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At least 55 records · Page 3Linked to original sources
PubMed2005

Phosphorylation-independent activity of atypical response regulators of Helicobacter pylori.

The genome of the gastric pathogen Helicobacter pylori harbors a remarkably low number of regulatory genes, including three and five open reading frames encoding two-component histidine kinases and response regulators, respectively, which are putatively involved in transcriptional regulation. Two of the response regulator genes, hp1043 and hp166, proved to be essential for cell growth, and inactivation of the response regulator gene hp1021 resulted in a severe growth defect, as indicated by a small-colony phenotype. The sequences of the receiver domains of response regulators HP1043 and HP1021 differ from the consensus sequence of the acidic pocket of the receiver domain which is involved in the phosphotransfer reaction from the histidine kinase to the response regulator. Using a genetic complementation system, we demonstrated that the function of response regulator HP166, which is essential for cell growth, can be provided by a mutated derivative carrying a D52N substitution at the site of phosphorylation. We found that the atypical receiver sequences of HP1043 and HP1021 are not crucial for the function of these response regulators. Phosphorylation of the receiver domains of HP1043 and HP1021 is not needed for response regulator function and may not occur at all. Thus, the phosphorylation-independent action of these regulators differs from the well-established two-component paradigm.

Amino Acid Sequence↗
PubMed2002

Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism.

Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY-P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by alpha4, beta5, alpha5, and alpha1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.

Amino Acid Sequence↗
PubMed2004

Plant response regulators implicated in signal transduction and circadian rhythm.

The so-called 'response regulators' were originally discovered as common components of the widespread histidine (His)-->aspartate (Asp) phosphorelay signal transduction system in prokaryotes. Through the course of evolution, higher plants have also come to employ such prokaryotic response regulators (RRs) for their own signal transduction, such as the elicitation of plant hormone (e.g. cytokinin) responses. Furthermore, plants have evolved their own atypical variants of response regulators, pseudo response regulators (PRRs), which are used to modulate sophisticated biological processes, including circadian rhythms and other light-signal responses. Recent studies using the model plant Arabidopsis thaliana have begun to shed light on the interesting functions of these plant response regulators.

Arabidopsis↗
PubMed2006

Structural and functional studies of the response regulator HupR.

HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.

Amino Acid Sequence↗
PubMed2001

Synthesis and biochemical characterization of a phosphorylated analogue of the response regulator CheB.

CheB is a response regulator protein in the bacterial chemotaxis two-component signal transduction pathway. Methylesterase CheB functions together with methyltransferase CheR to modulate the level of glutamate methylation in transmembrane chemoreceptors in response to environmental stimuli. The level of glutamate methylation in turn indirectly controls the direction of flagellar rotation. Like most two-component response regulators, CheB is activated in vivo by phosphorylation of a single aspartate, Asp 56, in its regulatory domain. Extensive biochemical and crystallographic studies have been completed on the inactive, unphosphorylated form of CheB. Because of the inherent lability of aspartyl phosphate bonds and the intrinsic phosphatase activity of CheB, the activated, phosphorylated form of CheB cannot be isolated for further characterization. We present a synthetic scheme to prepare an analogue of phosphorylated CheB using site-specific mutagenesis and chemical modification strategies. Initially, the two native cysteines found in CheB were substituted by serines and a cysteine was substituted for Asp 56 to yield D56C/C207S/C309S CheB. The unique cysteine in the substituted form of CheB was modified by sodium thiophosphate, Na(3)SPO(3), using two sequential disulfide bond exchange reactions. The analogue, D56C/C207S/C309S CheB-SPO(3), contained a thiophosphate group covalently bonded to the protein through a disulfide linkage at residue 56. Mass spectrometry showed that the protein was singly modified. Reverse phase chromatography showed that greater than 95% of the protein was modified under optimized conditions and that the analogue had a half-life of 28 days. In in vitro methylesterase assays in the presence of Mg(2+), the analogue exhibited activity equivalent to that of fully phosphorylated C207S/C309S CheB. Thus, D56C/C207S/C309S CheB-SPO(3) is a stable analogue that may be useful for characterization of the active form of CheB.

Aspartic Acid↗
PubMed2005

Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA.

The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced <45 % as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.

DNA, Bacterial↗
PubMed2004

RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate.

Bacterial Proteins↗
PubMed2001

Interaction of the response regulator ARR4 with phytochrome B in modulating red light signaling.

The Arabidopsis thaliana response regulator 4, expressed in response to phytochrome B action, specifically interacts with the extreme amino-terminus of the photoreceptor. The response regulator 4 stabilizes the active Pfr form of phytochrome B in yeast and in planta, thus elevates the level of the active photoreceptor in vivo. Accordingly, transgenic Arabidopsis plants overexpressing the response regulator 4 display hypersensitivity to red light but not to light of other wavelengths. We propose that the response regulator 4 acts as an output element of a two-component system that modulates red light signaling on the level of the phytochrome B photoreceptor.

