(Received for publication, March 10, 1995; and in revised form, July 14, 1995)
From the
VanS is a two-component transmembrane sensory kinase that,
together with its response regulator VanR, activates the expression of
genes responsible for vancomycin resistance in Enterococcus faecium BM4147. In this report, we demonstrate that the cytoplasmic domain
of VanS (including residues Met to Ser
) is
capable of high level activation (>500 fold) of the Escherichia
coli response regulator PhoB in vivo in the absence of
its signaling kinases PhoR, CreC (PhoM), or acetyl phosphate synthesis. In vitro experiments carried out on the purified proteins
confirmed that the activation is due to efficient cross-talk between
VanS and PhoB, since phospho-VanS catalyzed transfer of its phosphoryl
group to PhoB with
90% transfer in 5 min at a 1:4 VanS/PhoB
stoichiometry. However, the rate of transfer was at least 100-fold
slower than that observed between phospho-VanS and VanR. The in
vivo activation of PhoB was used as a reporter system to identify
peptide fragments of VanS capable of interfering with activation by
VanS(Met
-Ser
), in order to identify an
interaction domain. A library of plasmids encoding fragments of
VanS(Met
-Ser
) was constructed using
transposon mutagenesis, and a subpopulation of these plasmids encoded
peptides that interfered with activation of PhoB by
VanS(Met
-Ser
). A minimal size fragment
(Met
-Ile
) was shown to be both
necessary and sufficient for potent inhibition (85%) of this
activation.
Vancomycin is a potent glycopeptide antibiotic that has been
used in the treatment of Gram-positive bacterial infections for over 30
years(1) . Vancomycin causes cell death by disrupting cell wall
biosynthesis; the drug binds to the terminal D-Ala-D-Ala moieties of the growing peptidoglycan
cell wall, thereby inhibiting the transpeptidation and
transglycosylation steps necessary for peptidoglycan cell wall
maturation(2) . This unique mode of action has made the drug
relatively immune to resistance problems; however, over the past
decade, an increasing number of highly resistant strains of Enterococcus have been identified in clinical isolates and
waste water systems(3) . Vancomycin resistance in these strains
requires five plasmid-borne genes: vanH, vanA, vanX, vanR, and vanS(4) . Three of
these gene products (VanH, VanA, and VanX) are enzymes that together
confer resistance by leading to the incorporation of modified cell wall
precursors in the peptidoglycan. VanH is a D-specific
-ketoacid reductase that converts pyruvate to D-lactate(5, 6) . VanA is a D-Ala-D-Ala ligase with altered specificity; it is
capable of utilizing the D-lactate produced by VanH for
efficient D-Ala-D-lactate depsipeptide
synthesis(7) . The D-Ala-D-lactate
depsipeptide, when incorporated into the peptidoglycan, confers
resistance to vancomycin because the drug has a 1000-fold reduced
affinity toward D-Ala-D-lactate than toward the
native D-Ala-D-Ala peptidoglycan
precursors(8) . Recently, VanX has been shown to be a D-, D-specific dipeptidase, which is competent in D-Ala-D-Ala hydrolysis but does not hydrolyze the D-Ala-D-lactate
depsipeptide(9, 10) , effectively reducing the pools
of the D-Ala-D-Ala dipeptide and shunting flux to D-Ala-D-lactate peptidoglycan termini. All together,
these enzymes provide an efficient alternate cell wall biosynthetic
pathway that results in the selective incorporation of D-Ala-D-lactate into the growing peptidoglycan cell
wall.
VanS and VanR comprise a two-component regulatory system for
transcriptional activation of the genes vanH, vanA,
and vanX(11, 12) . Two-component regulatory
systems are signal transduction pathways commonly used by prokaryotes
to sense and adapt to stimuli in the environment; as many as 50
different systems may exist in a simple bacterium such as Escherichia coli, as well as other
bacteria(13, 14) . In addition, analogous signal
transduction pathways have recently been identified in eukaryotic
organisms including yeast(15, 16) , plants (17) , and neurospora(18) . These systems are
characterized by a sensor kinase (often a transmembrane signaling
kinase such as VanS), which undergoes autophosphorylation on a
histidine residue, and this phosphoryl group is then transferred to an
aspartate residue on a response regulator protein (in this case, VanR),
which usually acts as a transcriptional activator. Like many signaling
kinases, VanS has an N-terminal domain with two transmembrane-spanning
segments believed to act as a signal sensing domain and a C-terminal
cytoplasmic ``transmitter'' domain with autophosphorylation
and phosphotransfer activities(13) . Biochemical studies on the
cytoplasmic domain of VanS(Met-Ser
)
have shown that it is readily autophosphorylated at a histidine residue
(His
) in the presence of ATP and that phospho-VanS is
capable of efficient phosphotransfer to an aspartate residue on VanR
(Asp
) (12) . Genetic studies have implicated VanR
as a transcriptional activator of the vancomycin resistance
genes(11) . These predictions have been borne out by in
vitro studies with the purified protein. Gel mobility shift and
DNase footprinting analyses have shown that phospho-VanR has an
enhanced binding affinity for the P
and P
promoter regions of
the vancomycin resistance operon(19) .
