A. We, G. Burgess, and F. Caprioli, Urs Jenal and Holger Sondermann for providing the plasmids for PleD, WspR, and RocR overexpression, and Silvia Fernicola for help with the in vitro inhibition studies. Funding for this study was provided by the Italian Foundation for Research on Cystic Fibrosis (project FFC#13, with the contribution of Delegazione Novara and Delegazione Cosenza-2), by the CHEM-PROFARMA-NET Research Program (Project RBPR05NWWC_004), and PRIN/FIRB Research Programs (Projects 20094BJ9R7 and RBFR10LHD1) of the Italian Ministry for University and Research, 2009.

I. Absah, W. Faubion, and . Jr, Concomitant Therapy with Methotrexate and Anti-TNF-?? in Pediatric Patients with Refractory Crohn??s Colitis: A Case Series, Inflammatory Bowel Diseases, vol.18, issue.8, pp.1488-1492, 2012.
DOI : 10.1002/ibd.21885

D. Antoniani, P. Bocci, A. Maciag, N. Raffaelli, and P. Landini, 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, vol.39, issue.4, pp.1095-1104, 2010.
DOI : 10.1007/s00253-009-2199-x

C. Attila, A. Ueda, and T. Wood, 5-Fluorouracil reduces biofilm formation in Escherichia coli K-12 through global regulator AriR as an antivirulence compound, Applied Microbiology and Biotechnology, vol.189, issue.3, pp.525-533, 2009.
DOI : 10.1007/s00253-009-1860-8

T. Baba, A. T. Hasegawa, M. Takai, Y. Okumura, Y. Baba et al., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection, Molecular Systems Biology, vol.170, p.8, 2006.
DOI : 10.1038/msb4100050

C. Baraquet, K. Murakami, M. Parsek, and C. Harwood, The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP, Nucleic Acids Research, vol.40, issue.15, pp.7207-7218, 2012.
DOI : 10.1093/nar/gks384

N. Bardonnet and C. Blanco, ???uidA-antibiotic-resistance cassettes for insertion mutagenesis, gene fusions and genetic constructions, FEMS Microbiology Letters, vol.93, issue.3, pp.243-248, 1992.
DOI : 10.1016/0378-1097(92)90469-5

M. Baumgart, B. Dogan, M. Rishniw, G. Weitzman, B. Bosworth et al., Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum, The ISME Journal, vol.62, issue.5, pp.403-418, 2007.
DOI : 10.1073/pnas.252529799

F. Blattner, G. Plumkett, . Iii, C. Bloch, N. Perna et al., The Complete Genome Sequence of Escherichia coli K-12, Science, vol.277, issue.5331, pp.1453-1474, 1997.
DOI : 10.1126/science.277.5331.1453

A. Boehm, M. Kaiser, H. Li, C. Spangler, C. Kasper et al., Second Messenger-Mediated Adjustment of Bacterial Swimming Velocity, Cell, vol.141, issue.1, pp.107-116, 2010.
DOI : 10.1016/j.cell.2010.01.018

E. Brouillette, M. Hyodo, Y. Hayakawa, D. Karaolis, and F. Malouin, 3',5'-Cyclic Diguanylic Acid Reduces the Virulence of Biofilm-Forming Staphylococcus aureus Strains in a Mouse Model of Mastitis Infection, Antimicrobial Agents and Chemotherapy, vol.49, issue.8, pp.3109-3113, 2005.
DOI : 10.1128/AAC.49.8.3109-3113.2005

M. Buckstein, J. He, and H. Rubin, Characterization of Nucleotide Pools as a Function of Physiological State in Escherichia coli, Journal of Bacteriology, vol.190, issue.2, pp.718-726, 2008.
DOI : 10.1128/JB.01020-07

P. Cotter and S. Stibitz, c-di-GMP-mediated regulation of virulence and biofilm formation, Current Opinion in Microbiology, vol.10, issue.1, pp.17-23, 2007.
DOI : 10.1016/j.mib.2006.12.006