Arabidopsis↗
PubMed2003

The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii.

In order to identify a potential regulator of virulence gene expression in Legionella pneumophila, the L. pneumophila homologue of the response regulator GacA, LetA, was identified and cloned, facilitating the generation of a L. pneumophila letA insertion mutant. The L. pneumophila letA insertion mutant was more sensitive to oxidative and acid stress than the wild-type. The letA mutant exhibited reduced infectivity and was defective for intracellular growth within Acanthamoeba castellanii. Transcription of the rpoS and dotA genes was reduced in the letA mutant. Our data indicate that the response regulator LetA functions as a regulator of the stationary-phase stress response in L. pneumophila and is required for efficient replication within A. castellanii.

Acanthamoeba↗
PubMed2003

The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli.

The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule.

Amino Acid Sequence↗
PubMed1992

Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria.

Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.

Amino Acid Sequence↗
PubMed1989

Collaboration of Th1 and Th2 T cell clones in specific antibody responses: regulation of the IgM response to phosphorylcholine.

Carrier (KLH)-specific type 1 T cell clones (Th1), which are defined by secretion of IL-2 and IFN-gamma but not IL-4, and type 2 (Th2) clones, which secrete IL-4, but not IL-2 or IFN-gamma, have been isolated and analyzed for their ability to collaborate in providing help for B cells to secrete phosphorylcholine-specific IgM antibodies. The resulting antibody responses exhibited a characteristic pattern suggesting two distinct regulatory interactions among the Th1, Th2, and B cells. At low doses of antigen, Th1 cells enhanced the helper function of the Th2 cells, an effect due primarily to IL-2. At high doses of antigen, Th1 cells or IFN-gamma inhibited Th2-dependent antibody responses. The inhibitory effect of Th1 or IFN-gamma affected primarily the hapten-carrier-linked portion of the response. The overall effect was a modulation of the antigen dose-response curve for antibody production, eliminating the sharp increases in dose response mediated by isolated T cell clones. The data suggest that collaborative interactions of Th1 and Th2 cells in antibody production may have important physiological consequences.

Animals↗
PubMed1999

The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE.

Synthesis of the OmpF porin of Escherichia coli is regulated in response to environmental and growth phase signals. In order to identify constituents of the various regulatory pathways involved in modulating ompF transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased ompF'-lacZ activity were identified as previously described. Mutations mapping to a previously identified gene of unknown function, lrhA, were obtained. We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of ompF. In addition to altered growth phase regulation of ompF, these lrhA mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels. We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease. LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE.

Adenosine Triphosphatases↗
PubMed1995

Regulative response of the cranial neural tube after neural fold ablation: spatiotemporal nature of neural crest regeneration and up-regulation of Slug.

After unilateral ablation of the avian cranial neural folds, the remaining neuroepithelial cells are able to replace the missing neural crest population (Scherson et al., 1993). Here, we characterize the cellular and molecular nature of this regulative response by defining: (1) the time and location of neural crest cell production by the neuroepithelium; (2) rostrocaudal axial differences in the regulative response; and (3) the onset of expression of Slug, a transcription factor present in premigratory and migrating neural crest cells. Using DiI and HNK-1 antibody labeling techniques, we find that neural crest regeneration occurs only after apposition of the remaining neuroepithelium with the epidermis, suggesting that the developmental mechanism underlying regeneration of the neural crest may recapitulate initial generation of the neural crest. The regulative response occurs maximally at the 3-5 somite stage, and slowly declines thereafter. Surprisingly, there are profound regional differences in the regenerative ability. Whereas a robust regulation occurs in the caudal midbrain/hindbrain, the caudal forebrain/rostral midbrain regenerates neural crest to a much lesser extent. After neural fold removal in the hindbrain, regenerated neural crest cells migrate in a segmental pattern analogous to that seen in unablated embryos; a decrease in regulative response appears to occur with increasing depth of the ablation. Up-regulation of Slug appears to be an early response after ablation, with Slug transcripts detectable proximal to the ablated region 5-8 hours after surgery and prior to emergence of neural crest cells. Both bilateral and unilateral ablations yield substantial numbers of neural crest cells, though the former recover less rapidly and have greater deficits in neural crest-derived structures than the latter. These experiments demonstrate that the regulative ability of the cranial neuroepithelium to form neural crest depends on the time, location and extent of neural fold ablation.

Animals↗
PubMed1997

Identification by PCR of genes encoding multiple response regulators.

Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component systems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators. Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria. Sequence analysis revealed that 22 DNA fragments, which clearly originated from response regulator genes, were amplified from Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis and Lactobacillus bulgaricus. In each of these four species the receiver module of putative response regulator genes, which do not seem to be related to any of the already characterized genes, was identified. This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.

