The unorthodox histidine kinases BvgS and EvgS are responsiveto the oxidation status of a quinone electron carrier Andreas Bock and Roy Gross Lehrstuhl fu¨r Mikrobiologie, Biozentrum, Un
Trang 1The unorthodox histidine kinases BvgS and EvgS are responsive
to the oxidation status of a quinone electron carrier
Andreas Bock and Roy Gross
Lehrstuhl fu¨r Mikrobiologie, Biozentrum, Universita¨t Wu¨rzburg, Am Hubland, Wu¨rzburg, Germany
The purified soluble forms of the histidine kinases BvgS and
EvgS of Bordetella pertussis and Escherichia coli,
respect-ively, are shown to be responsive to oxidized ubiquinone-0
(Q-0) in vitro The oxidized ubiquinone is a strong inhibitor
of kinase activity of both enzymes with half maximal
inhi-bition occurring at 11 lM(BvgS) and 4 lM(EvgS) Reduced
Q-0 has no effect on the histidine kinases Kinase activity can
reversibly be switched off and on by changing the oxidation
status of the quinone This inhibitory effect is due to a
decrease of the kinase activity of BvgS rather than an
increase of intrinsic phosphatase activities Other electron
carriers such as menadione (MK-3), NAD or FAD did not
have a significant effect on the kinase activities of BvgS and EvgS Nicotinic acid and sulfate ions, known to inhibit the histidine kinases in vivo, did not affect the purified truncated sensor proteins lacking their periplasmic domains in vitro Mutations introduced by site-directed mutagenesis into the putative PAS domain of BvgS caused a weak decrease of qui-none-dependent inhibition of autophosphorylation These data suggest that BvgS and EvgS are connected with the oxi-dation status of the cell via the link to the ubiquinone pool Keywords: BvgS; EvgS; histidine kinase; signal transduction; PAS domain; phosphorelay; ubiquinone
Two-component signal transduction systems are
wide-spread among prokaryotes, but they are also found in
eukaryotic organisms such as fungi and plants [1,2] The
BvgAS two-component system regulates the virulence
properties of Bordetella pertussis, the etiological agent of
whooping cough, and of close relatives such as B
parap-ertussisand B bronchiseptica that cause similar respiratory
diseases in man and animals, respectively [3,4] It controls
expression of a huge virulence regulon including the genes
encoding the pertussis and adenylate cyclase toxins and
several adhesins Much less is known about the EvgAS
system of Escherichia coli, but it appears to be involved in
the transcriptional regulation of drug efflux pumps [5] The
two systems are highly related in their primary structure and
domain composition The histidine kinases BvgS and EvgS
belong to the small group of the so-called unorthodox
sensor proteins because they harbour an intermediate
receiver and at the C-terminus a histidine-containing
phosphotransfer (HPt) domain in addition to the classical
transmitter domain [6–9] Under permissive conditions, the
BvgS and EvgS histidine kinases autophosphorylate at a
conserved histidine residue in their transmitter domains
Autophosphorylation is followed by an obligate
intramo-lecular phosphorelay to an aspartic acid in their receiver and
a histidine in their HPt domains Finally, the phosphate
from the HPt domain is transferred with high specificity to
an aspartic acid in the cognate response regulators BvgA
and EvgA, respectively [7,10] Both sensor proteins have large periplasmic domains which were believed to be the major signal input sites Very little is known so far about the relevant signals perceived by these proteins, although temperature and compounds such as nicotinic acid and sulfate ions were recognized to modulate kinase activity
in vivo[9,11] However, attempts to identify specific binding
of modulating compounds to B pertussis cells remained inconclusive [12]
Recently, the presence of a PAS domain in BvgS was predicted; it is located in the cytoplasmic portion between the transmembrane and transmitter domains [13] PAS is an acronym formed from the names of the proteins in which imperfect repeat sequences (PAS domains) were first identified, i.e the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM) [13] PAS domains are signalling domains that function as input modules able to perceive oxygen, redox potential, light, and some other stimuli [13] The part of the BvgS protein containing the putative PAS domain was previously called linker region and several mutations have been identified within this domain which led either to constitutive kinase activity or to an inactivation of the protein [14–17] This suggests that the BvgS protein may perceive intracellular signals characteristic for PAS domains such as the energy status of the cell or the oxygen concentration In this respect
it is interesting to note that the BvgAS system does not only control expression of virulence related functions but also several housekeeping genes including cytochrome c and cytochrome d oxidases of the respiratory chain and several putative dehydrogenases [4,18,19] Remarkably, in contrast
to the virulence genes, many of these metabolic functions are negatively controlled by the BvgAS system
Several properties of the BvgAS regulon are similar to those of another unorthodox two-component system, the ArcAB system of E coli [20,21] The unorthodox histidine
Correspondence to R Gross, Lehrstuhl fu¨r Mikrobiologie,
Biozen-trum, Universita¨t Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg,
Germany Fax: + 49 931 888 4402, Tel.: + 49 931 888 4403,
E-mail: roy@biozentrum.uni-wuerzburg.de
Abbreviations: HPt, histidine-containing phosphotransfer;
Q-0, ubiquinone-0; Q-8, ubiquinone-8; MK-8, menaquinone-8.
