(Received for publication, October 24, 1996)
From the Department of Molecular Microbiology and
§ Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine, St.
Louis, Missouri 63110
The PhoP/PhoQ two-component regulatory system governs several virulence properties in the Gram-negative bacterium Salmonella typhimurium. The PhoQ protein is a Mg2+ and Ca2+ sensor that modulates transcription of PhoP-regulated genes in response to the extracellular concentrations of these divalent cations. We have purified a 146-amino acid polypeptide corresponding to the periplasmic (i.e. sensing) domain of the PhoQ protein. Mg2+ altered the tryptophan intrinsic fluorescence of this polypeptide whereas Ba2+, which is unable to modulate transcription of PhoP-regulated genes, did not. Mg2+ was more effective than Ca2+ at repressing transcription of PhoP-activated genes in vivo. However, maximal repression was achieved when both cations were present. An avirulent mutant harboring a single amino acid substitution in the sensing domain of PhoQ exhibited lower affinity for Ca2+ but similar affinity for Mg2+. Cumulatively, these experiments demonstrate that Mg2+ can bind to the sensing domain of PhoQ and establish the presence of distinct binding sites for Mg2+ and Ca2+ in the PhoQ protein.
Two-component regulatory systems often mediate the adaptive response of bacteria to new environmental conditions (1-4). These systems generally consist of a sensor protein that, in response to specific chemical or physical signals, modifies the phosphorylation state of the second component, usually a transcription factor whose affinity for DNA is modulated by phosphorylation. Sensor proteins are usually conserved in their C-terminal, cytoplasmic domain, which mediates the phosphorylation/dephosphorylation of the cognate regulatory proteins (5). On the other hand, the N-terminal, periplasmic domain of sensors is often involved in signal sensing and, therefore, confers specificity to each system.
The PhoP/PhoQ two-component system governs several virulence properties in the Gram-negative bacterium Salmonella typhimurium (6, 7). The PhoQ protein is a Mg2+ sensor that, in the presence of millimolar concentrations of this divalent cation, represses transcription of some 25 different PhoP-regulated loci (8, 9). Several of these genes are essential for growth in low Mg2+ environments, consistent with Mg2+ deprivation being the regulatory signal that activates the PhoP/PhoQ system (9). Ca2+ and Mn2+ can replace Mg2+ to repress transcription of PhoP-activated genes, but other divalent cations, including Ni2+, Cu2+, and Ba2+, have no effect (8). The regulatory role of the PhoP/PhoQ system is not limited to Salmonella pathogenesis, because several PhoP-regulated loci are not essential for virulence in mice (10, 11), and phoP-hybridizing sequences have been detected in a wide variety of non-pathogenic Gram-negative species (12).
The PhoQ protein features two transmembrane regions, a long cytoplasmic
tail, and a large periplasmic domain rich in acidic residues that could
be involved in binding divalent cations (Ref. 13; Fig. 1). Several
lines of experimental evidence suggest that the regulatory effect of
Mg2+ results from direct conformational changes provoked in
the periplasmic domain of the PhoQ protein. First, a protein chimera in
which the periplasmic domain of PhoQ was replaced by the corresponding region of the osmolarity sensor EnvZ lost the capacity to respond to
Mg2+ (8). Second, Mg2+ modifies the trypsin
susceptibility of the PhoQ protein in vitro at the same
concentrations that are required to repress transcription of
PhoP-activated genes in vivo (8). Finally, the changes in trypsin susceptibility in the PhoQ protein were detected in
spheroplasts, which indicates that soluble components from the
periplasmic space are not necessary for the Mg2+-mediated
effect (8).
In this paper, we demonstrate that the sensing domain of the PhoQ protein specifically binds Mg2+. Furthermore, we establish that the PhoQ protein has distinct binding sites for Ca2+ and Mg2+, and we identify a mutant PhoQ protein that is differentially altered in its response to Ca2+. Our data indicate that the PhoP/PhoQ system mediates the response to changes in the environmental levels of Ca2+ and Mg2+ and suggest a model in which binding of these divalent cations to the periplasmic domain of the PhoQ protein promotes a conformation that is unfavorable for the activation of the PhoP protein.
