Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens

Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or virR_D57N onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.     

Domain Analysis of ArcS,the Hybrid Sensor Kinase of the Shewanella oneidensis MR-1 Arc Two-Component System,Reveals Functional Differentiation of Its Two Receiver Domains

In all species of the genus Shewanella, the redox-sensing Arc two-component system consists of the response regulator ArcA, the sensor kinase ArcS, and the separate phosphotransfer protein HptA. Compared to its counterpart ArcB in Escherichia coli, ArcS has a significantly different domain structure. Resequencing and reannotation revealed that in the N-terminal part, ArcS possesses a periplasmic CaChe-sensing domain bracketed by two transmembrane domains and, moreover, that ArcS has two cytoplasmic PAS-sensing domains and two receiver domains, compared to a single one of each in ArcB. Here, we used a combination of in vitro phosphotransfer studies on purified proteins and phenotypic in vivo mutant analysis to determine the roles of the different domains in ArcS function. The analysis revealed that phosphotransfer occurs from and toward the response regulator ArcA and involves mainly the C-terminal RecII domain. However, RecI also can receive a phosphate from HptA. In addition, the PAS-II domain, located upstream of the histidine kinase domain, is crucial for function. The results support a model in which phosphorylation of RecI stimulates histidine kinase activity of ArcS in order to maintain an appropriate level of phosphorylated ArcA according to environmental conditions. In addition, the study reveals some fundamental mechanistic differences between ArcS/HptA and ArcB with respect to signal perception and phosphotransfer despite functional conservation of the Arc system in Shewanella and E. coli.     

Role of the Sln1‐phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.     

Ligand and antagonist driven regulation of the Vibrio cholerae quorum-sensing receptor CqsS

Quorum sensing, a bacterial cell–cell communication process, controls biofilm formation and virulence factor production in Vibrio cholerae, a human pathogen that causes the disease cholera. The major V. cholerae autoinducer is (S)‐3‐hydroxytridecan‐4‐one (CAI‐1). A membrane bound two‐component sensor histidine kinase called CqsS detects CAI‐1, and the CqsS → LuxU → LuxO phosphorelay cascade transduces the information encoded in CAI‐1 into the cell. Because the CAI‐1 ligand is known and because the signalling circuit is simple, consisting of only three proteins, this system is ideal for analysing ligand regulation of a sensor histidine kinase. Here we reconstitute the CqsS → LuxU → LuxO phosphorylation cascade in vitro. We find that CAI‐1 inhibits the initial auto‐phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI‐1‐controlled. CAI‐1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities. Our data are consistent with a two‐state model for ligand control of histidine kinases.     

How to Switch Off a Histidine Kinase: Crystal Structure of Geobacillus stearothermophilus KinB with the inhibitor Sda

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-Å-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.     

A new class of signal transducer in His-Asp phosphorelay systems

Nitrate transport activity of the LtnT permease of the cyanobacterium Synechococcus elongatus is activated when LtnA, a response regulator without an effector domain, is phosphorylated by LtnB, a hybrid histidine kinase. We identified a protein (LtnC) that is required for activation of LtnT. LtnC consists of an N-terminal histidine-containing phosphoacceptor (HisKA) domain, a receiver domain, and a unique C-terminal domain found in some cyanobacterial proteins. Because LtnC lacks an ATP-binding kinase domain of a histidine kinase, it is incapable of autophosphorylation, but LtnC is phosphorylated by LtnA. The histidine residue in the HisKA domain but not the aspartate residue in the receiver domain is essential for phosphorylation of LtnC and activation of LtnT. LtnC phosphorylation leads to oligomerization of the protein. Fusion of the C-terminal domain of LtnC to glutathione S-transferase, which forms oligomers, also activates LtnT, suggesting that oligomerization of the LtnC C-terminal domain causes LtnT activation. These results indicate that the C-terminal domain of LtnC acts as an effector domain that directs the output of the signal from the phosphorelay system. The two-step (His-Asp-His) phosphorelay system, composed of the LtnB, LtnA, and LtnC proteins, is distinct from the known phosphorelay systems, namely, the typical two-component system (His-Asp) and the multistep phosphorelay system (His-Asp-His-Asp), because the HisKA domain of LtnC is the terminal phosphoacceptor that determines the signal output. LtnC is a new class of signal transducer in His-Asp phosphorelay systems that contains a HisKA domain and an effector domain.     

Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.     