Sensor kinases and response regulators of two-component regulatory systems share extensive sequence similarities to other family members, even of phylogenetically distant species(13) . These sequence similarities probably lead to structural similarities that are responsible for cross-reactivities between sensor kinases and response regulators of different systems. This phenomenon, called ``cross-talk,'' has been observed in vitro(20, 21) , and it has been implicated in complex phenotypes in vivo. For example, the E. coli phosphate (PHO) regulon is activated by two different sensor kinases in vivo, which may represent a special form of cross-talk called cross-regulation(22) . The sensor kinase PhoR activates the response regulator PhoB that in turn activates synthesis of bacterial alkaline phosphatase in response to limiting phosphate levels (Fig. S1A)(23) . In the absence of PhoR, the sensor kinase CreC or acetyl phosphate activates PhoB ( (24) and (25) and for reviews, see (22) and (26) ) Furthermore, cross-talk interactions have been used to identify new two-component regulatory genes by complementation of known sensor kinase mutants (E. coli envZ and/or phoR creC(27, 28, 29, 30) ). These results reinforce the concept that individual two-component systems share a common mechanism and suggest that it is likely that similar regions of the sensor kinases and response regulators are involved in the transmembrane signal sensing, autophosphorylation, and interprotein phosphorelay steps of these signal transduction pathways.
Figure S1: Scheme 1.
We are
working to define the regions of VanS involved in the signaling
process, including those involved in the recognition and
phosphorylation of its cognate response regulator VanR. Studies on the
chemotaxis sensor CheA have demonstrated that there are distinct
regions that are responsible for its autokinase and phosphotransfer
activities(31) , as well as regions that specifically interact
with the response regulator, CheY(32) . To perform a similar
molecular dissection of VanS, we developed a genetic method to screen
VanS fragments that retain structure/function elements necessary for
signal transduction. Since the potential health risks associated with
vancomycin-resistant Enterococcus mitigated against using the
native VanS/VanR signaling pathway as an assay for VanS function, we
constructed an artificial signal transduction pathway that involved in vivo activation of the E. coli response regulator
PhoB by VanS through cross-talk. In this system, all of the known
signaling pathways for activation of PhoB (PhoR, CreC, or acetyl
phosphate synthesis) have been eliminated by mutation(25) , and
therefore any activation by VanS can be quantified by measuring
bacterial alkaline phosphatase (encoded by phoA) (Fig. S1B). In this report, we demonstrate that the
cytoplasmic domain of VanS (residues Met to
Ser
) is capable of high level activation of E. coli PhoB both in vitro and in vivo, and we have used
this cross-reactivity as a reporter system to identify a domain of VanS
that inhibits PhoB activation by VanS.
Plasmid pAT89 carrying vanS was a generous gift from Dr. M. Arthur (Institut Pasteur). pBB100 was a generous gift from Barry Ballard (Harvard Medical School), and pBAD32 was a generous gift of Luz-Marie Guzman and Jon Beckwith (Harvard Medical School). Nucleotide sequencing was performed with the Sequenase kit (U. S. Biochemical Corp.). Oligonucleotides were prepared by Dr. Charles Dahl (Harvard Medical School). Antibiotics were from Sigma and (unless otherwise noted) were used at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 34 µg/ml; and kanamycin, 50 µg/ml.
