Comparison of the Pseudomonas aeruginosa and Escherichia coli PhoQ sensor domains: evidence for distinct mechanisms of signal detection

The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.     

Crystal structure of a functional dimer of the PhoQ sensor domain

The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.     

The connector SafA interacts with the multi-sensing domain of PhoQ in Escherichia coli

Sensor histidine kinases of two-component signal transduction systems (TCSs) respond to various environmental signals and transduce the external stimuli across the cell membrane to their cognate response regulators. Recently, membrane proteins that modulate sensory systems have been discovered. Among such proteins is SafA, which activates the PhoQ/PhoP TCS by direct interaction with the sensor PhoQ. SafA is directly induced by the EvgS/EvgA TCS, thus connecting the two TCSs, EvgS/EvgA and PhoQ/PhoP. We investigated how SafA interacted with PhoQ. Bacterial two-hybrid and reporter assays revealed that the C-terminal region (41-65 aa) of SafA activated PhoQ at the periplasm. Adding synthetic SafA(41-65) peptide to the cell culture also activated PhoQ/PhoP. Furthermore, direct interaction between SafA(41-65) and the sensor domain of PhoQ was observed by means of surface plasmon resonance. NMR spectroscopy of (15) N-labelled PhoQ sensor domain confirmed that SafA and Mg(2+) provoked a different conformational change of PhoQ. Site-directed mutagenesis studies revealed that R53, within SafA(41-65), was important for the activation of PhoQ, and D179 of the PhoQ sensor domain was required for its activation by SafA. SafA activated PhoQ by a different mechanism from cationic antimicrobial peptides and acidic pH, and independent of divalent cations and MgrB.     

The phosphatase activity is the target for Mg2+ regulation of the sensor protein PhoQ in Salmonella

The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. Environmental deprivation of Mg(2+) activates the PhoP/PhoQ signal transduction cascade, which results in an increased expression of genes necessary for survival inside the host. It was previously demonstrated that the interaction of Mg(2+) with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down-regulation of PhoP-activated genes. We have now examined the regulatory effect of Mg(2+) on the putative activities of the membrane-bound PhoQ. We demonstrated that Mg(2+) promotes a phospho-PhoP phosphatase activity in the sensor protein. This activity depends on the intactness of the conserved His-277, suggesting that the phosphatase active site overlaps the H box. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, because Mg(2+) did not stimulate the release of inorganic phosphate from phospho-PhoP in a fusion protein that lacks this sensing domain. These findings reveal that the sensor PhoQ harbors a phospho-PhoP phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg(2+)-triggered regulation of the PhoP/PhoQ system.     

Quantitative proteomic analysis of Salmonella enterica serovar Typhimurium under PhoP/PhoQ activation conditions

The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg(2+) concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg(2+) concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg(2+) concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg(2+) concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg(2+)-concentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella's adaptation to the host environment.     

Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg²⁺ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.     

Mutational analysis of the Escherichia coli PhoQ sensor kinase: differences with the Salmonella enterica serovar Typhimurium PhoQ protein and in the mechanism of Mg2+ and Ca2+ sensing

The PhoP-PhoQ two-component system plays a role in Mg2+ homeostasis and/or the virulence properties of a number of bacterial species. A Salmonella enterica serovar Typhimurium PhoQ sensor kinase mutant, in which the threonine at residue 48 in the periplasmic sensor domain is changed to an isoleucine, was shown previously to result in elevated expression of PhoP-activated genes and to affect mouse virulence, epithelial cell invasion, and sensitivity to macrophage killing. We characterized a complete set of proteins having amino acid substitutions at position 48 in the closely related Escherichia coli PhoQ protein. Numerous mutant proteins having amino acid substitutions with side chains of various sizes and characters displayed signaling phenotypes similar to that of the wild-type protein, indicating that interactions mediated by the wild-type threonine side chain are not required for normal protein function. Changes to amino acids with aromatic side chains had little impact on signaling in response to extracellular Mg2+ but resulted in reduced sensitivity to extracellular Ca2+, suggesting that the mechanisms of signal transduction in response to these two divalent cations are different. Surprisingly, the Ile48 protein displayed a defective phenotype rather than the hyperactive phenotype seen with the S. enterica serovar Typhimurium protein. We also describe a mutant PhoQ protein lacking the extracellular sensor domain with a defect in the ability to activate PhoP. The defect does not appear to be due to reduced autokinase activity but rather appears to be due to an effect on the stability of the aspartyl-phosphate bond of phospho-PhoP.     

Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs.     

Characterization of the catalytic activities of the PhoQ histidine protein kinase of Salmonella enterica serovar Typhimurium

Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2) or Mn2+ was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.     

Spatial distribution of cytoplasmic domains of the Mg(2+)-transporter MgtE, in a solution lacking Mg(2+), revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg(2+) transporter that controls cellular Mg(2+) concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg(2+)-free and Mg(2+)-bound forms. The Mg(2+)-binding sites lay at the interface of the 2 domains, making the Mg(2+)-bound form compact and globular. In the Mg(2+)-free structure, however, the domains are far apart, and the Mg(2+)-binding sites are destroyed. Therefore, it is unclear how Mg(2+)-free MgtE changes its conformation to accommodate Mg(2+) ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg(2+) in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg(2+)-bound crystal structure, facilitating efficient capture of Mg(2+) with increased intracellular Mg(2+) concentration, which is necessary to close the gate.     

Mg(2+) modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3

Extracellular Mg(2+) directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg(2+) presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg(2+)-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K(+) channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg(2+) on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg(2+)-sensitivity and mimicked the slowing of activation kinetics caused by Mg(2+) binding to the wild-type channel. These results suggest that Mg(2+) modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg(2+), indicating that D284 and D319 do not mediate the slowing of activation caused by Mg(2+) binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg(2+)-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg(2+) sensitivity but is in the vicinity of the binding site. We conclude that Mg(2+) binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K(+) channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.     

Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin alphaIIb cytoplasmic domain

Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the alphaIIb cytoplasmic domain of integrinalphaIIbbeta(3). We have previously demonstrated that CIB lacks structural stability in the absence of divalent metal ions but that it acquires a well-folded conformation upon addition of Ca(2+) or Mg(2+). Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca(2+)-bound CIB (Ca(2+)-CIB) and the Mg(2+)-bound protein (Mg(2+)-CIB) bind with high affinity and through a similar mechanism to alphaIIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca(2+)-CIB and Mg(2+)-CIB, but involve hydrophobic interactions in each case. Since the Mg(2+) concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the alphaIIb cytoplasmic domain independent of an intracellular Ca(2+) stimulus in vivo. This raises the question of whether CIB can act as a Ca(2+) sensor in alphaIIbbeta(3) signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in alphaIIbbeta(3), post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and alphaIIbbeta(3). Differences in NMR spectra do suggest, however, that Ca(2+)-binding to the Mg(2+)- CIB-alphaIIb complex induces subtle structural changes that could further modulate the activity of alphaIIbbeta(3).