The quorum regulatory cascade is poorly characterized in Vibrio parahaemolyticus, in part because swarming and virulence factors-the hallmarks of the organism-are repressed by this scheme of gene control, and quorum sensing seems to be silenced in many isolates. In these studies, we examine a swarming-proficient, virulent strain and identify an altered-function allele of the quorum regulator luxO that is demonstrated to produce a constitutively active mimic of LuxOϳP. We find that LuxO* affects the expression of three small regulatory RNAs (Qrrs) and the activity of a translational fusion in opaR, the output regulator. Tests for epistasis showed that luxO* is dominant over luxO and that opaR is dominant over luxO. Thus, information flow through the central elements of the V. parahaemolyticus quorum pathway is proven for the first time. Quorumsensing output was explored using microarray profiling: the OpaR regulon encompasses ϳ5.2% of the genome. OpaR represses the surface-sensing and type III secretion system 1 (T3SS1) regulons. One novel discovery is that OpaR strongly and oppositely regulates two type VI secretion systems (T6SS). New functional consequences of OpaR control were demonstrated: OpaR increases the cellular cyclic di-GMP (c-di-GMP) level, positively controls chitin-induced DNA competency, and profoundly blocks cytotoxicity toward host cells. In expanding the previously known quorum effects beyond the induction of the capsule and the repression of swarming to elucidate the global scope of genes in the OpaR regulon, this study yields many clues to distinguishing traits of this Vibrio species; it underscores the profoundly divergent survival strategies of the quorum On/Off phase variants.Many members of the Vibrionaceae are well known for their capacities to communicate and to control group activities such as biofilm formation, virulence, and luminescence via cell-tocell signaling (reviewed in reference 48). However, quorum sensing has not been intensively investigated in Vibrio parahaemolyticus. This ubiquitous marine organism is the leading worldwide cause of seafood-borne gastroenteritis (43,47,60). Although its pathogenic strategies are not well understood, V. parahaemolyticus possesses a powerful arsenal of potential virulence factors, including proteases, hemolysins, two type VI secretion systems (T6SS1 and T6SS2), and two type III secretion systems (T3SS1 and TSS2) (37). The two T3SS, which are specially designed to inject effector virulence factors into eukaryotic host cells, have garnered much attention recently and have been shown to play distinct and critical roles in the pathogenicity of the organism (9, 27). Another hallmark of the organism is a marked proficiency at surface colonization, which is determined by its vigorous capacity to swarm and form robust biofilms (reviewed in reference 39). Our lack of knowledge about quorum sensing in V. parahaemolyticus stems in part from the fact that the archetypal V. parahaemolyticus strains appear defective in cell density-dependent regulation. S...