An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints.     

A genomic analysis of two-component signal transduction in Streptococcus pneumoniae

A genomics-based approach was used to identify the entire gene complement of putative two-component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram-positive pathogen highlights the potential of two-component signal transduction as a multicomponent target for antibacterial drug discovery.     

Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria.     

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.     

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.     

Identification of a novel two component system in Thermotoga maritima. Complex stoichiometry and crystallization

Two-component signal transduction systems, comprised of histidine kinase and its cognate response regulator, are the predominant mechanism by which microorganisms sense and respond to changes in many different environmental conditions. Different Thermotoga maritima histidine kinases have been used as prototypes; among them, the orphan TM0853 has been presented as a structural model of class I histidine kinases. We used phosphotransfer assays to identify TM0468 as the partner response regulator of TM0853. Since full-length TM0853 can be produced as a soluble protein in Escherichia coli, it was used to analyze the union stoichiometry in an intact two-component system for the first time. We demonstrate that TM0853, or its cytoplasmic catalytic portion, form a 1:1 complex with TM0468 with native PAGE. The complex band is unique, even in the presence of an excess of each individual protein, indicating that the union is cooperative. We corroborated these findings by using ultracentrifugation assays. Therefore, we propose that the general mode of interaction in an orthodox two-component system may be the stoichiometric and cooperative complex between a dimeric histidine kinase and two response regulators. Finally, we have been able to produce protein crystals of the complex between the cytoplasmic portion of TM0853 and TM0468 that diffract to 2.8 A Bragg spacing.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.     

The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.     

Mapping the interaction sites of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> phytochrome FphA with the global regulator VeA and the White Collar protein LreB

Aspergillus nidulans senses red and blue-light and employs a phytochrome and a Neurospora crassa White Collar (WC) homologous system for light perception and transmits this information into developmental decisions. Under light conditions it undergoes asexual development and in the dark it develops sexually. The phytochrome FphA consists of a light sensory domain and a signal output domain, consisting of a histidine kinase and a response regulator domain. Previously it was shown that the phytochrome FphA directly interacts with the WC-2 homologue, LreB and another regulator, VeA. In this paper we mapped the interaction of FphA with LreB to the histidine kinase and the response regulator domain at the C-terminus in vivo using the bimolecular fluorescence complementation assay and in vitro by co-immunoprecipitation. In comparison, VeA interacted with FphA only at the histidine kinase domain. We present evidence that VeA occurs as a phosphorylated and a non-phosphorylated form in the cell. The phosphorylation status of the protein was independent of the light receptors FphA, LreB and the WC-1 homologue LreA.     

Cross-talk suppression between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli

Many bacteria possess large numbers of two-component signalling systems, which are composed of histidine kinase-response regulator pairs. The high level of sequence similarity between some systems raises the possibility of undesired cross-talk between a histidine kinase and a non-cognate response regulator. Although molecular specificity ensures that phospho-transfer occurs primarily between correct partners, even a low level of inappropriate cross-talk could lead to unacceptable levels of noise or interference in signal transduction. To explore mechanisms that provide insulation against such interference, we have examined cross-talk between the histidine kinase CpxA and non-cognate response regulator OmpR in Escherichia coli. Our results show that there are two mechanisms that suppress cross-talk between these two proteins, which depend on the corresponding cognate partners CpxR and EnvZ and on the bifunctional nature of the histidine kinases CpxA and EnvZ. When cross-talk is detectable, we find it is independent of CpxA stimulus. We also show that cross-talk suppression leads to mutational robustness, i.e. it masks the effects of mutations that would otherwise lead to increased cross-talk. The mechanisms that provide insulation against interference described here may be applicable to many other two-component systems.     

Molecular modeling and functional analysis of the AtoS-AtoC two-component signal transduction system of Escherichia coli

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.     

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.     

Detection of a mammalian histone H4 kinase that has yeast histidine kinase-like enzymic activity

A well characterized histidine kinase purified from yeast has been shown to phosphorylate histone H4 on a histidine residue. This enzyme is unlike the two-component histidine kinases predominantly found in prokaryotes. Until now, a histidine kinase similar to this yeast enzyme has not been purified from a mammalian source. By using a purification scheme similar to that used to purify the yeast histidine kinase, a protein fraction with histone H4 kinase activity has been isolated from porcine thymus. The yeast histidine kinase was shown to be detectable using an in-gel kinase assay system and using this system, four major bands of histone H4 kinase activity were apparent in the porcine thymus preparation. Through the use of immunoprecipitation, alkaline hydrolysis and subsequent phosphoamino acid analysis it has been demonstrated that this partially purified kinase fraction is capable of phosphorylating histone H4 on histidine. In conclusion, an preparation has been made from porcine thymus that contains histone H4 kinase activity and at least one of the kinases present in this preparation is a histidine kinase.     

Interaction Analysis of a Two-Component System Using Nanodiscs

Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems.     

The hybrid histidine kinase DokA is part of the osmotic response system of Dictyostelium.

We have used PCR to identify a Dictyostelium homolog of the bacterial two-component system. The gene dokA codes for a member of the hybrid histidine kinase family which is defined by the presence of conserved amino acid sequence motifs corresponding to an N-terminal receptor domain, a central kinase and a C-terminal response regulator moiety. Potential function of the regulator domain was demonstrated by phosphorylation in vitro. dokA mutants are deficient in the osmoregulatory pathway, resulting in premature cell death under high osmotic stress. Under less stringent osmotic conditions, cells grow at a normal rate, but development at the multicellular stage is altered. dokA is a member of a family of histidine kinase-like genes that play regulatory roles in eukaryotic cell function.