Project

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Mechanisms of cyclic di-GMP signaling

Applicant Jenal Urs
Number 127433
Funding scheme Sinergia
Research institution Biozentrum der Universität Basel Systembiologie
Institution of higher education University of Basel - BS
Main discipline Experimental Microbiology
Start/End 01.02.2010 - 31.01.2014
Approved amount 1'164'294.00
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All Disciplines (4)

Discipline
Experimental Microbiology
Biochemistry
Structural Research
Biophysics

Keywords (17)

cyclic di-GMP; diguanylate cyclase; phosphodiesterase; GGDEF; EAL; PilZ; bacteria; signaling; biofilm; Caulobacter crescentus; X-ray crystallography; single molecule FRET; bioinformatics; structural modeling; c-di-GMP; signal transduction; chronic infection

Lay Summary (English)

Lead
Lay summary
Living cells employ small diffusible molecules, so-called second messengers, to signal environmental cues from sensory proteins to cellular receptors. Only recently, it has become apparent that bacteria utilize the cyclic di-nucleotide c-di-GMP as a ubiquitous second messenger to switch between rapidly growing single cells and a quiescent life style, called biofilm. In pathogenic bacteria, this switch is often accompanied by the transition from an acute to a chronic phase of infection. This makes c-di-GMP signal transduction an attractive target for novel antibiotics that interfere with bacterial persistence. The cellular concentration of c-di-GMP is the result of the opposing activities of diguanylate cyclases that synthesize c-di-GMP from two GTP molecules, and phosphodiesterases that degrade the compound. These two key enzymatic activities regulate c-di-GMP and thus the state of the various c-di-GMP receptors and their associated activities within the cell.To uncover the molecular mechanisms of the c-di-GMP signaling network, this Sinergia project aims at combining in vivo studies with pathogenic and non-pathogenic bacterial model systems with the analysis of the isolated and purified proteins of the network. This involves their enzymatic and biophysical characterization, 3D-structure determination by X-ray crystallography and the study of their dynamic properties by fluorescence energy transfer measurements. Furthermore, the vast amount of bioinformatic data available will be exploited for structure prediction and the identification of yet unrecognized members of the network. The results will further our general knowledge about c-di-GMP mediated cell signaling and behavior and contribute important information towards the successful control of chronic infections by animal and human pathogens.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger c-di-GMP
Sundriyal Amit, Massa Claudia, Samoray Dietrich, Zehender Fabian, Sharpe Timothy, Jenal Urs, Schirmer Tilman (2014), Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger c-di-GMP, in Journal of biological Chemistry, 289(10), 6978-6990.
Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction
Steiner Samuel, Lori Christian, Boehm Alex, Jenal Urs (2013), Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction, in EMBO Journal, 32(3), 354-368.
A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins
Nesper Jutta Reinders Alberto Glatter Timo Schmidt Alexander Jenal Urs (2012), A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins, in Journal of Proteomics, 75, 4874-4878.
Efficient Enzymatic Production of the Bacterial Second Messenger c-di-GMP by the Diguanylate Cyclase YdeH from E. coli
Zähringer Franziska, Massa Claudia, Schrimer Tilman (2011), Efficient Enzymatic Production of the Bacterial Second Messenger c-di-GMP by the Diguanylate Cyclase YdeH from E. coli, in Applied Biochemistry and Biotechnology, 163(1), 71-79.
Solution Structure of the PilZ Domain Protein PA4608 Complex with Cyclic di-GMP Identifies Charge Clustering as Molecular Readout
Habazettl Judith, Allan Martin, Jenal Urs, Grzesiek Stephan (2011), Solution Structure of the PilZ Domain Protein PA4608 Complex with Cyclic di-GMP Identifies Charge Clustering as Molecular Readout, in Journal of Biological Chemistry, 286(16), 14304-14314.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
EMBO workshop on alpha-proteobacteria Talk given at a conference Role of second messenger signaling in cell polarity and behavior 30.03.2014 Marburg, Germany Jenal Urs;
36th Annual Infectious Diseases Symposium Talk given at a conference Signaling in infection: A central role for nucleotide second messengers 13.03.2014 Luzern, Switzerland Jenal Urs;
EMBO Members Meeting Talk given at a conference Role of second messenger signaling in bacterial growth and behavior 23.10.2013 Heidelberg, Germany Jenal Urs;
Nobel Conference on Biofilms Talk given at a conference Bacterial surface recognition: The INs and OUTs of the flagellar motor 28.08.2013 Stockholm, Sweden Jenal Urs;
Annual Meeting of the Swiss Society for Microbiology Talk given at a conference Signaling in Bacterial Cell Polarity 27.06.2013 Interlaken, Switzerland Jenal Urs;
Namur Research College Symposium Talk given at a conference Of time, space and speed: Cyclic di-GMP signaling in bacterial cell growth and behavior 19.04.2013 Namur, Belgium Jenal Urs;
Bacterial Networks 2013 Talk given at a conference The INs and OUTs of the flagellar motor 16.03.2013 Poltusk, Poland, Poland Jenal Urs; Nesper Jutta;
GRC (Gordon Research Conference) Microbial Stress Response Talk given at a conference Activation of EPS biogenesis through second messenger mediated protein-protein crosslinking 15.07.2012 South Hadley, USA, United States of America Jenal Urs;
General Meeting of the American Society for Microbiology (ASM) Talk given at a conference Cyclic di-GMP signaling in Caulobacter crescentus 16.06.2012 San Francisco, USA, United States of America Jenal Urs;
Translational Medicine Talk given at a conference Biofilm Mediated Bacterial Persistence and Chronic Infections 07.06.2012 Basel, Schweiz, Switzerland Jenal Urs;
GRC (Gordon Research Conference) on Signal Transduction in Microorganisms (STIM) Talk given at a conference Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction 21.01.2012 Ventura, USA, United States of America Jenal Urs;