(Received 15 February 2002, revised 7 May 2002,
accepted 30 May 2002)
Trang 2kinase ArcB together with the response regulator ArcA
regulates the expression of many genes which are involved
in the adaptation of the bacteria during the switch between
aerobic and anaerobic growth conditions The ArcAB
regulon comprises factors involved in electron transport, the
citrate cycle, the glyoxylate shunt and lipid metabolism [21]
Recently, it was shown that the oxidized form of quinones
such as ubiquinone and menadione (MK-3) specifically
interfere with the kinase activity and switch the enzyme off,
whereas the reduced quinones remained without effect [22]
Interestingly, the unorthodox ArcB protein shows a domain
structure that is very similar to those of the BvgS and EvgS
proteins, but it lacks the extensive periplasmic domain
Because of to their striking structural similarities to ArcB
and to the genes regulated by these two-component systems,
in the present study we investigated whether quinones may
also be a relevant signal for the BvgS and EvgS histidine
kinases
M A T E R I A L S A N D M E T H O D S
Phosphorylation assays
Over-expression and purification of the histidine kinases
BvgS and EvgS was described recently [7] Briefly, both
proteins were expressed with an N-terminal His-tag which is
fused in front of the putative PAS domains of the two
proteins thus substituting for the transmembrane and
periplasmic domains [7] Prior to their analysis the protein
solutions were dialysed to remove dithiothreitol and
phenylmethanesulfonyl fluoride from the reaction
mix-tures The phosphorylation assays were carried out as
described previously using [c-32P]ATP (specific activity
> 5000 CiÆmmol)1) [23,24] The purified proteins were
incubated at concentrations of 1 lM(BvgS and EvgS) or
between 4.7 and 7.1 lM(BvgS-H643A, BvgS-Del, BvgS-C4)
at room temperature in reaction buffer containing 50 mM
Tris pH 7.5, 50 mMKCl, 10 mMMgCl2, 50 nM[c-32P]ATP,
0.2 mM ATP Reactions were stopped by the addition of
sample buffer (60 mMTris pH 6.8, 10% glycerol, 2% SDS,
5% 2-mercaptoethanol, 0.05% Bromophenol blue) After
separation of the phosphorylated proteins by SDS/PAGE
[25] the gels were dried at 80C for 90 min and exposed for
several hours at room temperature Quantitative
densitom-etry was performed using a PhosphorImager (Molecular
Dynamics) according to standard protocols Relative
phos-phorylation values were calculated with respect to the
strongest signal on a gel set as 100%
To investigate the effect of inhibitory compounds, the
kinases were incubated for 2.5 min in the absence or
presence of increasing concentrations of ubiquinone-0 (Q-0)
(1 lM to 5 mM), MK-3 (1 lM to 1 mM), oxidized and
reduced FAD and NAD (1 lM to 5 mM), nicotinic acid
(1 mMto 10 mM) or MgSO4(1 lMto 50 mM) In the case of
