The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP
© Hunter et al.; licensee BioMed Central Ltd. 2014
Received: 7 May 2013
Accepted: 24 January 2014
Published: 4 February 2014
Diguanylate cyclases (DGCs) regulate biofilm formation and motility in bacteria by synthesizing the second messenger cyclic di-GMP (c-di-GMP) in response to environmental stimuli. DGC enzymatic activity is believed to be dependent on the presence of a GG(D/E)EF active site motif, however approximately 25% of known DGCs contain a degenerate active site. The Vibrio cholerae protein VCA0965 contains an AGDEF active site and is presumed to be an inactive DGC.
Ectopic expression of VCA0965 in V. cholerae causes a 3-fold reduction in flagellar-based motility. Additionally, an RXXD allosteric inhibition mutant of VCA0965 strongly inhibited motility and stimulated biofilm formation. This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP. In support of this, ectopic expression of VCA0965 and VCA0965 containing a mutation in its RXXD motif significantly increased the intracellular c-di-GMP levels in V. cholerae and Escherichia coli. Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro. Systematic mutation of the first amino acid in the AGDEF motif of VCA0965 revealed that glycine, methionine, and histidine also produced an active DGC capable of inhibiting motility and increasing the intracellular concentration of c-di-GMP in V. cholerae.
Based on these results, we conclude that VCA0965 is capable of c-di-GMP synthesis and that the first amino acid of the GG(D/E)EF motif is more tolerant of substitutions than currently appreciated.
KeywordsDiguanylate cyclase Phosphodiesterase Cyclic di-GMP Biofilm GGDEF
Cyclic di-GMP (c-di-GMP) is a bacterial second messenger that is widely utilized by bacteria, with more than 80% of sequenced bacteria predicted to use this signal [1, 2]. C-di-GMP controls a variety of phenotypes, including biofilm formation, motility, and virulence in multiple bacteria [3–8]. In Vibrio cholerae, high levels of c-di-GMP induce biofilm formation, reduce swimming motility, and inhibit colonization in a murine infection model [9, 10]. These c-di-GMP associated behaviors are enacted through c-di-GMP binding to a variety of receptor proteins [5, 11–13] and, potentially to one of two classes of riboswitches [14, 15]. Intracellular levels of c-di-GMP are controlled through synthesis from two molecules of GTP by diguanylate cyclases (DGC)  and degradation to pGpG or GMP by phosphodiesterases (PDE) [6, 17].
DGCs are characterized by the presence of a conserved GGDEF domain composed of approximately 200 amino acids . These domains are believed to require the specific amino acid sequence GG(D/E)EF in their active site (referred to as A-site here) in order to retain their enzymatic activity. In addition to their active site motif, approximately 53% of GGDEFs contain an RXXD motif . The RXXD motif is a feedback inhibition site (referred to as the I-site here) located near the active site, which specifically binds to dimeric c-di-GMP to non-competitively inhibit enzyme activity . EAL domains are one of the two enzymatic domains that contain c-di-GMP specific phosphodiesterase (PDE) activity, the other being the HD-GYP domain [6, 17]. Approximately 67% of DGCs are multi-domain proteins, containing at least one partner domain, with the most common partner domain being the EAL domain . Interestingly, in more than 40% of these EAL-GGDEF proteins (and in 24% of DGCs in general) the GGDEF active site is degenerate at one or more amino acids, suggesting that many of these proteins are incapable of c-di-GMP synthesis .
One explanation for the high frequency of degenerate DGCs is that these proteins in some cases act as c-di-GMP receptors by binding to c-di-GMP at either their degenerate active site or their RXXD inhibition site. These degenerate DGCs respond to c-di-GMP in several ways. C-di-GMP may mediate participation of the DGC in a regulatory cascade, as does PopA from C. crescentus, PelD from Pseudomonas aeruginosa, and CdgA from Bdellovibrio bacteriovorus which bind c-di-GMP to regulate cell cycle progression, biofilm formation, and predation, respectively [20–22]. Binding of c-di-GMP may be required for proper localization of the protein akin to both FimX from P. aeruginosa and SgmT from Myxococcus xanthus[23, 24]. Additionally, degenerate DGCs may also retain other roles independent of c-di-GMP binding. CdpA in V. cholerae requires its degenerate GGDEF domain, but not c-di-GMP, to retain its PDE activity, and the highly degenerate GGDEF domain of YybT from Bacillus subtilis exhibited ATPase activity [10, 25]. In one case, a degenerate DGC has been shown to be active as the Pectobacterium atrosepticum DGC ECA3270 retains DGC activity despite having a degenerate SGDEF active site motif . Interestingly, evidence is accumulating that DGCs and PDEs themselves form protein complexes, and it is intriguing to speculate degenerate DGCs impact these processes [27, 28].
