Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.13.3 (histidine kinase)
 2,405 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a "patch" near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and P1 signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6K gamma attP "genome targeting" suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.
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PMID:Altered recognition mutants of the response regulator PhoB: a new genetic strategy for studying protein-protein interactions. 896 56

A temperature-sensitive lethal mutant of Staphylococcus aureus was found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded by yycFG; orthologues of yycFG could be identified in the genomes of Bacillus subtilis and other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either the yycF or the yycG coding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of the ermB resistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.
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PMID:Role in cell permeability of an essential two-component system in Staphylococcus aureus. 1036 39

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.
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PMID:Natural genetic transformation of Streptococcus mutans growing in biofilms. 1120 87

Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic 'suicide attack' system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications.
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PMID:A fungicide-responsive kinase as a tool for synthetic cell fate regulation. 2613 83

This study reports the introduction of egfp or mCherry markers to the Sp245, Sp7, and M40 wild-type strains of Azospirillum brasilense and the hhkB (encoding for a putative hybrid histidine kinase) minus mutant an isogenic strain of A. brasilense Sp245 to monitor colonization of wheat (Triticum aestivum). Two plasmids were constructed: (1) the pJMS-2 suicide plasmid derived from pSUP202 and harboring the mCherry gene expressed under the constitutive kanamycin resistance promoter to create a cis tag and (2) the broad-range plasmid pMP2449-5 that carries the mCherry gene under the lac promoter, which is derived from the plasmid pMP2444; to create the in trans tag. The stability of the plasmids encoding egfp and mCherry were confirmed in vitro for seven days of bacterial growth, and then, the A. brasilense strains harboring the plasmids were studied under nonselective conditions for adherence to seeds and, at seven or 14 days post-inoculation, for wheat root colonization. The utility of the labeled strains was proven by observation, using fluorescence microscopy and confocal laser scanning microscopy (CLSM) in wheat plants inoculated with the labeled strains and compared with the CFU g-1 for seed and wheat root. The method was suitable for observation of the in situ formation of mini-colonies, enabled visualization of bacterial colonization sites on large root fragments, and showed adherence to germinated seeds and root colonization of all strains by cell counts and direct microscopic examination. Thus, we are able to quantify the structures of the biofilms formed by each strain.
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PMID:Versatile use of Azospirillum brasilense strains tagged with egfp and mCherry genes for the visualization of biofilms associated with wheat roots. 3017 3