menadione, the reaction mixtures contained 15% (v/v)
ethanol, because MK-3 was dissolved in ethanol as a stock
solution All compounds were obtained from Sigma
For the determination of the effect of Q-0 on the
autophosphorylation kinetics of BvgS and EvgS the kinases
were incubated for 15 s, 30 s, 1 min, 2 min or 4 min before
the addition of sample buffer Reaction mixtures contained
20 lMQ-0 and/or 500 lMdithionite (sodium hydrosulfite)
For experiments investigating the reversibility of the
quinone effect on BvgS, the kinase was first incubated in the presence of Q-0 (5 mM) Then, after an additional 4.5 min, Q-0 was reduced by the addition of 25 mM
dithionite
For characterization of the autodephosphorylation of BvgS, the kinase was first incubated for 5 min under standard conditions in the presence of [c-32P]ATP as described above Then 5 mMQ-0 and/or 16 mM nonradio-active ATP were added After the addition of Q-0 and/or ATP samples were taken immediately and after 0.5, 1, 2, 4,
8, 16 and 32 min; the reaction was stopped by the addition
of sample buffer The half-life of phosphorylated BvgS was determined as described previously [24]
All experiments were performed at least three times independently Statistical analysis was performed using MicrosoftEXCEL97 andSIGMAPLOTfor Windows, version 5.00
Site-directed mutagenesis The BvgS mutants were constructed using the Quik-ChangeTM site-directed mutagenesis kit (Stratagene) In the case of BvgS-C4 a previously described constitutive BvgS mutant was reconstructed [16] In the case of BvgS-H and BvgS-Del the putative quinone binding site in the BvgS PAS domain was the target for mutagenesis [26] The previously described plasmid pQE-BvgS-TRO [7] was used as a template and the following oligonucleotides were synthes-ized for mutagenesis.: For the construction of the mutant bvgSalleles the oligonucleotides bvgS-C4-a (5¢-GCTGGG CGAACTCAAGAGCATCATCGGCGG-3¢) and bvgS-C4-b (5¢-CCAGCCGCCGATGATGCTCTTGAGTTCG CC-3¢), bvgS-Qmut1 (5¢-GCCCGCGAAATGGCCGAG TTCCTGCTC-3¢) and bvgS-Qmut2 (5¢-GAGCAGGAAC TCGGCCATTTCGCGGGC-3¢) and bvgS-Del-a (5¢-GA CCCGGCGCTGACGCGCGTGGCC-3¢) and bvgS-Del-b
used All proteins used in this study are shown schematically
in Fig 1
Hpt
R T PAS TM
BvgS* C
BvgS-H643A C
H643A F583
BvgS-C4 C
G688S
BvgS-Del C
∆ 639-647 >
Fig 1 Structural organization of BvgS derivatives used in this study.
On the top the domain structure of the wild-type BvgS protein is shown BvgS* shows the organization of the soluble truncated BvgS derivative referred to as control protein throughout this study The positions of the mutations introduced in the three BvgS derivatives BvgS-H643A, BvgS-Del and BvgS-C4 are shown TM, transmem-brane domain; PAS, PAS domain; T, transmitter; R, receiver; Hpt, histidine-containing phosphotransfer domain.