Here we investigate the degenerate V. cholerae DGC VCA0965. We examined the ability of all 40 V. cholerae DGCs to inhibit motility in semisolid agar, and we determined that VCA0965 was active in this assay. This result was surprising as VCA0965 is a DGC that encodes a degenerate AGDEF active site. Rather than functioning as a receptor for c-di-GMP, our results suggest that VCA0965, despite its degenerate active site motif, is capable of c-di-GMP synthesis.
Bacterial strains and culture conditions
Strain and plasmid list
Strain or plasmid
Source or citation
Wild type strain
pEVS143 backbone, overexpression of QrgB (Vibrio harveyi DGC) under Ptac promoter
pEVS143 backbone, overexpression of VCA0965 under Ptac promoter
pEVS143 backbone, overexpression of VCA0965 E287A under Ptac promoter
pEVS143 backbone, overexpression of VCA0965 R267A, D270A under Ptac promoter
pEVS143 backbone, overexpression of VCA0965 R267A, D270A, E287A under Ptac promoter
pET28b backbone, overexpression of VCA0965 (180–396) with a 6 histidine C terminal tag
Minimum biofilm eradication concentration (MBEC) and flow cytometry biofilm assays
MBEC plates were prepared by inoculating 150 μL of LB/well with a 1 to 1000 dilution from overnight V. cholerae cultures. Wells were supplemented with 100 μg mL-1 kanamycin and 0.1 mM IPTG as needed. Plates were incubated at 37°C with gentle aeration for 7 hours before washing in PBS, fixing with 95% ethanol and staining with 0.41% crystal violet in 10% ethanol. Crystal violet was eluted in 160 μL of 95% ethanol. The optical density at 600 nm (OD600) was determined using a Spectra Max M5 spectrometer (Molecular Devices). To avoid saturation, the elutions were also diluted 1 to 10 and the resultant OD600 was measured. If the OD600 of the undiluted elution exceeded 1, then the OD600 was calculated from the 1 to 10 dilution. Flow cytometry cultures were prepared by diluting an overnight culture 1:100 in LB. Cultures were supplemented with kanamycin and IPTG as needed and grown at 35°C and 220 rpm for 4 hours. Flow cytometry analysis was performed as described .
Determination of the intracellular concentration of c-di-GMP
Cultures were prepared by diluting an overnight culture of V. cholerae 1:1000 into LB and grown to an OD600 between 0.6 and 1.0. Cultures were supplemented with kanamycin and IPTG as needed. Samples were extracted and analyzed by liquid chromatography coupled with tandem mass spectromentry (LC-MS/MS) as previously described  using an Acquity Ultra Performance liquid chromatography system (Waters) coupled with a Quattro Premier XE mass spectrometer (Waters). The concentration of c-di-GMP was determined by quantifying an 8-point standard curve of chemically synthesized c-di-GMP (Biolog) ranging from 1.9 nM to 250 nM.
