The long term goal is to develop an understanding concerning the molecular biology of Agrobacterium tumefaciens especially as it pertains to how this organism, interacts with plant cells to induce crown gall tumors. This interaction is interesting in that it appears that plant cell or tumor factors play a role in the regulation of genetic expression for certain bacterial traits. Among these are determinants encoded on the Ti plasmid required for conjugal transfer of this virulence factor. It is known that the tumor effectors for this regulation are the opines that the bacterium engineers the tumor cells to synthesize. It is also known that these opines most likely play a nutritional role in the ecology and natural history of the pathogen; virulent agrobacteria are specifically able to utilize the compounds as carbon and energy sources. The genetic determinants for such utilizatiion are also encoded on the Ti plasmid. The basis of this proposal is an investigation into the regulatory relationships between the Ti plasmid determinants for opine degradation and conjugal transfer. The loci for conjugal opine catabolism will be analyzed by recombinant DNA clonings, saturation insertional mutagenesis and production of specific deletions. Gene-enzyme relationship will be established by determining catabolic activities in well-mapped mutants. Determinants involved in conjugal transfer will be mapped by clone analysis and also by the brute force isolation of mutations induced by insertion elements. Once located, transcription and its control by opines will be determined for these two sets of genes by the use of transcription fusion insertion probes. The key problem of regulation will be approached by isolating mapping and comparing transfer-constitutive and opine constitutive mutants. The repressor gene, in both its mutant and wild-type form will be cloned and its influence on the regulation of Tra and opine catabolic loci determined. This work will provide important information concerning the location, organization and regulation of Ti plasmid genes. The work is equally important in that it constitutes an investigation into a set of traits, the expression of which is regulated by effectors associated with a pathogen-host interaction. This is clearly relevant to any interaction specific to an organism and its parasites.
