Identification and Characterization of Cyclic Diguanylate Signaling Systems Controlling Rugosity in Vibrio cholerae

Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
Journal of bacteriology (Impact Factor: 2.81). 10/2008; 190(22):7392-405. DOI: 10.1128/JB.00564-08
Source: PubMed


Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.

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    • "Immediately after each soxBDAG operon were predicted transcriptional regulators of the XRE family (CPS_2483 and CPS_4037) that could also be involved in differential regulation of sarcosine metabolic activity. Another class of regulators, acting via cyclic diguanylate (c-di-GMP), have been shown to control motility, attachment, EPS production and biofilm formation in a number of Gammaproteobacteria, including various species of Pseudomonas (Gjermansen et al. 2006), Vibrio (Beyhan et al. 2008; Ferreira et al. 2008), and Shewanella (Thormann et al. 2006). C. psychrerythraea 34H encodes 65 regulatory elements associated with c-di-GMP—more than 90 % of all sequenced Gammaproteobacteria. "
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    ABSTRACT: Colwellia is a genus of mostly psychrophilic halophilic Gammaproteobacteria frequently isolated from polar marine sediments and sea ice. In exploring the capacity of Colwellia psychrerythraea 34H to survive and grow in the liquid brines of sea ice, we detected a duplicated 37 kbp genomic island in its genome based on the abnormally high G + C content. This island contains an operon encoding for heterotetrameric sarcosine oxidase and is located adjacent to several genes used in the serial demethylation of glycine betaine, a compatible solute commonly used for osmoregulation, to dimethylglycine, sarcosine, and glycine. Molecular clock inferences of important events in the adaptation of C. psychrerythraea 34H to compatible solute utilization reflect the geological evolution of the polar regions. Validating genomic predictions, C. psychrerythraea 34H was shown to grow on defined media containing either choline or glycine betaine, and on a medium with sarcosine as the sole organic source of carbon and nitrogen. Growth by 8 of 9 tested Colwellia species on a newly developed sarcosine-based defined medium suggested that the ability to catabolize glycine betaine (the catabolic precursor of sarcosine) is likely widespread in the genus Colwellia. This capacity likely provides a selective advantage to Colwellia species in cold, salty environments like sea ice, and may have contributed to the ability of Colwellia to invade these extreme niches.
    Extremophiles 05/2013; 17(4). DOI:10.1007/s00792-013-0543-7 · 2.31 Impact Factor
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    • "However, while the I-site of the GGDEF domain in Orf53 is conserved, not all the amino acid residues of the A-site are conserved, suggesting that it is not a functional DGC (Fig. 1C). Instead, it could act as a c-di-GMP sensor like PelD or CdgG (Lee et al., 2007; Beyhan et al., 2008). Interestingly , members of the SXT/R391 family are not the only mobile elements bearing genes that may influence the intracellular concentration of c-di-GMP in bacteria. "
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    ABSTRACT: In Vibrio cholerae, the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) increases exopolysaccharides production and biofilm formation and decreases virulence and motility. As such, c-di-GMP is considered an important player in the transition from the host to persistence in the environment. c-di-GMP level is regulated through a complex network of more than 60 chromosomal genes encoding predicted diguanylate cyclases (DGCs) and phosphodiesterases. Herein we report the characterization of two additional DGCs, DgcK and DgcL, encoded by integrating conjugative elements (ICEs) belonging to the SXT/R391 family. SXT/R391 ICEs are self-transmissible mobile elements that are widespread among vibrios and several species of enterobacteria. We found that deletion of dgcL increases the motility of V. cholerae, that overexpression of DgcK or DgcL modulates gene expression, biofilm formation and bacterial motility, and that a single amino acid change in the active site of either enzyme abolishes these phenotypes. We also show that DgcK and DgcL are able to synthesize c-di-GMP in vitro from GTP. DgcK was found to co-purify with non-covalently bound flavin mononucleotide (FMN). DgcL's enzymatic activity was augmented upon phosphorylation of its phosphorylatable response-regulator domain suggesting that DgcL is part of a two-component signal transduction system. Interestingly, we found orthologues of dgcK and dgcL in several SXT/R391 ICEs from two species of Vibrio originating from Asia, Africa and Central America. We propose that besides conferring usual antibiotic resistances, dgcKL-bearing SXT/R391 ICEs could enhance the survival of vibrios in aquatic environments by increasing c-di-GMP level.
    Environmental Microbiology 11/2009; 12(2):510-23. DOI:10.1111/j.1462-2920.2009.02094.x · 6.20 Impact Factor
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    • "Little is known about the binding partners of GGDEF and EAL domain-containing proteins and the targets of c-di-GMP, and how the signal is transmitted. Genetic and phenotypic analyses provided invaluable information regarding proteins that affect similar pathways and clearly demonstrated signaling specificity and hierarchy (Beyhan et al., 2008; Kader et al., 2006; Kulasakara et al., 2006; Sommerfeldt et al., 2009). "
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    ABSTRACT: Bacterial pathogenesis involves social behavior including biofilm formation and swarming, processes that are regulated by the bacterially unique second messenger cyclic di-GMP (c-di-GMP). Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding signal transmission and the targets of c-di-GMP. FimX, a protein from Pseudomonas aeruginosa that governs twitching motility, belongs to a large subfamily containing both GGDEF and EAL domains. Biochemical and structural analyses reveals its function as a high-affinity receptor for c-di-GMP. A model for full-length FimX was generated combining solution scattering data and crystal structures of the degenerate GGDEF and EAL domains. Although FimX forms a dimer in solution via the N-terminal domains, a crystallographic EAL domain dimer suggests modes for the regulation of FimX by c-di-GMP binding. The results provide the structural basis for c-di-GMP sensing via degenerate phosphodiesterases.
    Structure 09/2009; 17(8):1104-16. DOI:10.1016/j.str.2009.06.010 · 5.62 Impact Factor
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