title: The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein.
abstract: Many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs studied to date have also been found to signal for a specific c-di-GMP-related process, and although recent studies have suggested that physical interaction between DGCs and target proteins may provide this signaling fidelity, the importance of the I-site has not yet been incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, we show that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity.
  
  
  
  Some bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of which signal to specific receptors using the same small molecule, c-di-GMP. Signaling specificity in these networks may be partially driven by physical interactions between DGCs and their receptors, in addition to the autoinhibitory site of DGCs preventing the overproduction of c-di-GMP. In this study, we show that disruption of the autoinhibitory site of a DGC in Pseudomonas fluorescens can result in the loss of interactions with its target receptor and reduced biofilm formation, despite increased production of c-di-GMP. Our findings implicate the autoinhibitory site as both an important feature for signaling specificity through the regulation of c-di-GMP production and a necessary element for the physical interaction between a diguanylate cyclase and its receptor.
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Kurt M
authors.initials: KM
authors.lastname: Dahlstrom
authors.affiliation: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: Krista M
authors.initials: KM
authors.lastname: Giglio
authors.affiliation: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: Holger
authors.initials: H
authors.lastname: Sondermann
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA georgeo@dartmouth.edu.
authors.forename: George A
authors.initials: GA
authors.lastname: O'Toole
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D009154:Mutation; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase