The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate-inducible expression of Pho regulon genes in Bacillus subtilis

PhoP–PhoR, one of three two-component systems known to be required to regulate the pho regulon in Bacillus subtilis , directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli , was autophosphorylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue. Phospho–PhoR phosphorylated its cognate response regulator, PhoP in vitro . B. subtilis phoR was placed in the Bacillus chromosome under the control of the P _spac promoter, which is IPTG inducible. The wild-type phoR , under either native promoter or P _spac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild-type phoR , or phoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR sequences or various extended N-terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild-type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C-terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate-limited conditions, and that the regulation for repression of APase under phosphate-replete conditions remains intact.     

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.     

Topology of the PhoR protein of Escherichia coli and functional analysis of internal deletion mutants

The PhoR protein of Escherichia coli K-12 belongs to a family of structurally related sensor-kinases that regulate responses to environmental stimuli. These proteins are often located in the inner membrane with two membrane-spanning segments that are separated by a periplasmic domain, which is supposed to sense the environmental stimuli. However, the hydrophobicity plot of PhoR suggests a somewhat different topology in which a large periplasmic domain is lacking and an extended cytoplasmic domain is present besides the kinase domain. In protease-accessibility experiments and by using phoR-phoA gene fusions, the topology of PhoR was investigated and the absence of a large periplasmic domain was confirmed. Furthermore, the function of the extended cytoptasmic domain was studied by creating internal deletions. The mutations in this domain resulted in a constitutive expression of the pho regulon, indicating that the mutant PhoR proteins are locked in their kinase function. We propose that this extended cytoplasmic domain functions by sensing an internal signal that represses the kinase function of the PhoR protein.     

PhoR1-PhoP1, a third two-component system of the family PhoRP from Myxococcus xanthus: role in development

The pair PhoR1-PhoP1 is the third two-component system of the family PhoRP reported in M. xanthus. PhoR1 is a histidine kinase anchored to the membrane through a transmembrane domain located in the amino-terminal portion of the protein. As a result, 93% of the protein is located in the cytoplasm. This topology is unusual in the PhoR-type histidine kinases. PhoP1 is a response regulator with a helix-loop-helix motif typical of the DNA-binding proteins. Although the operon phoPR1 is expressed during vegetative growth, it peaks during development. The expression levels of this operon are higher in phosphate-containing media than in those in which the nutrient is absent. A deletion mutant in this system exhibits a delay in aggregation and the formation of fruiting bodies larger than those of the wild-type strain. The expression of the operon is autoregulated. This system is also partially responsible for the expression of Mg-independent acid and neutral phosphatases, but it is not required for the expression of alkaline phosphatases.     

phoR1, a gene encoding a new histidine protein kinase Myxococcus xanthus

A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins. Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical. As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones. The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR. This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa. The M. xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing. A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain. A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetic basis for the stimulatory effect of phosphorylation on the methylesterase activity of CheB

Response regulators are activated to elicit a specific cellular response to an extracellular stimulus via phosphotransfer from a cognate sensor histidine kinase to a specific aspartate residue. Phosphorylation at the conserved aspartate residue modulates the activity of the response regulator. Methylesterase CheB is a two-domain response regulator composed of a regulatory domain and an effector domain with enzymatic activity. CheB functions within the bacterial chemotaxis pathway to control the level of chemoreceptor methylation. In its unphosphorylated state, the regulatory domain inhibits methylesterase activity of the effector domain. Phosphorylation of the regulatory domain leads to an enhancement of methylesterase activity through a relief of inhibition and a stimulatory effect on catalysis. CheB is a useful model protein for understanding the effects of phosphorylation of the regulatory domain on interdomain interactions and stimulation of enzymatic activity of the effector domain. Kinetic analyses of CheB activation indicate that the basis for the nearly 100-fold methylesterase activation upon phosphorylation is due to a change in the catalytic rate constant for the methylesterase reaction. It is also shown that the P2 domain of histidine kinase CheA inhibits the methylesterase activity of CheB and that this inhibition is decreased upon phosphorylation of CheB. Finally, studies of methylesterase catalysis by the free catalytic domain in the presence and absence of the regulatory domain have enabled detection of an association between the two domains in the absence of the linker.     

The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis

Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Mechanism of regulation of the bifunctional histidine kinase NtrB in Escherichia coli

NtrB is the bifunctional histidine kinase for nitrogen regulation. Dependent on the availability of nitrogen, it either autophosphorylates and serves as the phosphodonor for its cognate response regulator, NtrC, or, it promotes the rapid dephosphorylation of NtrC-P. The activity of NtrB depends on the interaction of two subdomains within its transmitter domain, the H-domain and the kinase domain. Both phosphotransfer activity and phosphatase activity reside in the H-domain. When separately expressed, this domain acts as a phosphatase. Interaction with the kinase domain results in the inhibition of the phosphatase activity and the phosphorylation of the conserved histidine of the H-domain.     

