Fuqua 9723837 Bacteria monitor and respond rapidly to changes in the physical and chemical character of their environment. It is now clear that many bacteria also have evolved mechanisms by which they measure their own population growth, a phenomenon generally described as quorum sensing. A wide range of bacteria, often pathogens or symbionts of animals or plants, monitor cell density by production and perception of a diffusible bacterial pheromone, an acylated homoserine lactone called an autoinducer (AI). The plant pathogen Agrobacterium tumefaciens regulates conjugal transfer of its primary virulence factor, the Ti (tumor-inducing) plasmid, in response to such an acylated homoserine lactone cell density signal. This project is directed towards understanding the mechanism of AI-mediated quorum sensing in A. tumefaciens, with the long term aim of applying this knowledge towards disrupting or manipulating cell density-dependent processes in the diverse and rapidly expanding collection of bacteria that employ autoinducers and quorum sensing. At high cell densities A. tumefaciens conjugally transfer the Ti plasmid to recipient agrobacteria. Cell density-dependence of Ti plasmid conjugation is mediated by a LuxI-LuxR type quorum-sensor and the Agrobacterium autoinducer (AAI), N-3-oxo(octanoyl)-L-homoserine lactone. The A. tumefaciens quorum-sensor consists of the AAI synthase TraI and the AAI-responsive transcriptional activator of Ti plasmid conjugal transfer (tra) genes, TraR. However, in contrast to the V. fischeri system, A. tumefaciens cell density responsiveness requires an additional regulatory protein called TraM. Tram is an integral component of the A. tumefaciens quorum sensor and strains carrying null mutations in tram conjugate irrespective of donor density. Tram inhibits transcriptional activation of TraR-dependent target genes. Preliminary genetic evidence suggests that Tram may interfere with the ability of TraR to interact with AAI, the tra gene target promoters, or both. The go al of this research is to understand the role of TraM as a component of the A. tumefaciens quorum sensor-specifically how TraM inhibits activation of TraR-dependent target genes. Using complementary genetic and biochemical approaches it will be determined whether (i) TraM occludes tra promoters from TraR, (ii) degrades and/or sequesters AAI, or (iii) physically associates with TraR thereby limiting its activity. A related area that is being investigated is the regulation of traM expression, and the importance of this regulation for control of Ti plasmid conjugal transfer. In dissecting the TraM mechanism of action, insights will be gained into the function of TraR itself, and by extension other LuxR-type proteins. Understanding the inhibitory function of TraM may provide models for modulating the activity of LuxR-type proteins in general. In a broader sense, this study will contribute to the understanding of multicellular behavior in prokaryotic organisms as well as the areas of signal transduction and transciptional regulation..
