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Anetzberger, Claudia (2012): Heterogeneity and timing in the quorum sensing system of Vibrio harveyi. Dissertation, LMU München: Fakultät für Biologie



Bacteria produce and excrete signaling molecules, so called autoinducers, which allow them to monitor their population density and/or their environment in a process best known as quorum sensing. The Gram-negative marine bacterium Vibrio harveyi regulates certain virulence factors like type III secretion, siderophore production, and exoproteolytic activity as well as biofilm formation and bioluminescence using quorum sensing. The bacterium produces three different autoinducers: HAI-1, a N-(3-hydroxybutyryl)-D-homoserine lactone, AI-2, a furanosylborate diester, and CAI-1, a (Z)-3-aminoundec-2-en-4-one. The autoinducers are recognized by the hybrid sensor kinases LuxN, LuxQ and CqsS. All information is transferred to the phosphotransfer protein LuxU and the response regulator LuxO via phosphorelay and further transduced into the copy number of the master regulator LuxR. LuxR induces/represses a multitude of genes/operons (>100) including the lux-operon responsible for the production of bioluminescence. In order to understand how single cells behave within an autoinducer-activated community, autoinducer-induced processes were investigated in a homogeneous environment over time. Analysis of wild type single cells with respect to bioluminescence revealed that even at high cell densities only 70% of the cells of a population were bright. Moreover, fractionation of the population was found for autoinducer-controlled promoters (of genes coding for bioluminescence, exoproteolytic activity, and type III secretion) using reporter strains containing promoter::gfp fusions. These results indicated phenotypic heterogeneity of a genetic homogeneous population and were independent of the used cultivation medium, temperature or strain. An artificial increase of the autoinducer concentrations resulted in an all-bright cell population similar as observed for a luxO deletion mutant. Both, wild type and deletion mutant switched to biofilm formation at high cell density. However, the capability of the mutant to produce biofilm was significantly reduced. These data suggest that a population of the non-differentiating bacterium Vibrio harveyi takes advantages of division of labor. In addition, a temporal variation of the autoinducer concentrations over time was found. The extracellular concentrations of the three autoinducers and quorum sensing-regulated functions of Vibrio harveyi were monitored in a growing culture. In the early and mid-exponential growth phase only AI-2 was detectable and bioluminescence was induced. In the late exponential growth phase both, HAI-1 and AI-2 reached their maximum values, bioluminescence stayed high and exoproteolytic activity was induced. The stationary phase was characterized by equal concentrations of HAI-1 and AI-2, exoproteolytic activity reached its maximum, and CAI-1 activity was detectable in the culture fluids. Furthermore, only a stable and mature biofilm was formed, when HAI-1 and AI-2 were present in the above described ratios over time. CAI-1 had no influence on the biofilm formation in Vibrio harveyi. These results demonstrate that not the cell density per se is important, but that autoinducers rather control the development of a Vibrio harveyi population.