Structural and Functional Analysis of a Quorum-Sensing Inhibition Mechanism

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Bacteria can exhibit social behaviors, one of which is the process known as quorum sensing. Quorum sensing regulates a range of functions, often those involved with host interactions such as pathogenesis and symbiosis, in response to bacterial population density via signal molecules. Once the signal molecule (e.g. acylated homoserine lactones or AHLs for the Proteobacteria) reaches a threshold concentration, it effectively binds and activates a transcription factor(s), which regulates expression of genes responsible for collective bacterial activities. Although the role of AHL quorum sensing in microbial systems is relatively well accepted, mechanistic studies of the regulatory circuitry, including production and diffusion of AHLs, activation and inhibition of AHL-responsive transcription factors, and control of gene expression, are still at an early stage. The aim of this project is to examine the structural and biochemical basis of quorum sensing in Agrobacterium tumefaciens, a model plant pathogen and a premier system for mechanistic studies of quorum sensing, using a range of genetic, biochemical and structural methods particularly protein X-ray crystallography. In A. tumefaciens quorum sensing requires the AHL-responsive TraR transcription factor, and under non-inducing conditions this protein is inhibited by the TraM anti-activator through formation of a highly stable heterocomplex. The objectives of this research are as follows. First, structural and functional studies of TraM, based on the PIs' recently solved crystal structure of TraM, will be continued. These efforts will dissect the biochemical and functional complexity of TraM activity. Second, the TraM inhibitory mechanism will be studied by determining the structure of the anti-activator heterocomplex (TraM-TraR-AHL) using X-ray crystallography, complemented with biochemical analysis of this complex using biophysical and kinetic techniques. Third, functional studies will be performed to test the predictions revealed through the co-crystal structure on TraM inhibition of TraR function. These studies will represent the first structural studies on mechanistic aspects of transcription factor inhibition in a quorum sensing pathway, and the first kinetic characterizations on the molecular events of TraM antagonism on TraR. Results from this work will provide a comprehensive structure-function understanding of TraM-TraR interaction. These findings could lead to novel approaches for limiting the spread of infective A. tumefaciens in agricultural situations by the deliberate intervention into their quorum sensing. These studies will also have significant impact in the area of microbial cell-to-cell communication, and may help develop novel approaches such as the design of chemical compounds that interfere with bacterial communication, to control and combat microbial invasion. This work will also provide abundant opportunities for the involvement of undergraduates and the training of graduate students, integrating with the rich educational environment at Indiana University.