An overnight culture of E. coli JM109(DE3) (Promega) containing the pVCA0965(180–396) vector was prepared and used to inoculate LB broth supplemented by 100 μg mL-1 kanamycin. The culture was grown at 37°C with shaking until an OD600 between 0.7-0.8 was reached. The cells were then induced by addition of IPTG to a concentration of 1 mM. The induced cells were incubated between 16 and 18 hours at 16°C with shaking. Following induction, cells were collected via centrifugation in a Sorvall RC-5B Superspeed Centrifuge for 10 minutes at 2,678 g and 4°C. The pellet was then resuspended in lysis buffer (25 mM Tris-Cl (pH 8.0), 500 mM NaCl, 5 mM 2-mercaptoethanol, 20 μg/mL DNase, one tablet Roche protease inhibitor kit) and lysed using a M-110P processor (Microfluidcs) 3 times at 20,000 psi. Novagen Ni-NTA His-bind resin was equilibrated in binding buffer (25 mM Tris-Cl (pH 8.2), 500 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol) and added to the lysate. Resin and lysate were incubated for 60 minutes at 4°C with constant end over end rotation. After incubation, lysate and resin were applied to a column and allowed to flow to pack. The resin was washed three times with small amounts of binding buffer. The protein was then step eluted by the addition of imidazole elution buffer (25 mM Tris-Cl (pH 8.2), 500 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol) with 50 mM, 100 mM, 200 mM, or 300 mM imidazole. VCA0965 was determined to elute in the 200 mM and 300 mM imidazole fractions. These fractions were concentrated on Amicon Ultra Centrifugal Units by centrifugation at 16,000 g for 20 minutes at 4°C. Concentrated fractions were washed once with dialysis buffer (30 mM Tris-Cl (pH 7.6), 100 mM NaCl) and then stored at −80°C in 20% glycerol.
Determination of DGC activity
Purified proteins were diluted 1/25 and mixed with 0.015 mM GTP in reaction buffer (41 mM NaCl, 24 mM Tris-Cl, 1.25 mM MgCl2) for 24 hours at room temperature. Samples were then boiled for five minutes until precipitate formed and centrifuged at 15,871 g for 10 min. to remove precipitated proteins. The concentration of c-di-GMP was determined using LC-MS/MS as described above.
Census of DGCs that regulate motility in Vibrio cholerae
Vibrio cholerae strain C6706str2 encodes 40 GGDEF domain containing proteins which potentially function as DGCs. As c-di-GMP represses motility in V. cholerae, we determined which of the 40 GGDEFs repress motility in laboratory conditions by individually overexpressing each from a Ptac promoter on a plasmid and measuring flagellar based swimming motility in 0.5% agar plates. We chose to survey the activity of all DGCs using a heterologous expression strategy, rather than mutagenesis analysis, because this approach ensures that each DGC is expressed in the conditions examined. Our results indicated that 23 of the GGDEF encoding proteins significantly repressed motility while 7 significantly increased motility (Figure 1). All 7 of the proteins that increase motility contain both a GGDEF domain and an EAL domain. Therefore, we hypothesize that these proteins likely possess net PDE activity resulting in decreased intracellular c-di-GMP. Interestingly, VCA0965 repressed motility 4.6-fold, yet it is predicted to be enzymatically inactive due to a non-canonical AGDEF active site. For this reason, we sought to further understand the role of VCA0965 in the regulation of c-di-GMP controlled behaviors in V. cholerae.
The active site of VCA0965 is required for motility inhibition
VCA0965-I induces biofilm formation
To confirm these results, we measured biofilm formation using flow cytometry as previously described . This assay measures aggregate formation in liquid cultures, which is driven by extracellular polysaccharide production. Analysis of biofilms by flow cytometry was indeed similar to the results of the MBEC assay in that WT VCA0965 was unable to stimulate aggregate formation, but the VCA0965-I mutant strongly stimulated aggregate formation in an A-site dependent manner (Figure 3B).
VCA0965 synthesizes c-di-GMP
Determining the plasticity of the first amino acid in the active site of VCA0965
The activity of the glycine, histidine, and methionine substitutions was further examined by determining if induction of these proteins significantly increased intracellular c-di-GMP in V. cholerae. Indeed, all three of these VCA0965 mutants significantly increased c-di-GMP when induced compared to the uninduced controls; the histidine and methionine mutations were statistically significant (p < 0.05 using a one-tailed Student’s t-test) while the glycine substitution bordered on statistical significance with a p-value of 0.067 (Figure 6B). The Ptac promoter used to induce these proteins is leaky accounting for the variation in c-di-GMP levels in the uninduced controls. All four active VCA0965 variants increased c-di-GMP levels by 3-fold. These results indicate that the first position of the VCA0965 active site is somewhat flexible, maintaining activity with four different amino acids.
