. The protein was rapidly phosphorylated (the
phosphorylated species reached a maximum within a few minutes) (Fig.
3). This finding is in agreement with the results of a previous study using BarA associated with everted
membrane vesicles (11). When the protein was incubated with
[
-32P]ATP or [
-32P]GTP, no labeling
occurred (data not shown), demonstrating that BarA is an
ATP-dependent kinase.
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Fig. 3.
Testing the autophosphorylation activity of
BarA. Purified 'BarA (~100 pmol) was incubated with
[ -32P]ATP in a total volume of 40 µl. At the
indicated times, a 5-µl sample was withdrawn for subsequent SDS-PAGE
analysis and quantitation by PhosphorImager. A, an
autoradiogram of the protein bands in the gel. B, the time
course of autophosphorylation of the purified protein.
-32P]ATP for 10 min to form a pool of
'BarA-P, and purified His6-UvrY (hereafter referred to as UvrY) was
added to the reaction mixture (Fig. 4). A
kinetic analysis revealed that during the initial period of the
reaction, UvrY rapidly became labeled, whereas 'BarA-P rapidly lost the
label. Subsequently, the level of both UvrY-P and 'BarA-P increased
slowly during the course of the experiment. This result was expected,
because excess [
-32P]ATP was not separated from the
reaction mixture. However, at the end of the reaction, the level of
'BarA-P declined, an event indicative of autophosphatase activity
harbored by many tripartite sensor kinases (8, 30).
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Fig. 4.
Transphosphorylation of UvrY by BarA.
Purified 'BarA (100 pmol) was preincubated with
[ -32P]ATP for 45 min in a 40-µl reaction mixture to
generate a pool of 'BarA-P. At time zero, 2 µl of UvrY (100 pmol/ml)
was added, and 5-µl samples were withdrawn at the indicated time
intervals for analysis. A, autoradiogram of the dried gel.
B, the relative amount of 'BarA-P and UvrY-P as quantitated
by PhosphorImager analysis.
-32P]ATP as the phosphoryl group donor (Fig.
5A). However, in the presence
of 'BarA and [
-32P]ATP, UvrY become clearly labeled,
indicating that its transphosphorylation is catalyzed by 'BarA. The
possibility of 'BarA acting as a phosphoryl group donor for non-cognate
response regulators, a concept documented as cross-talk (31), was then
tested. Equimolar amounts of purified ArcA, PhoB, and CpxR were each
incubated with 'BarA in the presence of [
-32P]ATP.
'BarA was heavily labeled but failed to transphosphorylate any of the
tested non-cognate response regulators (Fig. 5A).
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Fig. 5.
Testing the specificity of the
transphosphorylation reaction. The indicated proteins or protein
combinations were incubated in 5-µl reaction mixtures with
[ -32P]ATP. After 5 min, the reactions were terminated
by the addition of 2× SDS sample buffer and subjected to SDS-PAGE on
15% gels. The protein concentrations (in pmol) were 50 'BarA, 100 UvrY, 100 ArcA, 100 PhoB, 100 CpxR, and 50 ArcB. A,
comparison of the ability of 'BarA to phosphorylate the UvrY, ArcA,
PhoB, and CpxR proteins. B, the ability of UvrY to receive
the phosphoryl group from the ArcB sensor kinase.
,
'-dipyridyl at 0.2 mM,
and the bacterial growth rate was monitored. Neither AKP023 nor the
AKP014 mutant strain showed any significant growth defect as compared
with the wild type strain (data not shown).
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Fig. 6.
Mutations in the barA and
uvrY genes lead to hydrogen peroxide
hypersensitivity. A, agar diffusion assay. An LB-agar
plate was divided into three equal sectors, where ~103
cells of MC4100 (wild type), AKP014 (barA::km),
and AKP023 (uvrY::cm) were spread, and a filter
paper disc impregnated with 0.1 mM
H2O2 was placed at the intersection. The plate
was incubated overnight at 37 °C, and the zone of growth inhibition
was recorded. B, cell survival assay: squares,
MC4100 (wild type), diamonds, AKP014
(barA::kan), and circles, AKP023
(uvrY::cm). Mid-exponentially growing cells were
divided into two equal portions, and one of the portions was challenged
with 1.0 mM hydrogen peroxide. Aliquots were removed at 10, 20, 40, and 60 min and plated out on LB-agar plates for measurement of
cell viability. The relative survival of the culture was determined by
comparing the viability of the treated cultures with that of the
untreated ones.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP but not with [
-32P]ATP or
[
-32P]GTP, and 'BarA-P acts as a phosphoryl group
donor to UvrY. Second, UvrY can receive the phosphoryl group from
BarA-P but not from the non-cognate sensor ArcB-P or ATP. Third, BarA
catalyzes the phosphorylation of UvrY but not the phosphorylation of
the non-cognate response regulators ArcA, PhoB, or CpxR. The ability to
demonstrate phosphotransfer with purified components also suggests that
there are no intermediate proteins involved in the signal transduction. Genetic evidence that BarA and UvrY mediate the same signal
transduction pathway is provided by the finding that both the
uvrY and the barA mutant strains exhibit a
hydrogen peroxide hypersensitive phenotype.
Genes and phenotypes regulated by UvrY, SirA, ExpA, VarA, and GacA
No phenotype has, so far, been associated with the E. coli UvrY protein, whereas BarA has recently been reported to protect E. coli against hydrogen peroxide-mediated stress. In this work (13), it was shown that BarA is involved in the induction of the sigma factor RpoS, which, in turn, activates expression of both HPI and HPII (Ref. 35 and references therein). Our present finding that inactivation of the uvrY gene results in the same hydrogen peroxide hypersensitive phenotype (Fig. 6) provides genetic evidence for a functional link between the BarA and UvrY proteins. Together with the phylogenetic and biochemical data presented, we can also conclude that the two proteins constitute a two-component system.
BarA has also been suggested to be important for the acquisition of iron via siderophores in the uropathogenic E. coli strain DS17 (12). However, our results demonstrate that a barA mutant of E. coli strain MC4100 grew equally well as the wild type strain in iron-limiting media, suggesting that the previously demonstrated growth defect of a barA mutant of E. coli strain DS17 (12) is strain-specific. It is noteworthy that GacA, the response regulator of the homologue two-component system of Pseudomonas sp., positively regulates the formation of siderophores in the phytopathogens Pseudomonas viridiflava (36) and Pseudomonas marginalis (36).
Physical attachment of bacteria to host cells via P-pili has
been suggested to induce expression of BarA (12), but as not all
E. coli strains express pyelonephritis-pili, other stimuli are also likely to act on BarA. The fact that genes involved in invasion and type III secretion in S. typhimurium are under
the control of the SirA response regulator (33) agrees well with a
model in which a cognate sensor is stimulated by contact with the host
cell. Although, several attempts to identify the environmental signals
that activate the GacS-GacA and ExpS-ExpA two-component were without
success (37), it seems reasonable to presume that similar
contact-dependent stimulation of the sensor kinase may apply generally to all kinases of this family. Studies directed toward
identifying specific genes under the control of the BarA/UvrY two-component system in different E. coli isolates and
elucidating the nature of the signal sensed by the BarA sensor kinase
are yet to be undertaken.
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ACKNOWLEDGEMENTS |
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We are grateful to E. C. C. Lin, S. Normark, B. Michel, O. Kwon, and C. A. Lee for discussions.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM-40993 from the NIGMS, National Institutes of Health and by grants from the Swedish Natural Science Research Council (NFR).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1926; Fax: 617-738-7664; E-mail: dimitris_georgellis@hms.harvard.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M001550200
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis.
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