Signal transduction through the Vibrio harveyi quorum-sensing cascade

Quorum sensing is a process of cell-to-cell communication by which bacteria measure fluctuations in cell population density via secreted signal molecules called autoinducers. As cell population density increases, the accumulation of autoinducers leads to synchronous changes in gene expression in the bacterial community. Vibrio harveyi, a Gram-negative marine bacterium, produces three autoinducers and employs three cognate hybrid histidine sensor kinases to detect and respond to them.;Of particular interest to this work is the autoinducer called AI-2, which is produced and detected by diverse species of bacteria to facilitate interspecies communication. In Vibrios, AI-2 is detected by a heterodimer of the periplasmic binding protein, LuxP, and the histidine sensor kinase, LuxQ. Despite the frequency with which bacteria employ histidine sensor kinases to sense their environment, how kinase activity is regulated by ligand binding is not well understood. The experiments described in this thesis reveal that AI-2 binding causes a major conformational change in LuxP, stabilizing a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. Investigation of LuxQ kinase activity confirmed that autophosphorylation occurs in trans within a LuxQ dimer, while subsequent phosphotransfer occurs in cis. Therefore, the asymmetric quaternary structure induced by AI-2 in the periplasm is proposed to disrupt cytoplasmic symmetry and repress LuxQ kinase activity by preventing in trans autophosphorylation. The HAMP linker was identified as the domain responsible for LuxQ dimerization, suggesting that periplasmic asymmetry is transduced to the LuxQ cytoplasmic domains by disrupting HAMP-mediated dimerization.;The activity of all three histidine sensor kinase receptors is integrated into a shared phosphorelay cascade that ultimately controls production of LuxR, the quorum-sensing master regulator. LuxR is an unusual member of the TetR protein superfamily because it is capable of activating and repressing a large number of genes. Using protein binding microarrays and a two-layered bioinformatics approach, LuxR was shown to bind a 21 bp consensus operator with dyad symmetry. The definition and subsequent characterization of the LuxR consensus sequence enabled a scan of the V. harveyi genome and identification of novel targets of LuxR, thus expanding the understanding of the quorum-sensing regulon.