ABSTRACTThe second messenger cyclic dimeric GMP (c-di-GMP) plays a central role in controlling decision-making processes that are vitally important for the environmental survival of the human pathogenVibrio parahaemolyticus. The mechanisms by which c-di-GMP levels and biofilm formation are dynamically controlled inV. parahaemolyticusare poorly understood. Here, we report the involvement of OpaR in controlling c-di-GMP metabolism and its effects on the expression of the trigger phosphodiesterase (PDE) TpdA and the biofilm-matrix related genecpsA. Our results revealed that OpaR negatively modulates the expression oftpdAby maintaining a baseline level of c-di-GMP. The OpaR-regulated PDEs ScrC, ScrG, and VP0117 enable the upregulation oftpdA, to different degrees, in the absence of OpaR. We also found that TpdA plays the dominant role in c-di-GMP degradation under planktonic conditions compared to the other OpaR-regulated PDEs. In cells growing on solid medium, we observed that the role of the dominant c-di-GMP degrader alternates between ScrC and TpdA. We also report contrasting effects of the absence of OpaR oncpsAexpression in cells growing on solid media compared to cells forming biofilms over glass. These results suggest that OpaR can act as a double-edged sword to controlcpsAexpression and perhaps biofilm development in response to poorly understood environmental factors. Finally, using anin-silicoanalysis, we indicate outlets of the OpaR regulatory module that can impact decision making during the motile-to-sessile transition inV. parahaemolyticus.IMPORTANCEThe second messenger c-di-GMP is extensively used by bacterial cells to control crucial social adaptations such as biofilm formation. Here, we explore the role of the quorum-sensing regulator OpaR, from the human pathogenV. parahaemolyticus, on the dynamic control of c-di-GMP signaling and biofilm-matrix production. We found that OpaR is crucial to c-di-GMP homeostasis in cells growing on Lysogeny Broth agar and that the OpaR-regulated PDEs TpdA and ScrC alternate in the dominant role over time. Furthermore, OpaR plays contrasting roles in controlling the expression of the biofilm-related genecpsAon different surfaces and growth conditions. This dual role has not been reported for orthologues of OpaR, such as HapR fromVibriocholerae. It is important to investigate the origins and consequences of the differences in c-di-GMP signaling between closely and distantly related pathogens to better understand pathogenic bacterial behavior and its evolution.