Identification of three new GGDEF and EAL domain-containing proteins participating in the Scr surface colonization regulatory network in Vibrio parahaemolyticus

Vibrio parahaemolyticus rapidly colonizes surfaces using swarming motility. Surface contact induces the surface sensing regulon including lateral flagellar genes, spurring dramatic shifts in physiology and behavior. The bacterium can also adopt a sessile, surface-associated lifestyle and form robust biofilms. These alternate colonization strategies are influenced reciprocally by the second messenger c-di-GMP. Although V. parahaemolyticus possesses 43 predicted proteins with the c-di-GMP-forming GGDEF domain, none have been previously been identified as contributors to surface colonization. We sought to explore this knowledge gap by using a suppressor transposon screen to restore swarming motility of a non-swarming, high c-di-GMP strain. Two diguanylate cyclases, ScrJ and ScrL, each containing tetratricopeptide repeat coupled GGDEF domains were demonstrated to contribute additively to swarming gene repression. Both proteins required an intact catalytic motif to regulate. Another suppressor mapped in lafV, the last gene in a lateral flagellar operon. Containing a degenerate phosphodiesterase (EAL) domain, LafV affected expression of multiple genes in the surface sensing regulon and required LafK, a primary swarming activator, to repress. Mutation of the signature EAL motif had little effect on LafV’s repressive activity, suggesting LafV belongs to the subclass of EAL-type proteins that are regulatory but not enzymatic. Consistent with these activities and their predicted effects on c-di-GMP, scrJ and scrL, but not lafV mutants affected transcription of the c-di-GMP-responsive, biofilm reporter cpsA::lacZ. Our results expand the knowledge of the V. parahaemolyticus GGDEF/EAL repertoire and their roles in this surface colonization regulatory network. Significance A key survival decision, in the environment or the host, is whether to emigrate or aggregate. In bacteria, c-di-GMP signaling almost universally influences solutions to this dilemma. In V. parahaemolyticus, c-di-GMP reciprocally regulates swarming and sticking (i.e., biofilm formation) programs of surface colonization. Key c-di-GMP degrading phosphodiesterases responsive to quorum and nutritional signals have been previously identified. c-di-GMP-binding transcription factors programming biofilm development have been studied. Here, we further develop the blueprint of the c-di-GMP network by identifying new participants involved in dictating the complex decision of whether to swarm or stay. These include diguanylate cyclases with tetratricopeptide domains and a degenerate EAL protein that serves, analogous to the negative flagellar regulator RflP/YdiV of enteric bacteria, to regulate swarming.