scholarly journals Acetylation of Response Regulator Protein MtrA in M. tuberculosis Regulates Its Repressor Activity

2021 ◽  
Vol 11 ◽  
Author(s):  
Krishna Kumar Singh ◽  
P. J. Athira ◽  
Neerupma Bhardwaj ◽  
Devendra Pratap Singh ◽  
Uchenna Watson ◽  
...  
Keyword(s):  
Response Regulator ◽  
Alveolar Epithelial Cells ◽  
Post Translational Modification ◽  
Alveolar Epithelial ◽  
Regulator Protein ◽  
Regulatory Effects ◽  
Genomic Regions ◽  
Response Regulator Protein ◽  
The Impact ◽  
Repressor Activity

MtrA is an essential response regulator (RR) protein in M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.

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 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.

scholarly journals Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway

2000 ◽  
Vol 279 (5) ◽  
pp. L994-L1002 ◽  
Author(s):  
John D. Lang ◽  
Phillip Chumley ◽  
Jason P. Eiserich ◽  
Alvaro Estevez ◽  
Thad Bamberg ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Cell Injury ◽  
Inflammatory Responses ◽  
Alveolar Epithelial Cells ◽  
No Production ◽  
Inflammatory Reactions ◽  
Alveolar Epithelial ◽  
And Function ◽  
The Impact

Ventilator strategies allowing for increases in carbon dioxide (CO2) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O2 −), nitric oxide (·NO), and their product peroxynitrite (ONOO−). The reaction of CO2 and ONOO− can yield the nitrosoperoxocarbonate adduct ONOOCO2 −, a more potent nitrating species than ONOO−. Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of ·NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO2 (hypercapnia) revealed a significant increase in ·NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-l-arginine. Our studies reveal that hypercapnia modifies ·NO-dependent pathways to amplify cell injury. These results affirm the underlying role of ·NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.

scholarly journals Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli

2016 ◽  
Vol 371 (1707) ◽  
pp. 20150492 ◽  
Author(s):  
Diana Di Paolo ◽  
Oshri Afanzar ◽  
Judith P. Armitage ◽  
Richard M. Berry
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Response Regulator ◽  
Single Molecule Level ◽  
Flagellar Motors ◽  
Regulator Protein ◽  
Response Regulator Protein

For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. This article is part of the themed issue ‘The new bacteriology’.

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