Trang 3R E S U L T S
Effect of quinones on the activity of BvgS and EvgS
In the genus Bordetella, ubiquinone-8 (Q-8) is the
predom-inant quinone [27], whereas the major quinones synthesized
by E coli are Q-8, menaquinone-8 (MK-8) and
demethyl-menaquinone-8 [28] The incubation of the BvgS and EvgS
histidine kinases with the soluble Q-8 analogue Q-0 resulted
in a strong inhibition of autophosphorylation of both
enzymes (Fig 2) In fact, half maximal inhibition of BvgS
and EvgS occurred at about 11 lMand 4 lM, respectively
(Fig 3) Reduction of the quinone by dithionite entirely
abolished its negative effect on BvgS and EvgS (Fig 2)
Whereas the autophosphorylation activity of EvgS could be
inhibited completely by the addition of 100 lMQ-0, BvgS
activity could not be entirely abrogated by Q-0 reaching a
minimal basal activity of 10% (Fig 3) The soluble MK-8
analogue MK-3 did not affect the kinase activities of BvgS
and EvgS, which contrasts with the results reported
previously for the ArcB histidine kinase of E coli (data
not shown) The effect of Q-0 on the enzyme activities of the
histidine kinases is probably reversible because
autophos-phorylation of BvgS could be reactivated when oxidized
Q-0 had been reduced by addition of dithionite (Fig 4),
although we can not entirely exclude a Q-0 independent
action of dithionite on the autokinase activity of BvgS
Ubiquinone acts on the histidine kinase
but not on intrinsic phosphatase activities
The negative effect on autophosphorylation observed after
addition of the oxidized ubiquinone to the histidine kinases
may either be the consequence of a decrease in the histidine
kinase activity, or, alternatively, of an increase in an intrinsic
autophosphatase activity present in the BvgS and EvgS
proteins [7,10] To investigate these two alternatives we
characterized the dephosphorylation kinetics of BvgS-P
The half-life of phosphorylated BvgS under the
experimen-tal conditions was estimated to be 12.3 min As shown in
Fig 5, the addition of oxidized Q-0 did not cause an increase in the dephosphorylation of BvgS-P, in contrast, a slight increase in the stability of the phosphoprotein could
be noted Moreover, no significant change in the stability of BvgA-P could be observed in the presence of BvgS and oxidized Q-0 (data not shown) These data suggest that in the case of BvgS probably its kinase activity but not its intrinsic phosphatases are affected by the presence of the quinone
Effect of FAD, NAD or modulating agents
on the BvgS histidine kinase activity
To investigate whether other electron carriers abundant in the cell have an impact on the activity of the BvgS and EvgS proteins, we added up to 5 mMFAD or 5 mMNAD to the reaction mixtures FAD was shown previously to bind to the PAS domain of the membrane signal transducer protein Aer of E coli [29] However, using physiologically relevant concentrations neither coenzyme had any significant effect
on the autophosphorylation activity of the two histidine kinases independent of their oxidation status (data not shown) Moreover, nicotinic acid and sulfate ions (which have long been known to inhibit BvgS and EvgS) at in vivo relevant concentrations did not influence the autophospho-rylation properties of the purified truncated BvgS and EvgS kinases in vitro (data not shown)
Effects of mutations in the BvgS PAS domain
on quinone sensing Similar to the ArcB histidine kinase of E coli, the presence of
a PAS domain was recently predicted in front of the transmitter domain of the BvgS protein [13] Similarly, the EvgS protein contains a sequence with characteristics of a PAS domain between its transmembrane and transmitter domains (data not shown) Previously, this part of the BvgS protein was termed linker and its relevance for BvgS function was recognized because mutations in this region were isolated which caused either an inactivation of the protein
time (min)
0 20 40 60 80 100 120
BvgS +Dt BvgS +Q-0 BvgS +Q-0 +Dt
+Q-0 +Dt +Q-0 +Dt none
A
0.25 0.50 1 2 4 (min)
+Q-0 +Dt +Q-0 +Dt none 0.25 0.50 1 2 4 (min)
time (min)
0 20 40 60 80 100 120
EvgS +Dt EvgS +Q-0 EvgS +Q-0 +Dt B
Fig 2 Effect of (Q-0) on the rate of
auto-phosphorylation of the BvgS (A) and EvgS (B)
histidine kinases The purified proteins were
incubated with [c-32P]ATP in the presence or
absence of Q-0 and/or dithionite On the
bottom panel autoradiograms of
representa-tive SDS/PAGE are shown In the top panel,
the increase with time of BvgS-P and EvgS-P
in the presence or absence of Q-0 and/or
dithionite is shown For this presentation the
relative amount of the phosphorylated
histi-dine kinases was determined by
Phosphor-Image analysis of the respective gels shown in
the bottom panel The maximal
phosphory-lation levels of BvgS and EvgS in the absence
of Q-0 and/or dithionite were taken as 100%
which correspond to P/protein values of
28.5% and 15.5%, respectively.
