The long-range goal of this lab is to continue developing the Agrobacterium-plant pathosystem as a model for studying molecular interactions between pathogenic bacteria and their hosts. After the successful oncogenic transformation of host plants, A. tumefaciens colonizes the transformed plant and uses tumor released compounds (opines) as nutrients. Under these conditions, the bacterium uses a quorum-sensing system composed of Tral, which synthesizes the bacterial pheromone N-3-oxooctanoylhomoserine lactone (OOHL) and TraR, which is an OOHL receptor and OOHL-dependent transcriptional regulator. We have purified both proteins and reconstituted their activities in vitro. We recently collaborated in solving the crystal structure of TraR, complexed with OOHL and DNA. We will take advantage of this structure to do structural studies of TraR, by doing alanine-scanning mutagenesis of the TraR surface, its OOHL binding determinants, and its DNA binding determinants. We will attempt to isolate positive control mutants, and will do a selection for constitutively active TraR mutants and for mutants able to detect heterologous autoinducers. We have shown that TraR synthesized in the absence of OOHL is rapidly targeted for proteolysis, probably by the CIp and Lon proteases. We will disrupt the Ion and the three clpP genes of Agrobacterium and measure the half life of the protein, and whether it still requires OOHL for stability and activity. We will overproduce the N-terminal domain of TraR in the present of C13 and N15 isotopes for collaborative studies of TraR folding. We will express the C-terminal domain in vivo in a form that dimerizes to check for DNA binding and for transcription activation. We have been to compare the biochemical properties of TraR to those of two other homologous proteins: CepR of Burkholderia cepacia, and YenR of Yersinia enterocolitica. CepR, requires its cognate pheromone for solubility, while YenR does not. In this respect, YenR is especially interesting, as we can purify this protein as an apo-protein or as an AHL complex and compare their properties. YenR binds to its binding site near the YenR promoter only in the apoprotein form, suggesting that the pheromone antagonizes protein function. We will do standard biochemical analysis of YenR will use several approaches to identify target genes of the YenR-Yenl regulon. In a final project, we will screen for AHL synthase genes from DNA that is purified from environmental samples and cloned into cosmids in E. coli. ? ?
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