Interactions between microbes and their hosts, including man, depend upon specific communication systems. Thus, bacteria, including pathogens, perceive their hosts by sensing chemical signals, and respond appropriately. Understanding these signaling pathways, and how the microbe and its host respond, could lead to strategies for preventing pathogenesis or fostering beneficial relationships. Agrobacterium tumefaciens which interacts with plants, provides an excellent model. As a part of this interaction, the bacterium responds to a plant signal by eliciting a second signal perceived by the entire bacterial population. This second, quorum-sensing signal controls transfer of the Ti plasmid, a virulence element, to other bacteria. The long term goal is to understand this hierarchical signaling process including how the quorum sensing signal, an acyl- homoserine lactone called AAI regulates the activity of TraR, the quorum-sensing transcriptional activator required for expression of the Ti plasmid conjugation system. There are three goals for the project period. First, primer extension, and DNA footprinting will be used to determine how AccR, the plant signal-responsive repressor regulates a divergent promoter system responsible for expression of TraR. This region contains several regulated promoters; one controlled by phosphorus availability and two or more controlled by AccR. Deletions will be used to identify cis acting elements responsible for these activities. Second, using wild-type and mutant alleles, novel genetic screens as well as in vitro assays with purified protein will be employed to examine the multimeric nature of TraR, the role played by AAI in converting TraR into an active activator, for forming multimers, for binding promoters, and for activating transcription. The nature of the interaction between unactivated TraR and inner membrane will be determined, as well as the role of AAI in converting TraR into a cytoplasmic protein. The role of Agrobacterium RpoA in gene activation by TraR will be assessed. If they interact regions of the two proteins involved in this interaction will be identified. Mutants of TraR affected in affinity for AAI will be isolated and used to probe the mechanism by which AAI activates the protein. Third, genetic screens, as well as in vitro interaction assays will be used to determine how the antiactivator, TraM, interacts with TraR. Using physiological and biochemical assays the role of TraM in targeting TraR for proteolytic degradation will be determined. The pathways for this proteolysis will be identified by isolating and characterizing Agrobacterium mutants that are unable to degrade TraR in the presence and absence of AAI.
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