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Query: UNIPROT:P06889 (Mol)
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Signal transduction by two-component regulatory systems involves phosphorylation of the receiver domain of a response regulator by the transmitter domain of the cognate histidine kinase. In the NtrBC system, phosphorylation of NtrC by NtrB results in transcriptional activation of nitrogen-regulated genes. We have used the yeast two-hybrid system to probe interactions between domains of the NtrB and NtrC proteins from Klebsiella pneumoniae. We constructed fusions from each of a series of proteins or protein domains to the activation and the DNA-binding domains of GAL4 and analysed expression of GAL1:lacZ and GAL1:HIS3 reporters in yeast. The DNA-binding domain of NtrC and the so-called sensor domain of NtrB appeared to provide the major determinants for dimerization of the fusion proteins. A strong and specific interaction was also shown between NtrB and NtrC, localized to the HN region of the NtrB transmitter module and to the NtrC receiver domain, whereas other domains of these proteins do not appear to contribute to the recognition specificity. The results presented here indicate that communication between two-component partners also involves protein-protein interactions that can be detected in vivo, suggesting that the yeast two-hybrid system is a powerful genetic tool for identifying functional partners of prokaryotic signal transduction pathways.
Mol Microbiol 2001 Apr
PMID:Two-hybrid analysis of domain interactions involving NtrB and NtrC two-component regulators. 1129 84

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold-inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a DeltaHik33 mutant using the DNA microarray technique. In wild-type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold-repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold-regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.
Mol Microbiol 2001 Apr
PMID:Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis. 1129 90

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.
Mol Microbiol 2001 May
PMID:The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa. 1140 99

Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density-dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid-log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis-activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S-P. Structural analysis suggested GBAP to be an 11-residue cyclic peptide containing a lactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C-terminal part of a 242-residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB-fsrC encoding FsrB and a putative histidine kinase FsrC and gelE-sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two-component regulatory system.
Mol Microbiol 2001 Jul
PMID:Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis. 1145 7

Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD during B. subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.
Mol Microbiol 2001 Nov
PMID:CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis. 1172 27

Competence for DNA uptake and genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system. A competence-stimulating polypeptide (CSP) is secreted by the bacteria and acts back on the cells via a transmembrane histidine kinase. This enzyme phosphorylates a response regulator that activates synthesis of a SigH-like protein. The new sigma factor enables expression of a set of proteins transcribed from a novel promoter. A mutation called trt had been found that circumvented this regulation. The mutant cells are constitutively competent; that is, they can be transformed at low cell densities, in the presence of proteases that attack CSP, or during growth at low pH. In this work, cells containing trt were shown to be competent even in the presence of a comAB mutation that blocks secretion of CSP. The trt mutation was localized to comD, the gene encoding the transmembrane histidine kinase. A DNA segment of the trt mutant corresponding to comCDE was cloned, and it was shown to contain the trt mutation by its ability to confer constitutive competence. A two-step assay, which was based on transfer of trt to a wild strain and screening for transformability in the presence of trypsin, served to locate the trt mutation precisely. It corresponds to a GC-->AT transition, which changes Asp299 in the histidine kinase to Asn. This alteration in the carboxyl terminal half of the protein, which is cytoplasmically located and contains the phosphorylase activity, presumably alters the enzyme conformation so that it is permanently activated, independent of signals from the transmembrane domain. These results may help illuminate the mechanism by which external signals affect kinase action in two-component regulatory systems, and they may be of practical value in facilitating genetic studies by rendering pneumococcal strains permanently competent.
Mol Microbiol 2001 Nov
PMID:Constitutive competence for genetic transformation in Streptococcus pneumoniae caused by mutation of a transmembrane histidine kinase. 1173 45

In Escherichia coli, the two-component regulatory system that controls the expression of outer membrane porins in response to environmental osmolarity consists of the sensor kinase EnvZ and the response regulator OmpR. Phosphorylated OmpR activates expression of the OmpF porin at low osmolarity, and at high osmolarity represses ompF transcription and activates expression of OmpC. We have characterized a substitution in the amino-terminal phosphorylation domain of OmpR, T83I, its phenotype is OmpF(-) OmpC(-). The mutant protein is not phosphorylated by small molecule phosphodonors such as acetyl phosphate and phosphoramidate, but it is phosphorylated by the cognate kinase EnvZ. Interestingly, the active site T83I substitution alters the DNA binding properties of the carboxyl-terminal effector domain. DNase I protection assays indicate that DNA binding by the mutant protein is similar to wild-type OmpR at the ompF promoter, but at ompC, the pattern of protection is different from OmpR. Our results indicate that all three of the OmpR binding sites at the ompC promoter must be filled in order to activate gene expression. Furthermore, it appears that OmpR-phosphate must adopt different conformations when bound at ompF and ompC. A model is presented to account for the reciprocal regulation of OmpF and OmpC porin expression.
J Mol Biol 2002 Jan 25
PMID:A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC. 1181 25

The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.
Mol Biol Cell 2002 Feb
PMID:The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p. 1185

The membrane-bound histidine kinase KdpD is a putative turgor sensor that regulates, together with the response regulator KdpE, expression of the kdpFABC operon. This operon encodes the high affinity K+-uptake system KdpFABC of Escherichia coli. Expression of kdpFABC is induced under K+ limiting growth conditions and in response to an osmotic upshift. Various structural features of KdpD and KdpE, which are important for stimulus perception and/or signal transduction were identified and are described here. Furthermore, various studies undertaken to elucidate the nature of the stimulus for KdpD result in a new model for KdpD stimulus perception. According to this, autophosphorylation activity of KdpD is not a result of changes in turgor per se. Instead, various--mainly intracellular parameters--that are related to changes of environmental conditions influence the activities of KdpD.
J Mol Microbiol Biotechnol 2002 May
PMID:Towards an understanding of the molecular mechanisms of stimulus perception and signal transduction by the KdpD/KdpE system of Escherichia coli. 1193 51

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.
J Mol Microbiol Biotechnol 2002 May
PMID:Mechanism of regulation of the bifunctional histidine kinase NtrB in Escherichia coli. 1193 52


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