Two kinds of plasmids were constructed to provide
controlled synthesis of VanS
(Met-Ser
). One was constructed using
pBB100, a derivative of pSPORT1 (Life Technologies, Inc.) containing a
synthetic merR gene(33) . pSPORT1 is a pMB1-based
vector carrying P
and encodes ampicillin
resistance and LacI. The other was constructed using the compatible
p15A-based vector, pBAD32. pBAD32 is a derivative of pBAD28 (34) that carries P
and encodes both
chloramphenicol resistance and AraC. The pSPORT and pBAD plasmids used
in this study are described in Table 1. Construction of plasmid
pSLF4 was accomplished using polymerase chain reaction with primer
(upstream) 5`-TACTTGGCGCAGGATATCAAAACGCCCCTTACATC-3` and the downstream
primer from the pSLF1 plasmid construction. The amplification product
of these primers was digested with HhaI and BamHI and
ligated with two purified fragments of pSLF3 obtained by digestion with NdeI, HhaI, and BamHI. Plasmids pSLF1,
pSLF4, and pSLF6 were confirmed by nucleotide sequencing of the vanS segment; all others were confirmed by mapping with
restriction enzymes.
Alkaline
phosphatase synthesis in BW20992 is due to activation of PhoB by VanS.
No activation was observed when a phoB or phoA mutant
was transformed with pSLF3, even under conditions of induction (Table 2). Furthermore, activation of PhoB by VanS was abolished
by PhoR in the presence of P (Table 2). This was
expected because PhoR inhibits activation of PhoB when P
is
in excess, presumably by facilitating the dephosphorylation of
phospho-PhoB. These results suggest that in strain BW21096, the net
dephosphorylation by PhoR is more efficient than the phosphotransfer to
PhoB from VanS (Fig. S2).
Figure S2: Scheme 2.
A further test of the cross-talk
between VanS and PhoB in vivo was performed using a
site-directed mutation of the presumed site of autophosphorylation in
VanS (His). Transformation of BW20992 with a plasmid
(pSLF4) encoding the H164Q form of
VanS(Met
-Ser
), resulted in no
increased activation of alkaline phosphatase synthesis (Table 2).
This is to be expected if activation of PhoB is due to phosphoryl
transfer from phospho-VanS. Taken together, these results are
consistent with an efficient level of cross-talk between VanS and PhoB in vivo, in which VanS activates the PHO regulon through
direct phosphorylation of the PHO response regulator PhoB. This level
of PhoB activation is similar to that observed with its partner sensor
kinase PhoR, as comparable strains synthesize about 600 units of
alkaline phosphatase upon activation of PhoB by PhoR under conditions
of P
limitation. (
)
Figure 1:
Phosphorylation of response
regulators by phospho-MBP-VanS. Lanes1-3,
proteins were incubated in 15 µl of phosphorylation buffer (50
mM Tris, 50 mM KCl, 1 mM MgCl,
pH 7.4) for 60 min at 37 °C with
[
-
P]ATP (final concentration, 1
mM; 50 µCi/pmol) and then quenched with 10 µl of SDS
stop solution. Final protein concentrations were as follows: VanR, 20
µM; PhoB, 20 µM; VanS, 5 µM. Lanes4 and 5, MBP-VanS (32 µM)
was incubated with [
-
P]ATP (final
concentration, 1 mM; 50 µCi/pmol) for 60 min in
phosphorylation buffer at 37 °C, and then 2 µl of this mixture
were added to 12.5 µl of VanR (lane4, 24
µM) or PhoB (lane5, 24 µM)
solutions in phosphorylation buffer. After 5 min at 30 °C, the
reactions were quenched with 10 µl of SDS stop buffer. Samples of
the quenched reactions (12.5 µl) were immediately loaded on a 12.5%
SDS-PAGE gel. The phosphorimage (Molecular Dynamics Inc.) of the gel is
shown.
Figure 2:
Time
Course of Phosphotransfer from P-MBP-VanS to (A)
PhoB and (B) VanR. Phosphorimages of SDS-PAGE gels of the
phosphorylated proteins are shown, conditions are described under
``Materials and Methods.'' Protein concentrations were as
follows: MBP-VanS, 10 µM; VanR, 20 µM; PhoB,
40 µM.