Agrobacterium tumefaciens↗
PubMed1995

Response regulators of bacterial signal transduction systems: selective domain shuffling during evolution.

Response regulators of bacterial sensory transduction systems generally consist of receiver module domains covalently linked to effector domains. The effector domains include DNA binding and/or catalytic units that are regulated by sensor kinase-catalyzed aspartyl phosphorylation within their receiver modules. Most receiver modules are associated with three distinct families of DNA binding domains, but some are associated with other types of DNA binding domains, with methylated chemotaxis protein (MCP) demethylases, or with sensor kinases. A few exist as independent entities which regulate their target systems by noncovalent interactions. In this study the molecular phylogenies of the receiver modules and effector domains of 49 fully sequenced response regulators and their homologues were determined. The three major, evolutionarily distinct, DNA binding domains found in response regulators were evaluated for their phylogenetic relatedness, and the phylogenetic trees obtained for these domains were compared with those for the receiver modules. Members of one family (family 1) of DNA binding domains are linked to large ATPase domains which usually function cooperatively in the activation of E. coli sigma 54-dependent promoters or their equivalents in other bacteria. Members of a second family (family 2) always function in conjunction with the E. coli sigma 70 or its equivalent in other bacteria. A third family of DNA binding domains (family 3) functions by an uncharacterized mechanism involving more than one sigma factor. These three domain families utilize distinct helix-turn-helix motifs for DNA binding. The phylogenetic tree of the receiver modules revealed three major and several minor clusters of these domains. The three major receiver module clusters (clusters 1, 2, and 3) generally function with the three major families of DNA binding domains (families 1, 2, and 3, respectively) to comprise three classes of response regulators (classes 1, 2, and 3), although several exceptions exist. The minor clusters of receiver modules were usually, but not always, associated with other types of effector domains. Finally, several receiver modules did not fit into a cluster. It was concluded that receiver modules usually diverged from common ancestral protein domains together with the corresponding effector domains, although domain shuffling, due to intragenic splicing and fusion, must have occurred during the evolution of some of these proteins. Multiple sequence alignments of the 49 receiver modules and their various types of effector domains, together with other homologous domains, allowed definition of regions of striking sequence similarity and degrees of conservation of specific residues. Sequence data were correlated with structure/function when such information was available.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed2002

Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus.

DivK is an essential response regulator in the Gram-negative bacterium Caulobacter crescentus and functions in a complex phosphorelay system that precisely controls the sequence of developmental events during the cell division cycle. Structure determinations of this single domain response regulator at different pH values demonstrated that the five-stranded alpha/beta fold of the DivK protein is fully defined only at acidic pH. The crystal structures of the apoprotein and of metal-bound DivK complexes at higher pH values revealed a synergistic pH- and cation binding-induced flexibility of the beta4-alpha4 loop and of the alpha4 helix. This motion increases the solvent accessibility of the single cysteine residue in the protein. Solution state studies demonstrated a 200-fold pH-dependent increase in the affinity of manganese for the protein between pH 6.0 and 8.5 that seems to involve deprotonation of an acido-basic couple. Taken together, these results suggest that flexibility of critical regions of the protein, ionization of the cysteine 99 residue and improved K(D) values for the catalytic metal ion are coupled events. We propose that the molecular events observed in the isolated protein may be required for DivK activation and that they may be achieved in vivo through the specific protein-protein interactions between the response regulator and its cognate kinases.

Amino Acid Sequence↗
PubMed2004

Co-regulation of acetylation and phosphorylation of CheY, a response regulator in chemotaxis of Escherichia coli.

CheY, a response regulator of the chemotaxis system in Escherichia coli, can be activated by either phosphorylation or acetylation to generate clockwise rotation of the flagellar motor. Both covalent modifications are involved in chemotaxis, but the function of the latter remains obscure. To understand why two different modifications apparently activate the same function of CheY, we studied the effect that each modification exerts on the other. The phosphodonors of CheY, the histidine kinase CheA and acetyl phosphate, each strongly inhibited both the autoacetylation of the acetylating enzyme, acetyl-CoA synthetase (Acs), and the acetylation of CheY. CheZ, the enzyme that enhances CheY dephosphorylation, had the opposite effect and enhanced Acs autoacetylation and CheY acetylation. These effects of the phosphodonors and CheZ were not caused by their respective activities. Rather, they were caused by their interactions with Acs and, possibly, with CheY. In addition, the presence of Acs elevated the phosphorylation levels of both CheA and CheY, and acetate repressed this stimulation. These observations suggest that CheY phosphorylation and acetylation are linked and co-regulated. We propose that the physiological role of these mutual effects is at two levels: linking chemotaxis to the metabolic state of the cell, and serving as a tuning mechanism that compensates for cell-to-cell variations in the concentrations of CheA and CheZ.

Acetylation↗