Trang 4(mutations in the PAS core domain) or a lack of response to
modulating compounds such as temperature, nicotinic acid
or sulfate ions (mutations in the b-scaffold domain or the
helical connector) [14–17] Interestingly, in the so-called
helical connector of the BvgS PAS domain a sequence with
similarities to a recently described quinone binding motif is
present consisting of a weak structural element of a triad of
residues [aliphatic-(X)3-H-(X)2,3-(L/T/S)] with the central
histidine residue on one side of an a-helical stretch [26] To investigate the functional relevance of the PAS domain for quinone sensing we generated three BvgS derivatives by site-directed mutagenesis carrying mutations either in the putative quinone binding motif (BvgS-H643A, BvgS-Del)
or containing a previously described mutation in the b-scaffold part of the PAS domain (BvgS-C4) (Fig 1) The latter mutation was found after a screen for B pertussis mutants exhibiting a constitutive phenotype, i.e not responding anymore to modulating compounds in vivo [16] However, all three mutations caused only mild effects
on quinone sensing (Fig 3) The most prominent effect was observed with the mutant BvgS-Del protein lacking the entire putative quinone binding site Even at Q-0 concen-trations > 100 lM the autophosphorylation activity of BvgS-Del could only be inhibited to 20% of its maximal activity, whereas, for comparison, the activity of the control protein BvgS decreased to 10% under the same conditions (Fig 3)
D I S C U S S I O N The data presented here provide strong evidence for a direct perception of redox signals by the BvgAS and EvgAS two-component signal transduction systems, as both sensory histidine kinases strongly respond to the presence of oxidized but not reduced ubiquinone (Q-0) The fact that menadione had no effect on BvgS is not surprising, because
in contrast with the situation in E coli MK-8 does not play
a role in B pertussis [27] However, in E coli ArcB
time (min)
20 30 40 50 60 70 80 90
+Q-0
-Q-0
Fig 5 Autodephosphorylation of BvgS in the presence or absence of Q-0 BvgS was phosphorylated by the addition of [c-32P]ATP After 5 min Q-0 (final concentration 5 m M ) and/or a 75-fold excess of nonradioactive ATP was added (time point 0 in the figure) At the indicated time points samples were taken and analysed On the x-axis time is shown in minutes; on the y-axis the log of the relative phosphorylation level at the various time points is shown On the right the autoradiographies of representative gels are shown.
BvgS-H643A
BvgS-Del BvgS-C4 BvgS EvgS
Q-0 [µM]
0 20 40 60 80 100
120
BvgS BvgS-H643A BvgS-Del BvgS-C4 EvgS
Fig 3 Inhibition of autophosphorylation of BvgS, EvgS and of mutated BvgS derivatives (BvgS-H643A, BvgS-Del, BvgS-C4) in the presence of different Q-0concentrations After SDS/PAGE analysis the relative amount of the phosphorylated histidine kinases was determined by Phos-phorImage analysis The figure shows representative autoradiographs of the samples after SDS/PAGE (right panel) and the results of their quantification by the PhosphorImager (left panel).
time (min)
0
20
40
60
80
100
addition of Dt
BvgS
Fig 4 Dependence of the kinase activity of BvgS on the redox state of
ubiquinone-0(Q-0) BvgS was incubated with [c-32P]ATP in the
pres-ence of 5 m M Q-0 for 4.5 min Then dithionite was added to a final
concentration of 25 m M The insert shows the autoradiography of a
representative gel.