A. Darfeuille-michaud, J. Boudeau, P. Bulois, C. Neut, A. Glasser et al., High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn???s disease, Gastroenterology, vol.127, issue.2, pp.412-421, 2004.
DOI : 10.1053/j.gastro.2004.04.061

K. Datsenko and B. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proceedings of the National Academy of Sciences, vol.97, issue.12, pp.6640-6645, 2000.
DOI : 10.1073/pnas.120163297

N. De, M. Pirruccello, P. Krasteva, N. Bae, R. Raghavan et al., Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR, PLoS Biology, vol.17, issue.3, p.67, 2008.
DOI : 10.1371/journal.pbio.0060067.st001

C. Deighton, O. Mahony, R. Tosh, J. Turner, C. et al., Management of rheumatoid arthritis: summary of NICE guidance European Crohn's and Colitis Organisation (ECCO) (2010) The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management, BMJ J Crohns Colitis, vol.338, issue.4, pp.28-62, 2009.

C. Dorel, O. Vidal, C. Prigent-combaret, I. Vallet, and P. Lejeune, in biofilm formation, FEMS Microbiology Letters, vol.178, issue.1, pp.169-175, 1999.
DOI : 10.1111/j.1574-6968.1999.tb13774.x

M. Fazli, O. Connell, A. Nilsson, M. Niehaus, K. Dow et al., The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia, Molecular Microbiology, vol.198, issue.2, pp.327-341, 2011.
DOI : 10.1111/j.1365-2958.2011.07814.x

R. Ferreira, D. Chodur, L. Antunes, M. Trimble, and L. Mccarter, Output Targets and Transcriptional Regulation by a Cyclic Dimeric GMP-Responsive Circuit in the Vibrio parahaemolyticus Scr Network, Journal of Bacteriology, vol.194, issue.5, pp.914-924, 2012.
DOI : 10.1128/JB.05807-11

M. Galperin, Bacterial signal transduction network in a genomic perspective+, Environmental Microbiology, vol.31, issue.6, pp.552-567, 2004.
DOI : 10.1128/JB.185.1.285-294.2003

M. Garavaglia, E. Rossi, P. Landini, P. Gray, G. Forrest et al., The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli Evidence for cyclic diguanylate as a vaccine adjuvant with novel immunostimulatory activities, PLoS One Cell Immunol, vol.7, issue.278, pp.113-119, 2012.

L. Gualdi, L. Tagliabue, S. Bertagnoli, T. Ieranò, D. Castro et al., Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli, Microbiology, vol.154, issue.7, pp.2017-2024, 2008.
DOI : 10.1099/mic.0.2008/018093-0

T. Ha, S. Morgan, W. Vaughn, I. Eto, and J. Baggott, Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay, Biochemical Journal, vol.272, issue.2, pp.339-342, 1990.
DOI : 10.1042/bj2720339

B. Hammer and B. Bassler, Distinct Sensory Pathways in Vibrio cholerae El Tor and Classical Biotypes Modulate Cyclic Dimeric GMP Levels To Control Biofilm Formation, Journal of Bacteriology, vol.191, issue.1, pp.169-177, 2009.
DOI : 10.1128/JB.01307-08

S. Haussler, Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa, Environmental Microbiology, vol.87, issue.6, pp.546-551, 2004.
DOI : 10.1046/j.1365-2958.1999.01339.x

Y. Hayakawa, R. N. Hirata, A. Hyodoa, M. Kawaia, and R. , A facile synthesis of cyclic bis(3??????5???)diguanylic acid, Tetrahedron, vol.59, issue.34, pp.6465-6471, 2003.
DOI : 10.1016/S0040-4020(03)01045-7

J. Hermon-taylor, Mycobacterium avium subspecies paratuberculosis, Crohn's disease and the Doomsday scenario, Gut Pathogens, vol.1, issue.1, p.15, 2009.
DOI : 10.1186/1757-4749-1-15

