Characterization of a response regulator protein that binds to Anabaena sp. strain L-31 kdp-promoter region

2008 ◽  
Vol 474 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Anand Ballal ◽  
Shree Kumar Apte
Keyword(s):  
Promoter Region ◽  
Response Regulator ◽  
Regulator Protein ◽  
Anabaena Sp ◽  
Response Regulator Protein

scholarly journals Unusual Heme Binding in the Bacterial Iron Response Regulator Protein: Spectral Characterization of Heme Binding to the Heme Regulatory Motif

Biochemistry ◽  
2011 ◽  
Vol 50 (6) ◽  
pp. 1016-1022 ◽  
Author(s):  
Haruto Ishikawa ◽  
Megumi Nakagaki ◽  
Ai Bamba ◽  
Takeshi Uchida ◽  
Hiroshi Hori ◽  
...  
Keyword(s):  
Response Regulator ◽  
Spectral Characterization ◽  
Heme Binding ◽  
Regulatory Motif ◽  
Regulator Protein ◽  
Iron Response Regulator ◽  
Response Regulator Protein

Rational design and molecular characterization of a chimaeric response regulator protein

2001 ◽  
Vol 310 (2) ◽  
pp. 283-290 ◽  
Author(s):  
Andreas Bock ◽  
Marcus Bantscheff ◽  
Anne-Laure Perraud ◽  
Karsten Rippe ◽  
Verena Weiss ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Rational Design ◽  
Response Regulator ◽  
Regulator Protein ◽  
Response Regulator Protein

Molecular Characterization of the Essential Response Regulator Protein YycF in Bacillus subtilis

2003 ◽  
Vol 6 (3-4) ◽  
pp. 155-163 ◽  
Author(s):  
Takafumi Watanabe ◽  
Yoshiki Hashimoto ◽  
Yoshihito Umemoto ◽  
Daisuke Tatebe ◽  
Eiji Furuta ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Molecular Characterization ◽  
Response Regulator ◽  
Regulator Protein ◽  
Response Regulator Protein

scholarly journals Candida albicans Response Regulator Gene SSK1 Regulates a Subset of Genes Whose Functions Are Associated with Cell Wall Biosynthesis and Adaptation to Oxidative Stress

2003 ◽  
Vol 2 (5) ◽  
pp. 1018-1024 ◽  
Author(s):  
Neeraj Chauhan ◽  
Diane Inglis ◽  
Elvira Roman ◽  
Jesus Pla ◽  
Dongmei Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signal Transduction ◽  
Hydrogen Peroxide ◽  
Cell Wall ◽  
Response Regulator ◽  
Cell Wall Biosynthesis ◽  
Regulator Protein ◽  
Two Component ◽  
Response Regulator Protein ◽  
Mutant Cells

ABSTRACT Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

scholarly journals Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

2008 ◽  
Vol 190 (19) ◽  
pp. 6467-6474 ◽  
Author(s):  
Oliwia Makarewicz ◽  
Svetlana Neubauer ◽  
Corinna Preusse ◽  
Rainer Borriss
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Promoter Region ◽  
Response Regulator ◽  
Dependent Manner ◽  
Coding Region ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Regulator Protein ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

scholarly journals Switched or Not?: the Structure of Unphosphorylated CheY Bound to the N Terminus of FliM

2006 ◽  
Vol 188 (21) ◽  
pp. 7354-7363 ◽  
Author(s):  
Collin M. Dyer ◽  
Frederick W. Dahlquist
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Active Role ◽  
Crystallographic Structure ◽  
Coupling Mechanism ◽  
Allosteric Communication ◽  
Regulator Protein ◽  
Target Binding ◽  
Response Regulator Protein

ABSTRACT Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF3 −-CheY, a phosphorylated CheY (CheY∼P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY∼P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM16]). While the peptide conformation and binding site are similar to those of the BeF3 −-CheY-FliM16 complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg2+-CheY-FliM16 complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF3 −-activated CheY. This structure makes clear the active role that the β4-α4 loop plays in the Tyr87-Tyr106 coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.

scholarly journals Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Varisa Huangyutitham ◽  
Zehra Tüzün Güvener ◽  
Caroline S. Harwood
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Cluster Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Regulator Protein ◽  
Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

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