| In a process called quorum sensing (QS), collectives of bacteria communicate and interact to coordinate gene expression throughout a population and carry out sophisticated behaviors in concert. This cell-to-cell communication mechanism enables bacteria to assess their population density through the production, secretion and detection of diffusible signal molecules called autoinducers (AIs). The free-living, bioluminescent marine bacterium Vibrio harveyi produces three different AIs and has been adopted as an ideal model system for studying the universal molecular processes underlying QS-dependent gene regulation.;The experiments described in this thesis aim to understand how V. harveyi accurately links, interprets and processes external AI levels to precisely control the internal regulation of downstream gene expression. This work characterizes five highly conserved small regulatory RNAs, called qrr1-5 (Quorum Regulatory RNA) that act at the core of the V. harveyi QS circuit. Mutational analysis reveals that the Qrrs function additively to control levels of LuxR, the master transcriptional regulator of QS target genes. This mechanism produces a gradient of LuxR that, in turn, enables differential regulation of QS-target genes. Other regulators appear to be involved in the control of V. harveyi qrr expression, which allows for the integration of additional sensory information to impinge on the regulation of QS gene expression.;This work also reveals certain design features of the V. harveyi QS circuit, in the form of multiple negative feedback regulation, that are required for the proper timing and expression level of QS target genes. Specifically, LuxR can activate the transcription of qrr2, qrr3, and qrr4, which leads to the rapid downregulation of luxR. Disruption of this feedback loop affects QS dynamics by delaying the transition from high to low cell density, and also decreases the cell density at which the population reaches a quorum. Two other feedback loops have been identified that control the levels of the central response regulator protein, LuxO. LuxO represses its own transcription, and this process does not require that LuxO be phosphorylated. The Qrrs also post-transcriptionally repress luxO translation, which results in a mechanism that prevents runaway expression of the qrr genes. Disruption of the two LuxO negative feedback loops causes increased LuxO∼P, which is ultimately translated downstream to affect LuxR protein levels, and the genes that are regulated by LuxR. Together, the multiple feedback loops described in this thesis function in coordination to control the precise timing of QS gene expression in response to changes in external AI concentrations, and ultimately to dictate the proper timing of QS transitions. Thus, the molecular underpinnings of network dynamics have been determined in V. harveyi and we have begun to understand how multiple feedback architectures may be beneficial for productive QS behaviors. |