Bioinformatic analysis of AGDEF active sites
Bioinformatic analysis of the frequency of the AG(D/E)EF active site motif
Number of proteins
Percentage of total GGDEFs
Percentage of total AG(D/E)EF proteins
Total GGDEF domain containing proteins analyzed
Proteins containing an AG(D/E)EF motif
Proteins containing an AG(D/E)EF motif and an RXXD motif
Proteins containing an AG(D/E)EF motif from Vibrio sp.
Known GGDEF domain containing proteins from Vibrio sp.
Proteins containing an RXXD and an XG(D/E)EF motif
Bioinformatic analysis of the N-terminal region of VCA0965
Number of proteins
Percentage of total GGDEFs
Percentage of total N-terminal analogs
Proteins containing homology to VCA0965′s N terminus
Proteins containing an AG(D/E)EF motif and homology to VCA0965′s N terminus
Proteins from Vibrio sp. containing homology to VCA0965′s N terminus
Proteins from Vibrio sp. containing homology to VCA0965′s N terminus and an AG(D/E)EF motif
Here we show that VCA0965, which encodes a degenerate AGDEF active site, is an active DGC that synthesizes c-di-GMP. Overexpression of VCA0965 represses motility by increasing c-di-GMP levels in V. cholerae, and the DGC domain of VCA0965 is capable of in vitro c-di-GMP synthesis. To our knowledge, this is the first description of an active DGC containing a naturally encoded AGDEF active site motif and only the second description of an active non-canonical DGC; the only other example being the SGDEF containing ECA3270 from Pectobacterium atrosepticum. ECA3270 regulates Type 1 secretion and was found to retain a reduced level of activity when the active site was mutated to AGDEF. Interestingly, we observed a loss of activity of VCA0965 when the first position was mutation to serine. This suggests that other amino acids in these DGCs have coevolved to tolerate these non-degenerate substitutions in the first position of their active site.
Retention of activity in ECA3270 and VCA0965 is surprising when the steric constraints of the active site are considered. Within the active site, the GGDEF motif is located in a hairpin . C-di-GMP bound at the active site extends over this hairpin, specifically the first G in the GGDEF motif. Although alanine is a conservative change compared to glycine, we were surprised to find that mutation of this alanine to the less conserved histidine and methionine amino acids maintained an active DGC. We would expect that larger amino acids at this position should create steric interactions detrimental to activity. It is not clear why these amino acids are tolerated over less drastic changes, such as valine, but we predict these differences are driven by specific amino acids flanking the active site in proximity to the hairpin. As both ECA3270 and VCA0965 tolerate non-canonical substitutions to the first position of their active sites, but the amino acids that are tolerated appear to be different, the flexibility of each DGC’s active site may follow its own “rules” and caution must be used when determining if noncanonical substitutions in the first residue of a given GGDEF motif render a DGC inactive.
Contrary to what might be expected based on the structure of the DGC domain, degeneracy at the first amino acid is quite common among DGCs. 23.7% of all DGCs are degenerate at one or more amino acids in the GGDEF motif . Of these, almost half vary at only one amino acid and most of these variances occur at the first amino acid . The frequency of variance at the first amino acid of the GGDEF motif despite the apparent steric limitations of the DGC structure suggest that there may be a high level of variance in DGC structure. Our in silico bioinformatic analysis of the AGDEF motif found that AG(D/E)EFs account for approximately 1.6 percent of DGCs, making this motif fairly common and suggesting that AGDEFs may have a variant structure that allows them to retain activity. How this variance impacts function remains to be addressed.
Finally, we found that although VCA0965 is able to repress motility and increase intracellular c-di-GMP levels when overexpressed, WT VCA0965 did not impact biofilm formation. However, the lack of biofilm induction is inconsistent as we have found that overexpression of VCA0965 led to an approximately 6-fold increase in biofilm formation in another study . When biofilm formation was measured using flow cytometry, no increase in biofilm formation was observed—a result corroborated by Massie et. al.. This inconsistency suggests that at the levels we are inducing VCA0965 it is only able to exert a slight effect on biofilm formation which is highly dependent on experimental conditions. These results are consistent with Massie et. al. who observed that some DGCs were able to induce biofilm formation at 12 µΜ c-di-GMP, the levels we determined are produced when VCA0965 is induced (Figure 4), while others did not . This differential induction of phenotypes by distinct DGCs at analogous concentrations of c-di-GMP is attributed to high-specificity signaling pathways. Therefore, it is not surprising for VCA0965 to have an intermediate phenotype. However, removing VCA0965 from feedback inhibition by mutation of the RXXD site increases the activity of VCA0965 allowing it to synthesize higher amounts of c-di-GMP and induce robust biofilm formation when overexpressed.
