TheunorthodoxhistidinekinasesBvgSandEvgSare responsive
to theoxidationstatusofaquinoneelectron carrier
Andreas Bock and Roy Gross
Lehrstuhl fu
¨
r Mikrobiologie, Biozentrum, Universita
¨
tWu
¨
rzburg, Am Hubland, Wu
¨
rzburg, Germany
The purified soluble forms ofthehistidinekinasesBvgS and
EvgS of Bordetella pertussis and Escherichia coli,respect-
ively, are shown to be responsiveto 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 l
M
(BvgS) and 4 l
M
(EvgS). Reduced
Q-0 has no effect on thehistidine kinases. Kinase activity can
reversibly be switched off and on by changing the oxidation
status ofthe quinone. This inhibitory effect is due to a
decrease ofthe kinase activity ofBvgS 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 ofBvgS 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 ofBvgS caused a weak decrease of qui-
none-dependent inhibition of autophosphorylation. These
data suggest that BvgSandEvgSare connected with the oxi-
dation statusofthe cell via the link tothe 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, andof close relatives such as B. parap-
ertussis and B. bronchiseptica that cause similar respiratory
diseases in man and animals, respectively [3,4]. It controls
expression ofa 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. ThehistidinekinasesBvgSand EvgS
belong tothe small group ofthe so-called unorthodox
sensor proteins because they harbour an intermediate
receiver and at the C-terminus a histidine-containing
phosphotransfer (HPt) domain in addition tothe classical
transmitter domain [6–9]. Under permissive conditions, the
BvgS andEvgShistidinekinases 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 ofa 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 ofthe 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 oftheBvgS 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 ofthe protein [14–17]. This
suggests that theBvgS protein may perceive intracellular
signals characteristic for PAS domains such as the energy
status ofthe 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 ofthe 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 ofthe BvgAS regulon are similar to
those of another unorthodox two-component system, the
ArcAB system of E.coli[20,21]. Theunorthodox histidine
Correspondence to R. Gross, Lehrstuhl fu
¨
r Mikrobiologie, Biozen-
trum, Universita
¨
tWu
¨
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)
Eur. J. Biochem. 269, 3479–3484 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03029.x
kinase ArcB together with the response regulator ArcA
regulates the expression of many genes which are involved
in the adaptation ofthe 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, theunorthodox ArcB protein shows a domain
structure that is very similar to those oftheBvgSand EvgS
proteins, but it lacks the extensive periplasmic domain.
Because ofto their striking structural similarities to ArcB
and tothe genes regulated by these two-component systems,
in the present study we investigated whether quinones may
also be a relevant signal for theBvgSandEvgS histidine
kinases.
MATERIALS AND METHODS
Phosphorylation assays
Over-expression and purification ofthehistidine kinases
BvgS andEvgS was described recently [7]. Briefly, both
proteins were expressed with an N-terminal His-tag which is
fused in front ofthe putative PAS domains ofthe 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-
32
P]ATP (specific activity
> 5000 CiÆmmol
)1
) [23,24]. The purified proteins were
incubated at concentrations of 1 l
M
(BvgS and EvgS) or
between 4.7 and 7.1 l
M
(BvgS-H643A, BvgS-Del, BvgS-C4)
at room temperature in reaction buffer containing 50 m
M
Tris pH 7.5, 50 m
M
KCl, 10 m
M
MgCl
2
,50 n
M
[c-
32
P]ATP,
0.2 m
M
ATP. Reactions were stopped by the addition of
sample buffer (60 m
M
Tris pH 6.8, 10% glycerol, 2% SDS,
5% 2-mercaptoethanol, 0.05% Bromophenol blue). After
separation ofthe phosphorylated proteins by SDS/PAGE
[25] the gels were dried at 80 °C 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 l
M
to 5 m
M
), MK-3 (1 l
M
to 1 m
M
), oxidized and
reduced FAD and NAD (1 l
M
to 5 m
M
), nicotinic acid
(1 m
M
to 10 m
M
)orMgSO
4
(1 l
M
to 50 m
M
). 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 ofthe effect of Q-0 on the
autophosphorylation kinetics ofBvgSandEvgSthe 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 l
M
Q-0 and/or 500 l
M
dithionite (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 m
M
). Then, after an additional
4.5 min, Q-0 was reduced by the addition of 25 m
M
dithionite.
For characterization ofthe autodephosphorylation of
BvgS, the kinase was first incubated for 5 min under
standard conditions in the presence of [c-
32
P]ATP as
described above. Then 5 m
M
Q-0 and/or 16 m
M
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
Microsoft
EXCEL
97 and
SIGMAPLOT
for Windows, version
5.00.