J. Hickman and C. Harwood, as a c-di-GMP-responsive transcription factor, Molecular Microbiology, vol.16, issue.2, pp.376-389, 2008.
DOI : 10.1111/j.1365-2958.2008.06281.x

L. Holland, O. Donnell, S. Ryjenkov, D. Gomelsky, L. Slater et al., A Staphylococcal GGDEF Domain Protein Regulates Biofilm Formation Independently of Cyclic Dimeric GMP, Journal of Bacteriology, vol.190, issue.15, pp.5178-5189, 2008.
DOI : 10.1128/JB.00375-08

M. Jabra-rizk, T. Meiller, C. James, and M. Shirtliff, Effect of Farnesol on Staphylococcus aureus Biofilm Formation and Antimicrobial Susceptibility, Antimicrobial Agents and Chemotherapy, vol.50, issue.4, pp.1463-1469, 2006.
DOI : 10.1128/AAC.50.4.1463-1469.2006

A. Kader, R. Simm, U. Gerstel, M. Morr, and U. Römling, Hierarchical involvement of various GGDEF domain proteins in rdar morphotype development of Salmonella enterica serovar Typhimurium, Molecular Microbiology, vol.180, issue.3, pp.602-616, 2006.
DOI : 10.1046/j.1365-2958.2001.02337.x

D. Karaolis, T. Means, D. Yang, M. Takahashi, T. Yoshimura et al., Bacterial c-di-GMP Is an Immunostimulatory Molecule, The Journal of Immunology, vol.178, issue.4, pp.2171-2181, 2007.
DOI : 10.4049/jimmunol.178.4.2171

A. Kaser, S. Zeissig, and R. Blumberg, Inflammatory Bowel Disease, Annual Review of Immunology, vol.28, issue.1, pp.573-621, 2010.
DOI : 10.1146/annurev-immunol-030409-101225

M. Krishnan, E. Manning, and M. Collins, Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis, Journal of Antimicrobial Chemotherapy, vol.64, issue.5, pp.1018-1023, 2009.
DOI : 10.1093/jac/dkp339

H. Kulasakara, V. Lee, A. Brencic, N. Liberati, J. Urbach et al., Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence, Proceedings of the National Academy of Sciences, vol.103, issue.8, pp.2839-2844, 2006.
DOI : 10.1073/pnas.0511090103

L. Lennard, The clinical pharmacology of 6-mercaptopurine, European Journal of Clinical Pharmacology, vol.5, issue.Suppl A, pp.329-339, 1992.
DOI : 10.1007/BF02220605

B. Levesque and S. Kane, Searching for the delta: 5- aminosalicylic acid therapy for Crohn's disease, Gastroenterol Hepatol (NY), vol.7, pp.295-301, 2011.

J. Moscoso, H. Mikkelsen, S. Heeb, P. Williams, and A. Filloux, The Pseudomonas aeruginosa sensor RetS switches Type III and Type VI secretion via c-di-GMP signalling, Environmental Microbiology, vol.77, issue.12, pp.3128-3138, 2011.
DOI : 10.1111/j.1462-2920.2011.02595.x

R. Paul, S. Weiser, N. Amiot, C. Chan, T. Schirmer et al., Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain, Genes & Development, vol.18, issue.6, pp.715-727, 2004.
DOI : 10.1101/gad.289504

R. Paul, S. Abel, P. Wassmann, A. Beck, H. Heerklotz et al., Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization, Journal of Biological Chemistry, vol.282, issue.40, pp.29170-29177, 2007.
DOI : 10.1074/jbc.M704702200

K. Paul, V. Nieto, W. Carlquist, D. Blair, and R. Harshey, The c-di-GMP Binding Protein YcgR Controls Flagellar Motor Direction and Speed to Affect Chemotaxis by a ???Backstop Brake??? Mechanism, Molecular Cell, vol.38, issue.1, pp.128-139, 2010.
DOI : 10.1016/j.molcel.2010.03.001