Our results highlight that the first amino acid of the GG(D/E)EF motif should be considered flexible in regards to DGC activity. In combination with the finding for ECA3270, a more accurate description of a functional active site should be (G/S/A)G(D/E)EF. However, whether enzymes which naturally encode a GG(D/E)EF will retain DGC activity upon substitution of the first glycine to serine or alanine remains to be determined. It is possible that intragenic epistatic interactions are necessary for these alterations of the first amino acid to remain functional. Additional non-canonical active site motifs should be assayed to determine if enzymatic activity of these DGCs is widespread. Furthermore, structural analysis of ECA3270 and VCA0965 can highlight how variance at the first amino acid of the GGDEF motif can accommodate DGC activity.
The research presented here shows that the AGDEF containing Vibrio cholerae DGC, VCA0965, is able to synthesize c-di-GMP to control motility and biofilm formation. Moreover, we found that addition of three other amino acids, glycine, methionine, and histidine, maintained DGC activity. Our results illustrate that the first residue of the GG(D/E)EF active site in DGCs is more flexible to substitutions than previously appreciated. As many DGCs encode non-canonical amino acids at this position, caution must be used when determining the activity of these enzymes. Whether or not substitution of non-canonical amino acids at the first position of the active site has important functional consequences on DGC activity remains to be explored.
This work was supported by the MSU Foundation and NIH grants K22AI080937, U19AI090872, and U54AI057153 to C.M.W. J.L.H. would like acknowledge support from the Professorial Assistantship program at MSU and the American Society for Microbiology Undergraduate Research Fellowship program. We also would like to thank the Rudolph Hugh Fellowship and the Russell B. DuVall Scholarship to B.J.K and the Russell B. DuVall Scholarship to J.L.H. from the Microbiology and Molecular Genetics department at MSU. The authors would like to thank the Michigan State Mass Spectrometry facility for assistance in quantifying c-di-GMP.
- Seshasayee AS, Fraser GM, Luscombe NM: Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity. Nucleic Acids Res. 2010, 38 (18): 5970-5981. 10.1093/nar/gkq382.PubMed CentralView ArticlePubMed
- Romling U, Galperin MY, Gomelsky M: Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev. 2013, 77 (1): 1-52. 10.1128/MMBR.00043-12.PubMed CentralView ArticlePubMed
- Simm R, Morr M, Kader A, Nimtz M, Romling U: GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol. 2004, 53 (4): 1123-1134. 10.1111/j.1365-2958.2004.04206.x.View ArticlePubMed
- Bellows LE, Koestler BJ, Karaba SM, Waters CM, Lathem WW: Hfq-dependent, co-ordinate control of cyclic diguanylate synthesis and catabolism in the plague pathogen Yersinia pestis. Mol Microbiol. 2012, 86 (3): 661-674. 10.1111/mmi.12011.PubMed CentralView ArticlePubMed
- Ryjenkov DA, Simm R, Romling U, Gomelsky M: The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria. J Biol Chem. 2006, 281 (41): 30310-30314. 10.1074/jbc.C600179200.View ArticlePubMed
- Ryan RP, Fouhy Y, Lucey JF, Crossman LC, Spiro S, He YW, Zhang LH, Heeb S, Camara M, Williams P, et al.: Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl Acad Sci USA. 2006, 103 (17): 6712-6717. 10.1073/pnas.0600345103.PubMed CentralView ArticlePubMed
- Kazmierczak BI, Lebron MB, Murray TS: Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa. Mol Microbiol. 2006, 60 (4): 1026-1043. 10.1111/j.1365-2958.2006.05156.x.PubMed CentralView ArticlePubMed