S.
typhimurium strains EG9065 (psiD::MudJ) and
EG9564 (psiD::MudJ pho-24) have been
described (8). Escherichia coli strain EG9649 is BL21[DE3]
carrying plasmid pT7-7Qp. Plasmid pT7-7Qp, which harbors the DNA region
encoding the sensor domain of PhoQ under the control of the T7 10
promoter, was constructed in two steps. First, a DNA fragment
comprising the region encoding the sensing domain of PhoQ (codons
45-190) was generated by the polymerase chain reaction using primers
F05, 5
-AGGAATTCGCCATGGATAAAACCACCTTTC-3
, and F06
5
-TGCAAGCTTACATATAGGAGCGTTTTAG-3
, and plasmid pEG9071 (14) as
template. The polymerase chain reaction product was digested with
NcoI and HindIII and cloned between the
NcoI and HindIII sites of plasmid pMON5907 (a
generous gift of Stephen Lee (15)) to form pEG7242. The primary
sequence of phoQ in plasmid pEG7242 was confirmed by DNA
sequence analysis. Second, pEG7242 plasmid DNA was digested with
NcoI, the 5
-protruding ends were filled in with the Klenow
fragment of DNA polymerase and dNTPs and then digested with
HindIII. The resulting 449-base pair fragment was purified
and cloned between the NdeI (filled in) and
HindIII sites of plasmid pT7-7 to form pT7-7Qp. Plasmid
pT7-7Qp harbors an ATG initiation codon 11 base pairs downstream of the
ribosome binding site in pT7-7. The ATG is followed by the DNA sequence corresponding to codons 45-190 of phoQ and by the
translation termination codon TAA. Bacteria were grown at 37 °C in
Luria broth (LB (16)) or in N-minimal medium containing 0.1% casamino
acids, 38 mM glycerol, and the indicated concentrations of
MgCl2 and CaCl2 (17). Ampicillin and
isopropyl-
-D-thiogalactopyranoside were used at 50 µg/ml and 0.7 mM, respectively.
The sensor domain
of the S. typhimurium PhoQ protein (residues 45-190
preceded by an N-terminal Met; PhoQp) was purified from E. coli strain EG9649. Expression of PhoQp was achieved by addition of 0.7 mM isopropyl--D-thiogalactopyranoside
to induce the DE3-encoded T7 RNA polymerase. Cells were pelleted,
resuspended in 10 mM Tris (pH 8.0), and subjected to
sonication. Cell debris was removed by centrifugation, and ammonium
sulfate was added (55% saturation) to the supernatant. Following
centrifugation, the pellet was resuspended in 10 mM Tris
(pH 8.0), and the supernatant was combined with ammonium sulfate (90%
saturation), and following centrifugation, the pellet was resuspended
in 10 mM Tris (pH 8.0). Both resuspended pellets were
passed through an Econo-Pac 10DG desalting column (Bio-Rad) in 100 mM NaCl, 10 mM Tris (pH 8.0) and then combined. The PhoQp fragment was further purified on a Waters high performance liquid chromatography system (flow rate (0.3 ml/min) and protein monitoring at 280 nm) using several chromatographic columns. First, we
used Waters Protein Pak 300 SW 7.5 mm × 30-cm gel filtration column. Fractions containing PhoQp (as determined by Coomassie Blue
staining of SDS-polyacrylamide gel electrophoresis) were pooled and
passed through an Econo-Pac 10DG desalting column (Bio-Rad) in 20 mM NaCl, 10 mM Tris (pH 8.0). These fractions
were loaded onto an ion exchange column (Waters advanced purification
glass column AP-1), and PhoQp was eluted using a 20 mM to
1.0 M NaCl linear gradient in 10 mM Tris (pH
8.0). The PhoQp-containing fractions, which eluted at 510 mM NaCl, were pooled and passed through an Econo-Pac 10DG
desalting column (Bio-Rad) in 10 mM
KH2PO4, 10 mM Tris (pH 6.9). These
fractions were loaded onto a Bio-Rad Bio-Scale ceramic hydroxyapatite
CHT-5 type I column. PhoQp was eluded with a 10-500 mM
KH2PO4 linear gradient in 10 mM
Tris (pH 6.9). The PhoQp-containing fractions, which eluted at 410 mM KH2PO4, were pooled and passed
through an Econo-Pac 10DG desalting column (Bio-Rad) in 10 mM Tris (pH 8.0).