E. Purcell, R. Mckee, S. Mcbride, C. Waters, and R. Tamayo, Cyclic Diguanylate Inversely Regulates Motility and Aggregation in Clostridium difficile, Journal of Bacteriology, vol.194, issue.13, pp.3307-3316, 2012.
DOI : 10.1128/JB.00100-12

F. Rivardo, R. Turner, G. Allegrone, H. Ceri, and M. Martinotti, Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens, Applied Microbiology and Biotechnology, vol.61, issue.3, pp.541-553, 2009.
DOI : 10.1007/s00253-009-1987-7

V. Robbe-saule, V. Jaumouille, M. Prevost, S. Guadagnini, C. Talhouarne et al., Crl Activates Transcription Initiation of RpoS-Regulated Genes Involved in the Multicellular Behavior of Salmonella enterica Serovar Typhimurium, Journal of Bacteriology, vol.188, issue.11, pp.3983-3994, 2006.
DOI : 10.1128/JB.00033-06

U. Römling and D. Amikam, Cyclic di-GMP as a second messenger, Current Opinion in Microbiology, vol.9, issue.2, pp.218-228, 2006.
DOI : 10.1016/j.mib.2006.02.010

U. Römling and C. Balsalobre, Biofilm infections, their resilience to therapy and innovative treatment strategies, Journal of Internal Medicine, vol.45, issue.6, pp.541-561, 2012.
DOI : 10.1111/joim.12004

D. Ryjenkov, R. Simm, U. Römling, and M. Gomelsky, The PilZ Domain Is a Receptor for the Second Messenger c-di-GMP, Journal of Biological Chemistry, vol.281, issue.41, pp.30310-30314, 2006.
DOI : 10.1074/jbc.C600179200

K. Sambanthamoorthy, R. Sloup, V. Parashar, J. Smith, E. Kim et al., Identification of Small Molecules That Antagonize Diguanylate Cyclase Enzymes To Inhibit Biofilm Formation, Antimicrobial Agents and Chemotherapy, vol.56, issue.10, pp.5202-5211, 2012.
DOI : 10.1128/AAC.01396-12

F. Shang, T. Xue, H. Sun, L. Xing, S. Zhang et al., The Staphylococcus aureus GGDEF Domain-Containing Protein, GdpS, Influences Protein A Gene Expression in a Cyclic Diguanylic Acid-Independent Manner, Infection and Immunity, vol.77, issue.7, pp.2849-2856, 2009.
DOI : 10.1128/IAI.01405-08

S. Shin and M. Collins, Thiopurine Drugs Azathioprine and 6-Mercaptopurine Inhibit Mycobacterium paratuberculosis Growth In Vitro, Antimicrobial Agents and Chemotherapy, vol.52, issue.2, pp.418-426, 2008.
DOI : 10.1128/AAC.00678-07

R. Simm, J. Fetherston, A. Kader, U. Römling, and R. Perry, Phenotypic Convergence Mediated by GGDEF-Domain-Containing Proteins, Journal of Bacteriology, vol.187, issue.19, pp.6816-6823, 2005.
DOI : 10.1128/JB.187.19.6816-6823.2005
URL : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251569

N. Sommerfeldt, A. Possling, G. Becker, C. Pesavento, N. Tschowri et al., Gene expression patterns and differential input into curli fimbriae regulation of all GGDEF/EAL domain proteins in Escherichia coli, Microbiology, vol.155, issue.4, pp.1318-1331, 2009.
DOI : 10.1099/mic.0.024257-0

C. Spangler, V. Kaever, and R. Seifert, Interaction of the Diguanylate Cyclase YdeH of Escherichia coli with 2',(3')-Substituted Purine and Pyrimidine Nucleotides, Journal of Pharmacology and Experimental Therapeutics, vol.336, issue.1, pp.234-241, 2011.
DOI : 10.1124/jpet.110.170993

S. Steiner, C. Lori, A. Boehm, and U. Jenal, Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein???protein interaction, The EMBO Journal, vol.19, issue.3, pp.354-368, 2012.
DOI : 10.1128/AEM.05814-11

V. Stocchi, L. Cucchiarini, M. Magnani, L. Chiarantini, P. Palma et al., Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells, Analytical Biochemistry, vol.146, issue.1, pp.118-124, 1985.
DOI : 10.1016/0003-2697(85)90405-1

C. Stover, X. Pham, A. Erwin, S. Mizoguchi, P. Warrener et al., Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen, Nature, vol.406, pp.959-964, 2000.

