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.     

Regulation of acid resistance by connectors of two-component signal transduction systems in Escherichia coli

Two-component signal transduction systems (TCSs), utilized extensively by bacteria and archaea, are involved in the rapid adaptation of the organisms to fluctuating environments. A typical TCS transduces the signal by a phosphorelay between the sensor histidine kinase and its cognate response regulator. Recently, small-sized proteins that link TCSs have been reported and are called "connectors." Their physiological roles, however, have remained elusive. SafA (sensor associating factor A) (formerly B1500), a small (65-amino-acid [65-aa]) membrane protein, is among such connectors and links Escherichia coli TCSs EvgS/EvgA and PhoQ/PhoP. Since the activation of the EvgS/EvgA system induces acid resistance, we examined whether the SafA-activated PhoQ/PhoP system is also involved in the acid resistance induced by EvgS/EvgA. Using a constitutively active evgS1 mutant for the activation of EvgS/EvgA, we found that SafA, PhoQ, and PhoP all contributed to the acid resistance phenotype. Moreover, EvgS/EvgA activation resulted in the accumulation of cellular RpoS in the exponential-phase cells in a SafA-, PhoQ-, and PhoP-dependent manner. This RpoS accumulation was caused by another connector, IraM, expression of which was induced by the activation of the PhoQ/PhoP system, thus preventing RpoS degradation by trapping response regulator RssB. Acid resistance assays demonstrated that IraM also participated in the EvgS/EvgA-induced acid resistance. Therefore, we propose a model of a signal transduction cascade proceeding from EvgS/EvgA to PhoQ/PhoP and then to RssB (connected by SafA and IraM) and discuss its contribution to the acid resistance phenotype.     

PhoP can activate its target genes in a PhoQ-independent manner

The PhoP/PhoQ two-component system controls the extracellular magnesium depletion response in Salmonella enterica. Previous studies have shown that PhoP is unable to up-regulate its target genes in the absence of PhoQ function. In this work, we demonstrate that PhoP overexpression can substitute for PhoQ- and phosphorylation-dependent activation. Either a high concentration of PhoP or activation via phosphorylation stimulates PhoP self-association.     

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.     

Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofluoride

The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg(2+). Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation.     

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.     

The H box-harboring domain is key to the function of the Salmonella enterica PhoQ Mg2+-sensor in the recognition of its partner PhoP

In two-component signaling systems, the transduction strategy relies on a conserved His-Asp phosphoryl exchange between the sensor histidine kinase and its cognate response-regulator, and structural and functional consensus motifs are found when comparing either the diverse histidine kinases or response regulators present in a single cell. Therefore, the mechanism that guarantees the specific recognition between partners of an individual pair is essential to unequivocally generate the appropriate adaptive response. Based on sequence alignments with other histidine kinases, we dissected the Salmonella enterica Mg2+-sensor PhoQ in different subdomains and examined by in vivo and in vitro assays its interaction with the associated response regulator PhoP. This signal transduction system allows Salmonella to withstand environmental Mg2+ limitation by triggering gene expression that is vital throughout the infective cycle in the host. Using resonant mirror biosensor technology, we calculated the kinetic and equilibrium binding constants and determined that the His-phosphotransfer domain is essential for the PhoQ specific recognition and interaction with PhoP. Additionally, we show the role of this domain in the bimolecular transphosphorylation and provide evidence that this region undergoes dimerization.     

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.