Faculty Opinions recommendation of Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition.

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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Structure of a Diguanylate Cyclase from Thermotoga maritima: Insights into Activation, Feedback Inhibition and Thermostability

PLoS ONE ◽

10.1371/journal.pone.0110912 ◽

2014 ◽

Vol 9 (10) ◽

pp. e110912 ◽

Cited By ~ 18

Author(s):

Angeline Deepthi ◽

Chong Wai Liew ◽

Zhao-Xun Liang ◽

Kunchithapadam Swaminathan ◽

Julien Lescar

Keyword(s):

Feedback Inhibition ◽

Thermotoga Maritima ◽

Diguanylate Cyclase

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Signal Detection and Target Gene Induction by the CpxRA Two-Component System

Journal of Bacteriology ◽

10.1128/jb.185.8.2432-2440.2003 ◽

2003 ◽

Vol 185 (8) ◽

pp. 2432-2440 ◽

Cited By ~ 152

Author(s):

Patricia A. DiGiuseppe ◽

Thomas J. Silhavy

Keyword(s):

Signal Transduction ◽

Feedback Inhibition ◽

Response Regulator ◽

Negative Regulator ◽

Outer Membrane Lipoprotein ◽

Envelope Stress ◽

Two Component ◽

Two Component Signal Transduction ◽

Cpx Pathway ◽

Target Gene Induction

ABSTRACT The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors. CpxA resides in the inner membrane and has both kinase and phosphatase activities. CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes. Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification. Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE. Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent. We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA. Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points. Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress. Moreover, the cpxP promoter is more inducible than any other regulon member tested. Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone. Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.

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Cellular L-arginine pools modulate c-di-GMP turnover and biofilm formation in Pseudomonas putida

10.5194/biofilms9-130 ◽

2020 ◽

Author(s):

Laura Barrientos-Moreno ◽

María Antonia Molina-Henares ◽

María Isabel Ramos-González ◽

Manuel Espinosa-Urgel

Keyword(s):

Pseudomonas Putida ◽

Biofilm Formation ◽

Response Regulator ◽

Second Messenger ◽

Colony Morphology ◽

Diguanylate Cyclase ◽

Arginine Biosynthesis ◽

Amino Acid Transport System ◽

Metabolic Signal ◽

Biofilm Development

The intracellular second messenger cyclic diguanylate (c-di-GMP) is broadly conserved in bacteria, where it influences processes such as virulence, stress resistance and biofilm development. In the plant-beneficial bacterium Pseudomonas putida KT2440, the response regulator with diguanylate cyclase activity CfcR is the main contributor to c-di-GMP levels in the stationary phase of growth. When overexpressed, CfcR increases c-di-GMP levels and gives rise to a pleiotropic phenotype that includes enhanced biofilm formation and crinkly colony morphology. Our group has previously reported that insertion mutants in argG and argH, the genes that encode the last two enzymes in the arginine biosynthesis pathway, do not display the crinkly colony morphology phenotype and show decreased c-di-GMP levels even in the presence of cfcR in multicopy (Ramos-Gonz&#225;lez, M.I. et al. 2016. Front. Microbiol. 7, 1093). Here we present results indicating that L-arginine acts both as an environmental and as a metabolic signal that influences the lifestyles of P. putida through the modulation of c-di-GMP levels and changes in the expression of structural elements of biofilms. Exogenous L-arginine partially restores c-di-GMP levels in arginine biosynthesis mutants, a response that is transduced through CfcR and possibly (an)other diguanylate cyclase(s). At least three periplasmic binding proteins, each forming part of an amino acid transport system, contribute in different ways to the response to external L-arginine. We propose that the turnover of the second messenger c-di-GMP is modulated by the state of global arginine pools in the cell resulting both from anabolism and from uptake.

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The Cpx Envelope Stress Response Is Controlled by Amplification and Feedback Inhibition

Journal of Bacteriology ◽

10.1128/jb.181.17.5263-5272.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5263-5272 ◽

Cited By ~ 161

Author(s):

Tracy L. Raivio ◽

Daniel L. Popkin ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Virulence Factors ◽

Histidine Kinase ◽

Feedback Inhibition ◽

Response Regulator ◽

Regulatory System ◽

Concomitant Increase ◽

Envelope Stress ◽

Two Component

ABSTRACT In Escherichia coli, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane). It is comprised of the histidine kinase CpxA and the response regulator CpxR. This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors. Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms. Western blot and operon fusion analyses demonstrated that the cpxRA operon is autoactivated. Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription ofcpxRA. Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway. This repression is dependent on an intact CpxA sensing domain. The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.

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