N. Sudarsan, E. Lee, Z. Weinberg, R. Moy, J. Kim et al., Riboswitches in Eubacteria Sense the Second Messenger Cyclic Di-GMP, Science, vol.321, issue.5887, pp.411-413, 2008.
DOI : 10.1126/science.1159519

L. Tagliabue, D. Antoniani, A. Maci?g, P. Bocci, N. Raffaelli et al., The diguanylate cyclase YddV controls production of the exopolysaccharide poly-N-acetylglucosamine (PNAG) through regulation of the PNAG biosynthetic pgaABCD operon, Microbiology, vol.156, issue.10, pp.2901-2911, 2010.
DOI : 10.1099/mic.0.041350-0

R. Tamayo, J. Pratt, and A. Camilli, Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis, Annual Review of Microbiology, vol.61, issue.1, pp.131-148, 2007.
DOI : 10.1146/annurev.micro.61.080706.093426

I. Tiede, G. Fritz, S. Strand, D. Poppe, R. Dvorsky et al., CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes, Journal of Clinical Investigation, vol.111, issue.8, pp.1133-1145, 2003.
DOI : 10.1172/JCI16432

A. Ueda, C. Attila, M. Whiteley, and T. Wood, and fluorouracil is an antagonist, Microbial Biotechnology, vol.189, issue.1, pp.62-74, 2009.
DOI : 10.1111/j.1751-7915.2008.00060.x

A. Ueda and T. Wood, Connecting Quorum Sensing, c-di-GMP, Pel Polysaccharide, and Biofilm Formation in Pseudomonas aeruginosa through Tyrosine Phosphatase TpbA (PA3885), PLoS Pathogens, vol.179, issue.3, p.1000483, 2009.
DOI : 10.1371/journal.ppat.1000483.s008

X. Wang, H. Lünsdorf, I. Ehrén, A. Brauner, and U. Römling, Characteristics of Biofilms from Urinary Tract Catheters and Presence of Biofilm-Related Components in Escherichia coli, Current Microbiology, vol.17, issue.6, pp.446-453, 2010.
DOI : 10.1007/s00284-009-9563-z

P. Wassmann, C. Chan, R. Paul, A. Beck, H. Heerklotz et al., Structure of BeF3???-Modified Response Regulator PleD: Implications for Diguanylate Cyclase Activation, Catalysis, and Feedback Inhibition, Structure, vol.15, issue.8, pp.915-927, 2007.
DOI : 10.1016/j.str.2007.06.016

H. Weber, C. Pesavento, A. Possling, G. Tischendorf, and R. Hengge, network of Escherichia coli, Molecular Microbiology, vol.14, issue.4, pp.1014-1034, 2006.
DOI : 10.1046/j.1365-2958.2001.02337.x

G. Wermuth, Selective optimization of side activities: the SOSA approach, Drug Discovery Today, vol.11, issue.3-4, pp.160-164, 2006.
DOI : 10.1016/S1359-6446(05)03686-X

J. Whitney and P. Howell, Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria, Trends in Microbiology, vol.21, issue.2, 2012.
DOI : 10.1016/j.tim.2012.10.001

X. Zogaj, M. Nimtz, M. Rohde, W. Bokranz, and U. Römling, The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix, Molecular Microbiology, vol.32, issue.6, pp.1452-1463, 2001.
DOI : 10.1093/glycob/9.1.31