N-terminal truncation of VC0395_0300 protein from Vibrio cholerae does not lead to loss of diguanylate cyclase activity

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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Structural Biochemistry of a Vibrio cholerae Dinucleotide Cyclase Reveals Cyclase Activity Regulation by Folates

Molecular Cell ◽

10.1016/j.molcel.2014.08.001 ◽

2014 ◽

Vol 55 (6) ◽

pp. 931-937 ◽

Cited By ~ 28

Author(s):

Deyu Zhu ◽

Lijun Wang ◽

Guijun Shang ◽

Xue Liu ◽

Jing Zhu ◽

...

Keyword(s):

Vibrio Cholerae ◽

Cyclase Activity ◽

Activity Regulation ◽

Structural Biochemistry

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Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response

Microbiology ◽

10.1099/mic.0.037887-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2873-2886 ◽

Cited By ~ 23

Author(s):

Melissa M. Lacey ◽

Jonathan D. Partridge ◽

Jeffrey Green

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Stress Response ◽

Cell Surface ◽

Regulatory Networks ◽

Oxidative Stress Response ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Phosphodiesterase Activity ◽

K 12

The Escherichia coli K-12 yfgF gene encodes a protein with domains associated with cyclic di-GMP signalling: GGDEF (associated with diguanylate cyclase activity) and EAL (associated with cyclic di-GMP phosphodiesterase activity). Here, it is shown that yfgF is expressed under anaerobic conditions from a class II FNR (regulator of fumarate and nitrate reduction)-dependent promoter. Anaerobic expression of yfgF is greatest in stationary phase, and in cultures grown at 28 °C, suggesting that low growth rates promote yfgF expression. Mutation of yfgF resulted in altered cell surface properties and enhanced sensitivity when anaerobic cultures were exposed to peroxides. The purified YfgF GGDEF-EAL (YfgFGE) and EAL (YfgFE) domains possessed cyclic di-GMP-specific phosphodiesterase activity, but lacked diguanylate cyclase activity. However, the catalytically inactive GGDEF domain was required for YfgFGE dimerization and enhanced cyclic di-GMP phosphodiesterase activity in the presence of physiological concentrations of Mg2+. The cyclic di-GMP phosphodiesterase activity of YfgFGE and YfgFE was inhibited by the product of the reaction, 5′-phosphoguanylyl-(3′–5′)-guanosine (pGpG). Thus, it is shown that the yfgF gene encodes an anaerobic cyclic di-GMP phosphodiesterase that is involved in remodelling the cell surface of E. coli K-12 and in the response to peroxide shock, with implications for integrating three global regulatory networks, i.e. oxygen regulation, cyclic di-GMP signalling and the oxidative stress response.

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Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors

Molecular BioSystems ◽

10.1039/c4mb00366g ◽

2014 ◽

Vol 10 (11) ◽

pp. 2823-2826 ◽

Cited By ~ 20

Author(s):

Justin L. Burns ◽

D. Douglas Deer ◽

Emily E. Weinert

Keyword(s):

Enzyme Catalysis ◽

Cyclase Activity ◽

Oxygen Binding ◽

Diguanylate Cyclase ◽

Oligomeric State ◽

Oxygen Dissociation

Globin coupled sensors exist as a mixture of oligomeric states, which affect both enzyme catalysis and oxygen binding.

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Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors

Applied Microbiology and Biotechnology ◽

10.1007/s00253-009-2199-x ◽

2009 ◽

Vol 85 (4) ◽

pp. 1095-1104 ◽

Cited By ~ 82

Author(s):

Davide Antoniani ◽

Paola Bocci ◽

Anna Maciąg ◽

Nadia Raffaelli ◽

Paolo Landini

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Whole Cell ◽

Cell Assays

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Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.00281-15 ◽

2015 ◽

Vol 198 (1) ◽

pp. 77-90 ◽

Cited By ~ 26

Author(s):

Dorota Skotnicka ◽

Tobias Petters ◽

Jan Heering ◽

Michael Hoppert ◽

Volkhard Kaever ◽

...

Keyword(s):

Myxococcus Xanthus ◽

Lifestyle Changes ◽

Gliding Motility ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Type Iv Pilus ◽

Cell Agglutination ◽

Content Type ◽

Type Iv ◽

Altered Cell

ABSTRACTThe nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes.Myxococcus xanthusundergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP inM. xanthusand show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of thepilAgene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes inM. xanthusencoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility inM. xanthus.IMPORTANCEWe provide the first direct evidence thatM. xanthussynthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of thepilAgene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

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Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts

Life ◽

10.3390/life11010031 ◽

2021 ◽

Vol 11 (1) ◽

pp. 31

Author(s):

Federico Mantoni ◽

Chiara Scribani Rossi ◽

Alessandro Paiardini ◽

Adele Di Matteo ◽

Loredana Cappellacci ◽

...

Keyword(s):

Cyclase Activity ◽

Environmental Cues ◽

Structural Studies ◽

Diguanylate Cyclase ◽

Multidomain Proteins ◽

Cyclic Diguanylate ◽

Conformational Switch ◽

Allosteric Control ◽

Kinetic Profile ◽

Ggdef Domain

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

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Diguanylate Cyclases Control Magnesium-Dependent Motility of Vibrio fischeri

Journal of Bacteriology ◽

10.1128/jb.00728-06 ◽

2006 ◽

Vol 188 (23) ◽

pp. 8196-8205 ◽

Cited By ~ 35

Author(s):

Therese M. O'Shea ◽

Adam H. Klein ◽

Kati Geszvain ◽

Alan J. Wolfe ◽

Karen L. Visick

Keyword(s):

Vector Control ◽

Vibrio Fischeri ◽

Soft Agar ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Reverse Transcription Pcr ◽

Transposon Insertion ◽

Diguanylate Cyclases ◽

Significant Difference ◽

The Impact

ABSTRACT Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids into Escherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an Rf value consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels, while multicopy expression decreased them. Semiquantitative reverse transcription-PCR experiments revealed no significant difference in the amount of flagellin transcripts produced in either the presence or absence of Mg2+ by either vector control or mifA-overexpressing cells, indicating that the impact of magnesium and cyclic-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and cyclic di-GMP in the control of motility of V. fischeri.

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