Three Parallel Quorum-Sensing Systems Regulate Gene Expression in Vibrio harveyi

Many bacteria coordinate gene expression using a process of cell-cell communication termed quorum sensing. Quorum sensing involves the production of, detection of, and response to extracellular signaling molecules known as autoinducers. Quorum-sensing systems allow bacteria to monitor their population density and to induce or repress particular genes in response to changes in cell number. Specifically, as a bacterial population grows, the extracellular concentration of autoinducer increases. When a critical autoinducer concentration is achieved, the bacteria respond as a group to coordinately alter target gene expression. Quorum-sensing systems have been identified in both gram-negative and gram-positive species of bacteria, and they are used to regulate diverse functions, such as bioluminescence, conjugation, virulence, biofilm formation, and antibiotic production (reviewed in reference 24). Vibrio harveyi is a bioluminescent marine bacterium that lives in several milieus: it can exist free swimming in seawater, adhered to abiotic surfaces, as a constituent of biofilm consortia in marine animals, and in pathogenic associations with marine hosts (28). Two quorum-sensing systems have been identified in V. harveyi, and together they control functions such as bioluminescence (2, 3), type III secretion (TTS) (18), and siderophore (21), polysaccharide (21), and metalloprotease production (26) (Fig. ​(Fig.1A).1A). The System 1 autoinducer, N-(3-hydroxybutanoyl) homoserine lactone (HSL) (denoted HAI-1 for harveyi autoinducer 1), is produced by LuxM and is detected by LuxN (2, 6). The System 2 autoinducer, called AI-2,is 3A-methyl-5,6-dihydro-furo(2,3-d)(1,3,2)dioxaborole-2,2,6,6A-tetraol, and the unborated precursor is made by LuxS (7, 31, 35). Two proteins, LuxP and LuxQ, function together as the AI-2 sensor (3). LuxP is similar to periplasmic ribose binding proteins. Both LuxN and LuxQ are hybrid two-component proteins that contain sensor kinase and response regulator domains. The remainder of the signaling cascade is shared by System 1 and System 2 (Fig. ​(Fig.1A1A). FIG. 1. Models of the V. harveyi and V. cholerae quorum-sensing systems. (A) V. harveyi has three parallel quorum-sensing systems that regulate the genes encoding bioluminescence (Lux), TTS, a secreted metalloprotease (VhpA), and other quorum-sensing-activated ... In V. harveyi, at low cell density (i.e., in the absence of the autoinducers), LuxN and LuxQ autophosphorylate and transfer phosphate to the shared phosphotransferase protein LuxU, which in turn passes it to the response regulator, LuxO (12-14). Phosphorylated LuxO is active and, in conjunction with σ54, activates the expression of genes encoding small regulatory RNAs (sRNAs) that, together with the RNA chaperone Hfq, destabilize the mRNA encoding the activator protein LuxR (20, 21). LuxR is required for expression of the target genes in the quorum-sensing regulon (Fig. ​(Fig.1A)1A) (18, 20, 22, 32). Because LuxR is required for transcription of luxCDABE (encoding luciferase), no light is produced under this condition. At high cell density, (i.e., in the presence of autoinducers), LuxN and LuxQ switch from kinases to phosphatases (14). Phosphate flow through the system is reversed, draining LuxO of phosphate (12). Unphosphorylated LuxO is inactive and thus cannot promote the expression of the sRNAs, leading to translation of luxR mRNA (20). LuxR binds the luxCDABE promoter and activates its expression, and V. harveyi produces light. Many other genes are controlled by quorum sensing in V. harveyi. While all require LuxR for their regulation, some of them, like lux, are induced at high cell densities, while others are repressed at high cell densities (Fig. ​(Fig.1A)1A) (18, 26). Miller et al. identified and characterized two two-component quorum-sensing circuits controlling quorum sensing in the human pathogen Vibrio cholerae (Fig. ​(Fig.1B)1B) (25). Analysis of the completed genome of V. cholerae revealed that the V. harveyi-like System 2 (luxS and luxPQ) was present as well as luxU and luxO, and a genetic screen identified a novel autoinducer synthase named CqsA (for cholerae quorum-sensing autoinducer) and a cognate sensor named CqsS (for cholerae quorum-sensing sensor). The CqsA-dependent autoinducer is called CAI-1 (for cholerae autoinducer 1), and its structure has not been determined. The V. cholerae quorum-sensing cascade functions analogously to that of V. harveyi, and the downstream regulators LuxU, LuxO, sRNA, Hfq, and HapR (which is homologous to LuxR of V. harveyi) function identically to those of V. harveyi (Fig. ​(Fig.1B)1B) (20, 25). However, many of the functions controlled by quorum sensing in V. cholerae (for example, those specifying virulence determinants) are not identical to those regulated by quorum sensing in V. harveyi (for example, bioluminescence and type III secretion). LuxS and AI-2 production are widespread in the bacterial kingdom, and AI-2 is proposed to be used for interspecies communication (1). In contrast, LuxM and HAI-1 activity appear to be restricted to V. harveyi and the closely related bacterium Vibrio parahaemolyticus, indicating that this signal molecule is relatively species specific (1). The newly identified V. cholerae CAI-1 synthase, CqsA, is not similar to LuxM nor any other known autoinducer synthase. Additionally, LuxN does not recognize CAI-1 (M. B. Miller and B. L. Bassler, unpublished data). These findings led us to speculate that CAI-1 is a novel autoinducer. We wondered if other bacteria produce CAI-1 or if this signal is unique to V. cholerae. In this report we show that several vibrios, including V. harveyi, produce CAI-1 activity. Genetic analysis in V. harveyi reveals that the Cqs system is present and acts as a third quorum-sensing system that, in parallel with Systems 1 and 2, controls the quorum-sensing target genes. All three autoinducers display synergistic activity, and thus the V. harveyi circuit is a three-way coincidence detector for the signals HAI-1, AI-2, and CAI-1. However, the V. harveyi CAI-1-CqsS system appears to differ from Systems 1 and 2 by responding to autoinducer at much lower cell densities.