The identity, purity, and concentration of the fragment were analyzed
by electrospray mass spectrometry, quantitative amino acid analysis,
and N-terminal amino acid sequencing. The predicted molecular mass for
PhoQp was 17,042.2 daltons and that determined by electrospray mass
spectrometry was 17,040 daltons; the extinction coefficient determined
from the amino acid analysis was 280 = 1.94 cm2 mg
1. Electrospray mass spectrometry,
quantitative amino acid analysis, and N-terminal amino acid sequencing
were performed by the Protein Chemistry Laboratory of the Washington
University School of Medicine.
Circular dichroism studies of the PhoQp fragment were carried out in a Jasco J600A spectropolarimeter. The PhoQp sample was analyzed at a concentration of 0.25 mg/ml in 50 mM choline chloride, 10 mM Tris (pH 7.0) at 25 °C from 195 to 250 nm. Intrinsic fluorescence was measured using a PTI (Alphascan) spectrofluorometer at a constant excitation wavelength of 280 nm, and emission spectra were collected from 300 to 400 nm. These experiments were carried out with the PhoQp fragment at a concentration of 0.026 mg/ml in 10 mM Tris (pH 7.0) and varying concentrations of monovalent (choline chloride, KCl, and NaCl) and divalent salts (MgCl2, CaCl2, and BaCl2). The solution containing choline chloride was titrated with aliquots of the solution containing MgCl2, and the change in intrinsic fluorescence was monitored as a function of Mg2+ concentration. At each titration step the concentration of PhoQp and the ionic strength remained constant, thus the fluorescence changes were not due to changes in the ionic strength of the medium. A buffer blank was subtracted from the spectrum under all conditions.
-Galactosidase activity was
determined in overnight cultures of strains EG9065 and EG9564 as
described (16).
To examine the ability of the periplasmic domain of PhoQ to bind divalent cations, we purified a polypeptide consisting of residues 45-190 of the Salmonella PhoQ protein using a combination of chromatographic techniques as described under "Experimental Procedures." Amino acid analysis and N-terminal sequencing of the purified polypeptide revealed a composition that was consistent with that predicted from the nucleotide sequence of this portion of the phoQ gene with an additional N-terminal Met, which had been incorporated into the phoQ gene in the expression vector. Furthermore, electrospray mass spectrometry revealed a mass of 17,040 daltons for the purified polypeptide, which is consistent with a predicted mass of 17,042.2 daltons.
Mg2+ and Ca2+ Alter the Trp Fluorescence Pattern of the Sensing Domain of PhoQTo investigate the ability
of the periplasmic domain of PhoQ to bind divalent cations, we examined
the circular dichroism (CD) and fluorescence spectra of this
polypeptide, which harbors four Trp residues (Fig. 1).
The CD spectra of PhoQp suggested the presence of both -helix and
-sheet structures in this polypeptide. However, no changes were
detected in the CD spectra in the presence of MgCl2 (20 mM) or NaCl (50 mM). On the other hand, the
intrinsic fluorescence at 335.0 nm of PhoQp was significantly modified
in the presence of MgCl2 (Fig.
2A). Changes could be observed with as little
as 7 mM MgCl2. CaCl2 could also
modify the fluorescence spectra of PhoQp (data not shown) but
BaCl2, which is unable to repress transcription of
PhoP-regulated genes in vivo, hardly modified the intrinsic
fluorescence of this polypeptide when tested at concentrations up to
200 mM (Fig. 2B).
These results provide a direct demonstration that the periplasmic region of PhoQ specifically binds Mg2+ and Ca2+. Moreover, they are consistent with our previous findings that Mg2+ could alter the trypsinization pattern of the periplasmic domain of the PhoQ protein presented in spheroplasts. Furthermore, they are in agreement with those reported by Waldburger and Sauer (18), who found that divalent cations promoted stabilization to urea denaturation of a similar PhoQ fragment derived from E. coli. Waldburger and Sauer (18), however, could not detect changes in the fluorescence spectra of their PhoQ-derived fragment when MgCl2 (10 mM) was added. The differences in the fluorescence results may reflect the presence of different salts in the buffers used to fix the ionic strength: 0.2 M KCl in the Waldburger and Sauer's experiments and choline chloride in those reported here. Consistent with this hypothesis, we established that higher concentrations of MgCl2 were required to promote fluorescence changes in PhoQp when KCl was present at 100 mM (data not shown). On the other hand, choline, which is not predicted to compete with Mg2+ for binding to PhoQp due to its large size, did not change the intrinsic fluorescence of PhoQp when added up to 500 mM (data not shown). Alternatively or in addition, the differences in the fluorescence results could be due to the 28 amino acid differences that exist between the 146 residues that comprise the periplasmic regions of the Salmonella and E. coli PhoQ proteins.