Site-directed mutagenesis
The BvgS mutants were constructed using the Quik-
Change
TM
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 theBvgS PAS
domain was the target for mutagenesis [26]. The previously
described plasmid pQE-BvgS-TRO [7] was used as a
template andthe following oligonucleotides were synthes-
ized for mutagenesis.: For the construction ofthe mutant
bvgS alleles 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
(5¢-GGCCACGCGCGTCAGCGCCGGGTC-3¢)were
used. All proteins used in this study are shown schematically
in Fig. 1.
Hpt
N
BvgS
C
RT
PAS
TM
BvgS*
C
BvgS-H643A
C
H643A
F583
BvgS-C4
C
G688S
BvgS-Del
C
∆ 639-647
>
Fig. 1. Structural organization ofBvgS derivatives used in this study.
On the top the domain structure ofthe wild-type BvgS protein is
shown. BvgS* shows the organization ofthe soluble truncated BvgS
derivative referred to as Ôcontrol proteinÕ throughout this study. The
positions ofthe 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.
3480 A. Bock and R. Gross (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Effect of quinones on the activity ofBvgSand EvgS
In the genus Bordetella, ubiquinone-8 (Q-8) is the predom-
inant quinone [27], whereas the major quinones synthesized
by E.coliare Q-8, menaquinone-8 (MK-8) and demethyl-
menaquinone-8 [28]. The incubation oftheBvgSand 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 l
M
and 4 l
M
, respectively
(Fig. 3). Reduction ofthequinone by dithionite entirely
abolished its negative effect on BvgSandEvgS (Fig. 2).
Whereas the autophosphorylation activity ofEvgS could be
inhibited completely by the addition of 100 l
M
Q-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 ofBvgS 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 thehistidine kinase
but not on intrinsic phosphatase activities
The negative effect on autophosphorylation observed after
addition ofthe oxidized ubiquinone tothehistidine kinases
may either be the consequence ofa decrease in the histidine
kinase activity, or, alternatively, of an increase in an intrinsic
autophosphatase activity present in theBvgSand 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 ofthe phosphoprotein could
be noted. Moreover, no significant change in the stability of
BvgA-P could be observed in the presence ofBvgS and
oxidized Q-0 (data not shown). These data suggest that in
the case ofBvgS 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 theBvgShistidine kinase activity
To investigate whether other electron carriers abundant in
the cell have an impact on the activity oftheBvgSand EvgS
proteins, we added up to 5 m
M
FAD or 5 m
M
NAD to the
reaction mixtures. FAD was shown previously to bind to
the PAS domain ofthe membrane signal transducer protein
Aer of E.coli[29]. However, using physiologically relevant
concentrations neither coenzyme had any significant effect
on the autophosphorylation activity ofthe two histidine
kinases independent of their oxidationstatus (data not
shown). Moreover, nicotinic acid and sulfate ions (which
have long been known to inhibit BvgSand EvgS) at in vivo
relevant concentrations did not influence the autophospho-
rylation properties ofthe purified truncated BvgSand EvgS
kinases in vitro (data not shown).
Effects of mutations in theBvgS PAS domain
on quinone sensing
Similar tothe ArcB histidine kinase of E.coli, the presence of
a PAS domain was recently predicted in front of the
transmitter domain oftheBvgS 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 ofthe 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 ofthe protein
time (min)
01234
relative level BvgS-P
0
20
40
60
80
100
120
14
0
BvgS
BvgS +D
t
BvgS
+Q-0
BvgS +Q-0 +D
t
+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)
01234
relative level EvgS-P
0
20
40
60
80
100
120
140
EvgS
EvgS +D
t
EvgS
+Q-0
EvgS +Q-0 +D
t
B
Fig. 2. Effect of (Q-0) on the rate of auto-
phosphorylation oftheBvgS (A) andEvgS (B)
histidine kinases. The purified proteins were
incubated with [c-
32
P]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 ofthe phosphorylated histi-
dine kinases was determined by Phosphor-
Image analysis ofthe respective gels shown in
the bottom panel. The maximal phosphory-
lation levels ofBvgSandEvgS 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.
Ó FEBS 2002 Quinone sensing by histidinekinases (Eur. J. Biochem. 269) 3481
(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 oftheBvgS PAS domain a sequence with
similarities toa recently described quinone binding motif is
present consisting ofa weak structural element ofa 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 ofthe 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 ofthe PAS domain (BvgS-C4) (Fig. 1). The
latter mutation was found after a screen for B. pertussis
mutants exhibiting a ÔconstitutiveÕ 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 l
M
the autophosphorylation activity of
BvgS-Del could only be inhibited to 20% of its maximal
activity, whereas, for comparison, the activity ofthe control
protein BvgS decreased to 10% under the same conditions
(Fig. 3).