Trang 5autophosphorylation was inhibited by both oxidized
qui-nones [22], whereas EvgS was found to be responsive only to
Q-0 but not to MK-3 This differential behaviour of the two
histidine kinases of E coli may indicate interesting
differ-ences in the fine tuning of their activity under different
growth conditions For example, it is known that high
aeration and logarithmic growth of the bacteria leads to a
predominance of Q-8, whereas under anaerobic conditions
and in stationary phase MK-8 is the predominant quinone
[30] In contrast with the ArcB protein, the BvgS and EvgS
proteins contain extensive periplasmic domains This
indi-cates that via these domains both proteins may also be
engaged in the perception of additional stimuli possibly
derived from the environment such as different modulating
agents (e.g sulfate ions and nicotinic acid) In fact, the data
presented here demonstrate that such compounds do not
interfere directly with the cytoplasmic portion of the BvgS
histidine kinase
As already pointed out by Georgellis and coworkers [22],
the quinones are membrane localized electron transporters
which are interesting candidates as redox indicators for
sensor proteins such as ArcB, BvgS and EvgS As the
quinones are the only components of the respiratory chain
which apparently are free in their movement within the
membrane, they may easily come into close contact with
membrane anchored sensor proteins It is not yet known
which parts of the senor proteins interact with the quinones,
although this interaction must involve the cytoplasmic
domains present in the truncated proteins used for these
studies The PAS domains, which have previously been
proposed to be localized in the ArcB and BvgS linker
regions close to the transmembrane domains, appear to be
suitable devices for such quinone interaction sites [13] PAS
domains are known to monitor changes in redox potential,
oxygen, the overall level of energy of a cell, and it was
recently shown that they can bind small ligands such as
FAD and ATP [13,29,31] In fact, mutations in this domain
have been reported which cause either the inactivation of the
BvgS protein or its constitutive activity under modulating
conditions induced by temperature, nicotinic acid or sulfate
ions [14–17] Interestingly, the BvgS PAS domain contains a
sequence motif that comprises essential features of a recently
described quinone binding motif [26] However, mutations
in this motif or in the b-scaffold domain caused only
relatively mild effects on quinone sensing by BvgS This
indicates that the sequence motifs described here may
contribute to quinone sensing but there may be additional
quinone interaction sites In fact, in the case of BvgS a
second putative quinone binding motif is predicted in the
transmitter domain centred around the
autophosphoryla-tion site at His729 (data not shown) Interestingly, the ArcB
orthologue of Haemophilus influenzae lacks a PAS domain,
but nevertheless can complement an E coli arcB mutant to
the wild-type phenotype [32] Therefore, the function of the
PAS domains of these proteins in quinone sensing remains
obscure It is interesting to note that the PAS domain of the
phosphorelay histidine kinase A of Bacillus subtilis was
recently shown to be a catalytic ATP-binding domain [31]
As the PAS domain of BvgS contains a putative ATP
binding site and mutations in this motif were previously
shown to inactivate the protein [14] it is likely that there are
alternative intracellular signals which are perceived by BvgS
via its PAS domain
It is not known yet which stimuli are relevant for BvgS mediated regulation of virulence genes during infection [3,4] Due to the lack of relevant changes of the virulence properties of bvgS mutants not responsive anymore to modulating compounds such as nicotinic acid and sulfate ions in infection experiments it was recently suggested that BvgS-mediated modulation of the virulence regulon may not be relevant at least in the respective animal models [33] Because such a constitutive BvgS derivative (BvgS-C4) insensitive to modulating compounds is shown here to exhibit biochemical properties very similar to those of the control protein BvgS with regard to its responsiveness to oxidized quinones, it may well be that in the previously reported infection experiments modulation of BvgS activity
by relevant intracellular stimuli still occurred Therefore, in the future for constitutive bvgS mutants that are not responsive to quinones it will be necessary to investigate whether modulation of BvgS activity is relevant for the infectious process
Quinone sensing by BvgS brings our attention to the BvgAS regulated housekeeping genes, the characterization
of which has been largely neglected as compared with that
of the virulence genes [19] In fact, sensing of redox signals
by the BvgS protein provides an interesting link to the previously reported observations that cytochrome d and c oxidases are regulated in a reciprocal manner by the BvgAS system [4,18] and may have important implications for the scenario of virulence evolution in the genus Bordetella The strong link of the master regulator of virulence to basic metabolism of the bacteria suggests that this system may have already been used by an ancestor of the pathogenic bacteria Virulence genes acquired later in evolution may have been connected with the BvgAS regulon because their expression may be required under the same conditions which also lead to expression of cytochrome d oxidase and repression of cytochrome c oxidase In this respect it is interesting to note that a recently isolated environmental organism probably closely related to a common ancestor of the pathogenic Borde-tellae, Bordetella petrii, is the first member of the genus with a facultatively anaerobic metabolism [27] In con-trast, the pathogenic Bordetellae are characterized by an obligate aerobic metabolism with their growth opti-mum under microaerophilic conditions which may be a consequence of their permanent association with host organisms
A C K N O W L E D G E M E N T S
We would like to thank V Weiss for discussions and D Beier and
J Gross for carefully reading of the manuscript This work was supported by a grant from the Deutsche Forschungsgemeinschaft as part of the priority program Regulatory networks in bacteria and by the Fonds der Chemischen Industrie.
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