Genetic analysis,structural modeling,and direct coupling analysis suggest a mechanism for phosphate signaling in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Proper phosphate signaling is essential for robust growth of Escherichia coli and many other bacteria. The phosphate signal is mediated by a classic two component signal system composed of PhoR and PhoB. The PhoR histidine kinase is responsible for phosphorylating/dephosphorylating the response regulator, PhoB, which controls the expression of genes that aid growth in low phosphate conditions. The mechanism by which PhoR receives a signal of environmental phosphate levels has remained elusive. A transporter complex composed of the PstS, PstC, PstA, and PstB proteins as well as a negative regulator, PhoU, have been implicated in signaling environmental phosphate to PhoR.

Results

This work confirms that PhoU and the PstSCAB complex are necessary for proper signaling of high environmental phosphate. Also, we identify residues important in PhoU/PhoR interaction with genetic analysis. Using protein modeling and docking methods, we show an interaction model that points to a potential mechanism for PhoU mediated signaling to PhoR to modify its activity. This model is tested with direct coupling analysis.

Conclusions

These bioinformatics tools, in combination with genetic and biochemical analysis, help to identify and test a model for phosphate signaling and may be applicable to several other systems.

     

A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis

Bacterial cell wall homeostasis is an intricately coordinated process that ensures that envelope integrity is maintained during cell growth and division, but can also adequately respond to growth‐limiting conditions such as phosphate starvation. In Bacillus subtilis, biosynthesis of the two major cell wall components, peptidoglycan and anionic polymers, is controlled by a pair of paralogous two‐component systems, WalRK and PhoPR respectively. Favorable growth conditions allow for a fast rate of cell wall biosynthesis (WalRK‐ON) and the incorporation of the phosphate‐containing anionic polymer teichoic acids (PhoPR‐OFF). In contrast, growth‐restricted cells under phosphate‐limiting conditions reduce the incorporation of peptidoglycan building blocks (WalRK‐OFF) and switch from the phosphate‐containing teichoic acids to the phosphate‐free anionic polymer teichuronic acid (PhoPR‐ON). Botella et al. (2014) deepen our knowledge on the PhoPR system by identifying one signal that is perceived by its histidine kinase PhoR. In fast‐growing cells, intracellular intermediates of teichoic acid biosynthesis are sensed by the cytoplasmic Per‐Arnt‐Sim domain as an indicator of favorable conditions, thereby inhibiting the autokinase activity of PhoR and keeping the system inactive. Depletion of teichoic acid building blocks under phosphate‐limiting conditions relieves this inhibition, activates PhoPR‐dependent signal transduction and hence the switch to teichuronic acid biosynthesis.     

Re-examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli

In Escherichia coli , EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR. OmpR-phosphate regulates the expression of the porin genes, ompF and ompC . To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted. Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity. The periplasmic domain of EnvZ was also replaced with the non-homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis . Osmoregulation of OmpF and OmpC production in cells containing the PhoR–EnvZ hybrid protein was indistinguishable from that in cells containing wild-type EnvZ. Identical results were obtained with an envZ – pta/ack strain, which could not synthesize acetyl phosphate. Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study. These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.     

The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli

Many Proteobacteria possess the paralogous PTS^Ntr, in addition to the sugar transport phosphotransferase system (PTS). In the PTS^Ntr phosphoryl‐groups are transferred from phosphoenolpyruvate to protein EIIA^Ntr via the phosphotransferases EI^Ntr and NPr. The PTS^Ntr has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS^Ntr plays a role in potassium homeostasis. In particular, EIIA^Ntr binds to and stimulates activity of a two‐component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high‐affinity K⁺ transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS^Ntr. The pho regulon, which comprises more than 30 genes, is activated by the two‐component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA^Ntr are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild‐type level in mutants deficient for EIIA^Ntr phosphorylation suggesting that non‐phosphorylated EIIA^Ntr modulates pho. Protein interaction analyses reveal binding of EIIA^Ntr to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA^Ntr is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.     

Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia

AtsR is a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia that negatively regulates quorum sensing and virulence factors such as biofilm production, type 6-secretion, and protease secretion. Here we elucidate the mechanism of AtsR phosphorelay by site-directed mutagenesis of predicted histidine and aspartic acid phosphoacceptor residues. We demonstrate by in vitro phosphorylation that histidine 245 and aspartic acid 536 are conserved sites of phosphorylation in AtsR, and we also identify the cytosolic response regulator AtsT (BCAM0381) as a key component of the AtsR phosphorelay pathway. Monitoring the function of AtsR and its derivatives in vivo by measuring extracellular protease activity and swarming motility confirmed the in vitro phosphorylation results. Together we find that the AtsR receiver domain plays a fine-tuning role in determining the levels of phosphotransfer from its sensor kinase domain to the AtsT response regulator.