Self-organised

Title Date Place
Bacterial Locomotion and Signal Transduction 19.01.2013 Tucson, USA, United States of America
Bacterial Networks 2010 04.09.2010 San Feliu, Spanien, Spain

Awards

Title Year
Elected EMBO Member 2012
ERC Advanced Investigator Grant 2012
Fellow of the American Academy of Microbiology 2011

Associated projects

Number Title Start Funding scheme
147090 Cyclic di-GMP signaling in cell behavior and reproduction 01.04.2013 Project funding (Div. I-III)
150814 Purchase of a 900 MHz high-resolution NMR instrument 01.12.2013 R'EQUIP
138414 Molecular mechanisms of c-di-GMP signaling and of target AMPylation 01.02.2012 Project funding (Div. I-III)
139230 High-performance IT Infrastructure for Computational Biology 01.02.2012 R'EQUIP
145023 Advanced Imaging System for Biomolecular Crystallization Screening 01.12.2012 R'EQUIP
108186 Mechanisms of c-di-GMP signaling in Caulobacter crescentus development and biofilm formation 01.04.2005 Project funding (Div. I-III)
105587 Structure and function of prokaryotic membrane permeases and pores and of a two-component response regulator 01.10.2004 Project funding (Div. I-III)
130469 Global analysis of C-DI-GMP signaling in Caulobacter crescentus 01.04.2010 Project funding (Div. I-III)

Abstract

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that emerges as a regulatory mastermind in orchestrating multicellular behavior, virulence, and biofilm-mediated persistence in a wide variety of bacteria. The cellular concentration of c-di-GMP is the result of the opposing activities of diguanylate cyclases (DGCs), which catalyze the condensation of two GTP molecules, and phosphodiesterases (PDEs) that degrade c-di-GMP to pGpG. C-di-GMP mediates downstream signaling by binding to different molecular switches, which in turn can influence cellular behavior on the transcriptional, translational, or allosteric level. The broad importance of this novel signaling molecule in pathogenic and non-pathogenic bacteria calls for a thorough analysis of the molecular mechanisms that control cellular levels of c-di-GMP and regulate its downstream targets. The c-di-GMP circuitry operating in most bacterial cells appears to be exceedingly complex. GGDEF (DGCs), EAL (PDEs), and PilZ (effectors) domain proteins belong to large protein families with multiple paralogs present in most bacteria, some of which have experienced functional diversification by adopting novel roles in cell signaling. To uncover structural, kinetic and dynamic properties of the c-di-GMP signaling network proteins, this project aims at a systematic analysis of its components on the atomic, molecular, and cellular level. A careful bioinformatics analysis will reveal conserved structural motifs of known members of the GGDEF, EAL, and PilZ protein families and will help identifying novel c-di-GMP signaling components. Crystal structure determination complemented by homology modeling will elucidate the detailed three-dimensional structures of representatives of the most important signaling components and its various regulatory conformations. These studies will be complemented by single molecule FRET experiments that address domain arrangements in solution and monitor the dynamics of conformational changes upon activation or ligand binding. Mechanistic models of catalysis and regulation that result from these studies will be validated and refined systematically and in a targeted manner by site directed mutagenesis and established in vitro and in vivo assays as readouts. In particular, we will make use of a well-developed model system for c-di-GMP signaling, Caulobacter crescentus, where cellular and molecular aspects are currently under intense investigation. Such an in depth analysis thrives on an interdisciplinary approach that combines different experimental skills and complementary methodology. Reaching a fundamental understanding of the basic mechanistic and quantitative aspects of c-di-GMP signaling components will lay the groundwork for a cell-wide systems level analysis of the c-di-GMP network, which eventually will lead to a full appreciation of the complex spatial and temporal control mechanisms involved in switching bacteria from motile and virulent single cells to persistent communities.
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