The yeast YPD1/SLN1 complex: insights into molecular recognition in two-component signaling systems

In Saccharomyces cerevisiae, a branched multistep phosphorelay signaling pathway regulates cellular adaptation to hyperosmotic stress. YPD1 functions as a histidine-phosphorylated protein intermediate required for phosphoryl group transfer from a membrane-bound sensor histidine kinase (SLN1) to two distinct response regulator proteins (SSK1 and SKN7). These four proteins are evolutionarily related to the well-characterized "two-component" regulatory proteins from bacteria. Although structural information is available for many two-component signaling proteins, there are very few examples of complexes between interacting phosphorelay partners. Here we report the first crystal structure of a prototypical monomeric histidine-containing phosphotransfer (HPt) protein YPD1 in complex with its upstream phosphodonor, the response regulator domain associated with SLN1.     

Design and Signaling Mechanism of Light-Regulated Histidine Kinases

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ∼ 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40°-60° rotational movement within an α-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.     

Spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity

Signal transduction proteins are often multi‐domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two‐component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so‐called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross‐talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.     

Determinants of homodimerization specificity in histidine kinases

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.     

The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p.

The Saccharomyces cerevisiae Sln1 protein is a ''two-component'' regulator involved in osmotolerance. Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.     

Phosphotransfer reactions of the CbbRRS three-protein two- component system from Rhodopseudomonas palustris CGA010 appear to be controlled by an internal molecular switch on the sensor kinase

The CbbRRS system is an atypical three-protein two-component system that modulates the expression of the cbb(I) CO(2) fixation operon of Rhodopseudomonas palustris, possibly in response to a redox signal. It consists of a membrane-bound hybrid sensor kinase, CbbSR, with a transmitter and receiver domain, and two response regulator proteins, CbbRR1 and CbbRR2. No detectable helix-turn-helix DNA binding domain is associated with either response regulator, but an HPt domain and a second receiver domain are predicted at the C-terminal region of CbbRR1 and CbbRR2, respectively. The abundance of conserved residues predicted to participate in a His-Asp phosphorelay raised the question of their de facto involvement. In this study, the role of the multiple receiver domains was elucidated in vitro by generating site-directed mutants of the putative conserved residues. Distinct phosphorylation patterns were obtained with two truncated versions of the hybrid sensor kinase, CbbSR(T189) and CbbSR(R96) (CbbSR beginning at residues T189 and R96, respectively). These constructs also exhibited substantially different affinities for ATP and phosphorylation stability, which was found to be dependent on a conserved Asp residue (Asp-696) within the kinase receiver domain. Asp-696 also played an important role in defining the specificity of phosphorylation for response regulators CbbRR1 or CbbRR2, and this residue appeared to act in conjunction with residues within the region from Arg-96 to Thr-189 at the N terminus of the sensor kinase. The net effect of concerted interactions at these distinct regions of CbbSR created an internal molecular switch that appears to coordinate a unique branched phosphorelay system.     

Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p

Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the αA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.     

Characterization of sporulation histidine kinases of Bacillus anthracis

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.     

The Group III Two-Component Histidine Kinase of Filamentous Fungi Is Involved in the Fungicidal Activity of the Bacterial Polyketide Ambruticin

We have shown that the plant pathogen Alternaria brassicicola exhibited very high susceptibility to ambruticin VS4 and to a lesser extent to the phenylpyrrole fungicide fludioxonil. These compounds are both derived from natural bacterial metabolites with antifungal properties and are thought to exert their toxicity by interfering with osmoregulation in filamentous fungi. Disruption of the osmosensor group III histidine kinase gene AbNIK1 (for A. brassicola NIK1) resulted in high levels of resistance to ambruticin and fludioxonil, while a mutant isolate characterized by a single-amino-acid substitution in the HAMP domain of the kinase only exhibited moderate resistance. Moreover, the natural resistance of Saccharomyces cerevisiae to these antifungal molecules switched to sensitivity in strains expressing AbNIK1p. We also showed that exposure to fludioxonil and ambruticin resulted in abnormal phosphorylation of a Hog1-like mitogen-activated protein kinase (MAPK) in A. brassicicola. Parallel experiments carried out with wild-type and mutant isolates of Neurospora crassa revealed that, in this species, ambruticin susceptibility was dependent on the OS1-RRG1 branch of the phosphorelay pathway downstream of the OS2 MAPK cascade but independent of the yeast Skn7-like response regulator RRG2. These results show that the ability to synthesize a functional group III histidine kinase is a prerequisite for the expression of ambruticin and phenylpyrrole susceptibility in A. brassicicola and N. crassa and that, at least in the latter species, improper activation of the high-osmolarity glycerol-related pathway could explain their fungicidal properties.