The PhoQ Protein Has Distinct Binding Sites for Ca2+ and Mg2+We had established previously that Ca2+ could repress expression of the PhoP-activated gene psiD with half-maximal repression attained at lower concentrations than those achieved with Mg2+ (8). However, the Ca2+-mediated effect could have resulted from the combined actions of Ca2+ and Mg2+, since the repressing role of Ca2+ had only been investigated in the presence of 8 µM Mg2+.
To establish the importance of Ca2+ and Mg2+ in
the regulation of the PhoP/PhoQ system, we examined the expression of
the psiD gene in the presence of different concentrations of
MgCl2, CaCl2 (with no added Mg2+),
or a mixture of these two salts. Repression of psiD
transcription was achieved at lower concentrations of Mg2+
than Ca2+, and maximal repression was attained when both
cations were present (Fig. 3A). An analysis
of these data is consistent with the existence of distinct binding
sites for Mg2+ and Ca2+ in the PhoQ protein
(see legend to Fig. 3). The apparent affinities of the PhoQ protein for
Mg2+ and Ca2+ were 1.4 ± 0.2 105 M1 and 4 ± 1 103 M
1, respectively. These two
sites do not appear to interact with one another because the repressing
effect in the presence of both cations was equivalent to the sum of the
individual repressing effects.
A Mutant PhoQ Protein That Is Less Responsive to Ca2+
A S. typhimurium strain harboring the
pho-24 allele overexpresses several PhoP-activated genes and
is attenuated for mouse virulence (19). We have established previously
that this mutant harbors a single amino acid substitution,
Thr48 Ile, in the periplasmic domain of the PhoQ
protein (8). To establish the sensitivity of the mutant PhoQ protein to
Mg2+ and Ca2+, we examined the transcriptional
activity of the psiD gene in a strain harboring a
chromosomal pho-24 allele. The mutant PhoQ protein had a
lower affinity for Ca2+ than the wild-type
(KaCa2+: 6 ± 1 102
M
1 versus 4 ± 1 103 M
1; Fig. 3B). On
the other hand, the affinity for Mg2+ was virtually
identical in the wild-type and mutant PhoQ proteins (KaMg2+: 1.5 ± 0.2 105 M
1 versus 1.4 ± 0.2 105 M
1); yet, the
transcriptional activity of the psiD gene at repressing concentrations of Mg2+ was higher in the pho-24
mutant than in the wild-type strain (Fig. 3B). Cumulatively,
these results indicate that Thr48 is required for normal
Ca2+ sensing and support a model in which the PhoQ protein
has distinct sites for Mg2+ and Ca2+.
The PhoQ protein represents the first and only example of a receptor that senses extracellular Mg2+ and Ca2+. In the PhoP/PhoQ signal transduction cascade, these cations act as primary signaling molecules rather than their familiar roles as cofactors (20) and second messengers (21). We have now provided direct evidence that Mg2+ and Ca2+ specifically bind to the sensing domain of the PhoQ protein. We would like to suggest that binding of divalent cations promotes a conformation in the PhoQ protein that is unfavorable for the activation of the PhoP protein, resulting in transcriptional repression of PhoP-activated genes.
The demonstration that the PhoQ protein harbors distinct sites for Mg2+ and Ca2+ and that these cations act independently to modulate PhoQ activity imply that both, Mg2+ and Ca2+, are physiologically relevant in the control of PhoP-regulated loci. This is further substantiated by the virulence attenuation that results from a single amino acid substitution in the periplasmic domain of the PhoQ protein that alters its response to Ca2+. Finally, the PhoQ protein may represent a new paradigm for divalent cation-binding proteins, since it does not have an EF-hand, a motif that is common among a large number of Ca2+-binding proteins (22), and shows no sequence similarity with the Ca2+-sensing receptor from mammalian parathyroid cells (23). Solving the crystal structure of the sensing domain of the PhoQ protein may reveal features of the divalent cation-binding sites that are not apparent from homology analysis and structural predictions of its primary amino acid sequence.