As expected, synthesis of the
VanS(Met-Ser
) under arabinose control
in plasmid pSLF5 resulted in high levels of alkaline phosphatase
synthesis (Table 2). Arabinose caused a
500-fold increase in
alkaline phosphatase activity. No detectable synthesis was observed in
the absence of arabinose, thus showing the tighter control of the P
promoter in comparison with P
. This tight control is further substantiated
in Fig. 3, where the amount of alkaline phosphatase activity is
shown to vary with the concentration of arabinose. A similar titration
curve has been observed for the expression of other genes in pBAD
vectors(34) . A linear increase in alkaline phosphatase
activity is observed with increasing concentrations up to about 100
µM arabinose. All experiments involving pBAD plasmids were
carried out using fructose as a carbon source in order to overcome
problems resulting from catabolite repression (data not shown).
Figure 3: Titration of PhoB Activation with pSLF5. Transformants of strain BW21469 carrying pSLF5 were grown on 2XYT agar containing chloramphenicol (100 µg/ml) and then inoculated in 5 ml of 2XYT broth containing chloramphenicol and grown to saturation overnight at 37 °C with aeration. A sample of the cell culture (200 µl) was diluted in 20 ml of TYE medium containing chloramphenicol, and 0.5-ml portions were subcultured in broth containing arabinose. Triplicate cultures were assayed for each concentration. Cell cultures were grown for 8 h at 37 °C with aeration and then assayed for alkaline phosphatase activity as described under ``Materials and Methods.''
Figure 4:
Schematic of inhibitory fragments of VanS
identified using the C-terminal truncation library. The VanS
cytoplasmic domain (Met-Ser
) is shown
schematically with the conserved regions as described by Parkinson and
Kofoid (13) annotated. Regions containing VanS sequence are
denoted as darkbars; lightregions represent sequences added as in-frame fusions from the transposon
insertions.
Plasmids
encoding four of these fragments were assayed for effects on alkaline
phosphatase synthesis in broth cultures in order to quantify the amount
of inhibition (Fig. 5). These plasmids were chosen as
representatives of the full range of VanS fragment sizes identified
from the genetic screen, ranging from 80 to 241 amino acids of VanS.
All of these plasmids demonstrated inhibition both in the presence or
absence of IPTG for induction of the fragment synthesis (Fig. 5). The inhibition observed in the absence of IPTG is
likely to be due to the moderate basal level of expression from the
pSPORT plasmid, which was also observed with the full-length VanS
construct (pSLF3, Table 2). In all cases, the inhibition was
greater with increased level of fragment synthesis, and the extent of
inhibition ranged from 50% to 85%. No correlation to the relative
amount of inhibition and the fragment size was observed.
Figure 5: Inhibition of alkaline phosphatase synthesis by C-terminal truncated fragments of VanS. Alkaline phosphatase assays were performed as described under ``Materials and Methods.'' Fragments denoted with an asterisk contain an 11-amino acid fusion from the transposon, those with contain a 27-amino acid fusion (see Table 3). Assays performed without induction (0 mM IPTG) of the fragment synthesis are openbars, data for induced (1 mM IPTG) fragments synthesis are represented by filledbars.
The two-component VanS and VanR proteins of vancomycin
resistance plasmids in Gram-positive Enterococci control
transcription of the structural genes vanH, vanA, and vanX that encode enzymes that produce a modified peptidoglycan
structure with 1000-fold lower affinity for vancomycin. Efforts to
study the interactions of VanS and VanR in E. coli await a
good method for transcriptional activation of the vanH and vanR promoters in Gram-negative bacteria. The studies reported
here establish that the cytoplasmic domain
(Met-Ser
) of the VanS sensory
autokinase can recognize the response regulatory component PhoB of the E. coli PHO regulon and drive transcriptional activation of phoA (encoding alkaline phosphatase). This heterologous
recognition between sensor and response regulator opens the possibility
of structure/function mutagenesis in both VanS and PhoB to screen for
mutual recognition domains and should also provide an assay for
evaluation of the full-length transmembrane VanS function, especially
in regard to the ligand(s) that promotes VanS activation, e.g. in E. coli mutants with a leaky outer membrane
envelope(39) .
The in vivo cross-talk between VanS
and PhoB was validated by in vitro analysis of phosphoryl
group transfer from the phosphohistidine form of MBP-VanS to PhoB with
the purified proteins. Comparison of the rates of phosphoryl transfer
from phospho-MBP-VanS to purified PhoB or VanR when the response
regulator proteins are in excess of VanS (1:4 molar ratio for PhoB, 1:2
molar ratio for VanR) indicates a 100:1 rate preference for homologous
(VanR) versus heterologous (PhoB) phosphorylation of the
response regulator. K and K
values are yet to be obtained to provide estimates of k
/K
catalytic efficiency
criteria. Both the in vivo and in vitro VanS/PhoB
assays may be applicable to screen for agents that would inhibit
two-component regulatory system signal transduction (40) and
ultimately inhibit vancomycin activation of the complex vanRSHAX operon.