- Newell PD, Monds RD, O’Toole GA: LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens p f0–1. Proc Natl Acad Sci USA. 2009, 106 (9): 3461-3466. 10.1073/pnas.0808933106.PubMed CentralView ArticlePubMed
- Tischler AD, Camilli A: Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol Microbiol. 2004, 53 (3): 857-869. 10.1111/j.1365-2958.2004.04155.x.PubMed CentralView ArticlePubMed
- Tamayo R, Schild S, Pratt JT, Camilli A: Role of cyclic Di-GMP during el tor biotype Vibrio cholerae infection: characterization of the in vivo-induced cyclic Di-GMP phosphodiesterase CdpA. Infect Immun. 2008, 76 (4): 1617-1627. 10.1128/IAI.01337-07.PubMed CentralView ArticlePubMed
- Hickman JW, Harwood CS: Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol. 2008, 69 (2): 376-389. 10.1111/j.1365-2958.2008.06281.x.PubMed CentralView ArticlePubMed
- Krasteva PV, Fong JC, Shikuma NJ, Beyhan S, Navarro MV, Yildiz FH, Sondermann H: Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP. Science. 2010, 327 (5967): 866-868. 10.1126/science.1181185.PubMed CentralView ArticlePubMed
- Srivastava D, Harris RC, Waters CM: Integration of Cyclic di-GMP and Quorum Sensing in the Control of vpsT and aphA in Vibrio cholerae. J Bacteriol. 2011, 193 (22): 6331-6341. 10.1128/JB.05167-11.PubMed CentralView ArticlePubMed
- Sudarsan N, Lee ER, Weinberg Z, Moy RH, Kim JN, Link KH, Breaker RR: Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science. 2008, 321 (5887): 411-413. 10.1126/science.1159519.View ArticlePubMed
- Lee ER, Baker JL, Weinberg Z, Sudarsan N, Breaker RR: An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science. 2010, 329 (5993): 845-848. 10.1126/science.1190713.PubMed CentralView ArticlePubMed
- Ryjenkov DA, Tarutina M, Moskvin OV, Gomelsky M: Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain. J Bacteriol. 2005, 187 (5): 1792-1798. 10.1128/JB.187.5.1792-1798.2005.PubMed CentralView ArticlePubMed
- Schmidt AJ, Ryjenkov DA, Gomelsky M: The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. J Bacteriol. 2005, 187 (14): 4774-4781. 10.1128/JB.187.14.4774-4781.2005.PubMed CentralView ArticlePubMed
- Galperin MY, Nikolskaya AN, Koonin EV: Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol Lett. 2001, 203 (1): 11-21. 10.1111/j.1574-6968.2001.tb10814.x.View ArticlePubMed
- Christen B, Christen M, Paul R, Schmid F, Folcher M, Jenoe P, Meuwly M, Jenal U: Allosteric control of cyclic di-GMP signaling. J Biol Chem. 2006, 281 (42): 32015-32024. 10.1074/jbc.M603589200.View ArticlePubMed
- Duerig A, Abel S, Folcher M, Nicollier M, Schwede T, Amiot N, Giese B, Jenal U: Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression. Genes Dev. 2009, 23 (1): 93-104. 10.1101/gad.502409.PubMed CentralView ArticlePubMed
- Whitney JC, Colvin KM, Marmont LS, Robinson H, Parsek MR, Howell PL: Structure of the cytoplasmic region of PelD, a degenerate diguanylate cyclase receptor that regulates exopolysaccharide production in Pseudomonas aeruginosa. J Biol Chem. 2012, 287 (28): 23582-23593. 10.1074/jbc.M112.375378.PubMed CentralView ArticlePubMed
- Hobley L, Fung RK, Lambert C, Harris MA, Dabhi JM, King SS, Basford SM, Uchida K, Till R, Ahmad R, et al.: Discrete Cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus. PLoS Pathog. 2012, 8 (2): e1002493-10.1371/journal.ppat.1002493.PubMed CentralView ArticlePubMed
- Huang B, Whitchurch CB, Mattick JS: FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J Bacteriol. 2003, 185 (24): 7068-7076. 10.1128/JB.185.24.7068-7076.2003.PubMed CentralView ArticlePubMed
- Petters T, Zhang X, Nesper J, Treuner-Lange A, Gomez-Santos N, Hoppert M, Jenal U, Sogaard-Andersen L: The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus. Mol Microbiol. 2012, 84 (1): 147-165. 10.1111/j.1365-2958.2012.08015.x.PubMed CentralView ArticlePubMed