DISCUSSION
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 tothe 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.coliMK-8 does not play
aroleinB. pertussis [27]. However, in E.coli ArcB
time (min)
0 5 10 15 20 25 30 35
relative level BvgS-P
20
30
40
50
60
70
80
90
100
BvgS +Q0
BvgS -Q0
0 0.5 1 2
4
816
32 (min)
0 0.5 1 2
4
816
32 (min)
+Q-0
-Q-0
Fig. 5. Autodephosphorylation ofBvgS in the presence or absence of Q-0. BvgS was phosphorylated by the addition of [c-
32
P]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 ofthe relative phosphorylation level at the various
time points is shown. On the right the autoradiographies of representative gels are shown.
0 1 2.5 5 10 20 50 100 200 (µM Q-0)
BvgS-H643A
BvgS-Del
BvgS-C4
BvgS
EvgS
Q-0 [µM]
0 50 100 150 200
relative phosphorylation (%)
0
20
40
60
80
100
120
BvgS
BvgS-H643A
BvgS-Del
BvgS-C4
EvgS
Fig. 3. Inhibition of autophosphorylation of BvgS, EvgSandof mutated BvgS derivatives (BvgS-H643A, BvgS-Del, BvgS-C4) in the presence of
different Q-0 concentrations. After SDS/PAGE analysis the relative amount ofthe phosphorylated histidinekinases was determined by Phos-
phorImage analysis. The figure shows representative autoradiographs ofthe samples after SDS/PAGE (right panel) andthe results of their
quantification by the PhosphorImager (left panel).
time (min)
0 1020304 00 5
relative level BvgS-P
0
20
40
60
80
100
addition of Dt
2
4
6.5 8.5 10.5 16 31 51 (min)
BvgS
Fig. 4. Dependence ofthe kinase activity ofBvgS on the redox state of
ubiquinone-0 (Q-0). BvgS was incubated with [c-
32
P]ATP in the pres-
ence of 5 m
M
Q-0 for 4.5 min Then dithionite was added toa final
concentration of 25 m
M
. The insert shows the autoradiography of a
representative gel.
3482 A. Bock and R. Gross (Eur. J. Biochem. 269) Ó FEBS 2002
autophosphorylation 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 ofthe two
histidine kinasesof E.colimay 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 ofthe 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, theBvgSand 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 ofthe 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, BvgSand EvgS. As the
quinones arethe only components ofthe 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 ofthe 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 andBvgS linker
regions close tothe 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 ofa 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, theBvgS PAS domain contains a
sequence motif that comprises essential features ofa 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 toquinone sensing but there may be additional
quinone interaction sites. In fact, in the case ofBvgS 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.coliarcBmutant 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 Aof Bacillus subtilis was
recently shown to be a catalytic ATP-binding domain [31].
As the PAS domain ofBvgS 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 tothe lack of relevant changes ofthe virulence
properties ofbvgS 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 ofthe virulence regulon may
not be relevant at least in the respective animal models [33].
Because such a ÔconstitutiveÕ 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 ofBvgS activity
by relevant intracellular stimuli still occurred. Therefore, in
the future for ÔconstitutiveÕ bvgS mutants that are not
responsive to quinones it will be necessary to investigate
whether modulation ofBvgS 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 theBvgS 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 ofthe master regulator of
virulence to basic metabolism ofthe 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 toa common ancestor ofthe pathogenic Borde-
tellae, Bordetella petrii, is the first member ofthe 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.
ACKNOWLEDGEMENTS
We would like to thank V. Weiss for discussions and D. Beier and
J. Gross for carefully reading ofthe manuscript. This work was
supported by a grant from the Deutsche Forschungsgemeinschaft as
part ofthe priority program ÔRegulatory networks in bacteriaÕ and by
the Fonds der Chemischen Industrie.
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3484 A. Bock and R. Gross (Eur. J. Biochem. 269) Ó FEBS 2002
. The unorthodox histidine kinases BvgS and EvgS are responsive
to the oxidation status of a quinone electron carrier
Andreas Bock and Roy Gross
Lehrstuhl. signal for the BvgS and EvgS histidine
kinases.
MATERIALS AND METHODS
Phosphorylation assays
Over-expression and purification of the histidine kinases
BvgS