The observed cross-reactivity between VanS and the E. coli PHO regulon provides an excellent in vivo assay for VanS function. This system may be exploited to
investigate the underlying mechanisms of VanS signaling, and it may
also provide a means for a detailed understanding of the molecular
interactions that govern the signaling processes in two-component
systems in general. Towards this goal, we performed two screens of a
genetic library of VanS fragments to identify those regions of VanS
involved in the transmembrane signaling and phospho-relay steps of the
signal transduction pathway. First, a search was conducted for VanS
fragments that retain the phosphotransfer activity to PhoB, and,
second, a search was conducted for VanS fragments that inhibit the
VanS/PhoB phosphoryl transfer. To this end, we prepared and screened
mutant libraries of the cytoplasmic VanS construct that were further
truncated from the C terminus by m transposon mutagenesis. In
the first search, we assayed the library for fragments capable of
complementing a VanS(Met
-Ser
) mutant
(H164Q) that would be expected to phosphorylate fragments in trans but is unable to undergo autophosphorylation. No subfragments of
the VanS(Met
-Ser
) construct were
detected that retained the ability to activate PhoB. This negative
screening result may not be surprising since fragment-based PhoB
activation would require efficient phosphorylation of the fragment in trans from the
VanS(Met
-Ser
) H164Q mutant as well as
efficient transfer of the phosphoryl group to PhoB.
To assess the
ability of VanS fragments to inhibit
VanS(Met-Ser
) interaction with PhoB
required simultaneous synthesis of both
VanS(Met
-Ser
) and VanS subfragments,
resulting from the m
transposon insertion library. Several
mutant plasmids were identified and sequenced that yielded a nested set
of fragments with progressive truncations from the C terminus. The
minimal size VanS fragment that retains inhibition contains 80 residues
(Met
-Ile
). There is precedent in the E. coli chemotaxis two-component regulatory system for
dominant-negative phenotypes resulting from the overexpression of the
sensory kinase CheA (32) and the serine chemoreceptor Tsr (41) in a ``domain liberation'' approach. In the VanS
case, this small 80-amino acid fragment retains His
that
is the autophosphorylation site and the phosphoryl donor (to VanR and
PhoB). While the small size (
9 kDa) augurs well for structural
determination of the dominant negative
VanS(Met
-Ile
) fragment, initial attempts to
overproduce it have led to insoluble protein that has so far resisted
functional renaturation. It will be important to determine whether the
80-amino acid inhibitory fragment is interfering with VanS
autophosphorylation from ATP, e.g. perhaps by
heterodimerization with VanS
(Met
-Ser
), or by reducing the levels
phospho-PhoB, or both. In order to test whether the fragments inhibit
the VanS/PhoB signaling by affecting the levels of phospho-PhoB in the
cell, e.g. by blocking the interaction with
VanS(Met
-Ser
) or by aiding in the
dephosphorylation of phospho-PhoB, we expressed the fragment under
conditions of constitutive PhoB activation by CreC. No effect was
observed on the PhoB activation under these conditions (data not
shown). These negative results imply that the fragments inhibit the
signal transduction pathway by interfering with the autophosphorylation
of VanS (Met
-Ser
). It remains to be
determined if further truncation from the N terminus of
VanS(Met
-Ser
) will lead to a dominant
negative phenotype. In this regard, the osmosensor kinase EnvZ (42) is thought to have a similar overall topology to VanS, and
a ``linker region'' between the transmembrane region and the
histidine phosphorylation site of EnvZ has an important role in
``locking in'' fixed signaling modes. Similar linker regions
have been noted in the related chemoreceptor proteins (14, 43) and the genetic hybrid protein
Taz-1(44) . This linker region is also retained in the
Met
-Ile
fragment of VanS.
In summary, these approaches may advance our understanding of homologous and heterologous sensor/response regulator recognition elements for binding and phosphotransfer; for antibiotic and antifungal agents that may disrupt these systems in bacteria, yeast, and fungi; and in particular the specific reversal of vancomycin resistance in pathogenic Gram-positive bacteria.