- Rao F, See RY, Zhang D, Toh DC, Ji Q, Liang ZX: YybT is a signaling protein that contains a cyclic dinucleotide phosphodiesterase domain and a GGDEF domain with ATPase activity. J Biol Chem. 2010, 285 (1): 473-482. 10.1074/jbc.M109.040238.PubMed CentralView ArticlePubMed
- Perez-Mendoza D, Coulthurst SJ, Humphris S, Campbell E, Welch M, Toth IK, Salmond GP: A multi-repeat adhesin of the phytopathogen, Pectobacterium atrosepticum, is secreted by a Type I pathway and is subject to complex regulation involving a non-canonical diguanylate cyclase. Mol Microbiol. 2011, 82 (3): 719-733. 10.1111/j.1365-2958.2011.07849.x.View ArticlePubMed
- Lindenberg S, Klauck G, Pesavento C, Klauck E, Hengge R: The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control. Embo J. 2013, 32 (14): 2001-2014. 10.1038/emboj.2013.120.PubMed CentralView ArticlePubMed
- Ryan RP, McCarthy Y, Andrade M, Farah CS, Armitage JP, Dow JM: Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris. Proc Natl Acad Sci USA. 2010, 107 (13): 5989-5994. 10.1073/pnas.0912839107.PubMed CentralView ArticlePubMed
- Thelin KH, Taylor RK: Toxin-coregulated pilus, but not mannose-sensitive hemagglutinin, is required for colonization by Vibrio cholerae O1 El Tor biotype and O139 strains. Infect Immun. 1996, 64 (7): 2853-2856.PubMed CentralPubMed
- Dunn AK, Millikan DS, Adin DM, Bose JL, Stabb EV: New rfp- and pES213-Derived Tools for Analyzing Symbiotic Vibrio fischeri Reveal Patterns of Infection and lux Expression In Situ. Appl Environ Microbiol. 2006, 72 (1): 802-810. 10.1128/AEM.72.1.802-810.2006.PubMed CentralView ArticlePubMed
- Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012, 9 (7): 671-675. 10.1038/nmeth.2089.View ArticlePubMed
- Massie JP, Reynolds EL, Koestler BJ, Cong J, Agostoni M, Waters CM: Quantification of high specificity signaling. Proc Natl Acad Sci USA. 2012, 109 (31): 12746-12751. 10.1073/pnas.1115663109.PubMed CentralView ArticlePubMed
- Beyhan S, Tischler AD, Camilli A, Yildiz FH: Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di-GMP level. J Bacteriol. 2006, 188 (10): 3600-3613. 10.1128/JB.188.10.3600-3613.2006.PubMed CentralView ArticlePubMed
- Waters CM, Lu W, Rabinowitz JD, Bassler BL: Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic Di-GMP levels and repression of vpsT. J Bacteriol. 2008, 190 (7): 2527-2536. 10.1128/JB.01756-07.PubMed CentralView ArticlePubMed
- Sambanthamoorthy K, Parashar V, Smith JM, Kim E, Sloup RE, Semmelhack MF, Neiditch MB, Waters CM: Identificatin of small molecules that antagonize diguanylate cyclase enzymes to inhibit biofilm formation. Antimicrob Agents Chemother. 2012, 56 (10): 5202-5211. 10.1128/AAC.01396-12.PubMed CentralView ArticlePubMed
- De N, Pirruccello M, Krasteva PV, Bae N, Raghavan RV, Sondermann H: Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biol. 2008, 6 (3): e67-10.1371/journal.pbio.0060067.PubMed CentralView ArticlePubMed
- Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, et al.: The Pfam protein families database. Nucleic Acids Res. 2012, 40 (Database issue): D290-301.PubMed CentralView ArticlePubMed
- Ryan RP, Fouhy Y, Lucey JF, Dow JM: Cyclic di-GMP signaling in bacteria: recent advances and new puzzles. J Bacteriol. 2006, 188 (24): 8327-8334. 10.1128/JB.01079-06.PubMed CentralView ArticlePubMed
- Chan C, Paul R, Samoray D, Amiot NC, Giese B, Jenal U, Schirmer T: Structural basis of activity and allosteric control of diguanylate cyclase. Proc Natl Acad Sci USA. 2004, 101 (49): 17084-17089. 10.1073/pnas.0406134101.PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.