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J Bacteriol. 2016 May 13;198(11):1595-603. doi: 10.1128/JB.00090-16. Print 2016 Jun 1.

The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein.

Author information

1
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
2
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
3
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA georgeo@dartmouth.edu.

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.

IMPORTANCE:

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.

PMID:
27002135
PMCID:
PMC4959289
DOI:
10.1128/JB.00090-16
[Indexed for MEDLINE]
Free PMC Article

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