Agrobacterium tumefaciens, a gram negative soil bacterium that can live in parasitic association with higher plants, will be used to understand how disease-causing microbes and their hosts interact, and may also provide fundamental insights about how cells and organisms gain and process information from their surroundings. Agrobacterium senses wounded plants susceptible to infection by perceiving a class of phenolic compounds, while it senses tumorous plant tissues that are a consequence of infection by perceiving a class of derivatized amino acids and sugars known as opines. Two proteins (VirA and VirG) are required for the first of these recognition systems, and we will test the hypotheses that one of these is an environmental sensor, while the other is a transcriptional activator. We will determine whether the putative environmental sensor has a binding site for inducers, and whether it (like several homologous proteins) is capable of autophosphorylation. We will also obtain point mutants in each of these proteins that are constitutively active. To facilitate all these studies, we will attempt to reconstruct this regulatory system in E. coli. We will also study a separate regulatory system which regulates the virG promoter, identifying both cis- and trans-acting elements. Another set of T-plasmid encoded genes is inducible by opines, and little is known about the proteins mediating this response. We will identify trans-acting factors required to regulate opine-inducible genes, and subclone and sequence them. Finally and most importantly we will identify and characterize new loci which are induced by opines. By analyzing their predicted amino acid sequence and by characterizing stains with mutations in each of these genes we will gain insights about their role in the maintenance of this parasitic association.
