Identification of Agrobacterium tumefaciens genes that direct the complete catabolism of octopine

Abstract

Agrobacterium tumefaciens R10 was mutagenized by using the promoter probe transposon Tn5-gusA7, and a library of approximately 5,000 transcriptional fusions was screened for octopine-inducible patterns of gene expression. Twenty-one mutants carrying strongly inducible gusA fusions, 20 of which showed defects in the catabolism of octopine or its metabolites, were obtained. One group of mutants could not use octopine as a carbon source, while a second group of mutants could not utilize arginine or ornithine and a third group could not utilize octopine, arginine, ornithine, or proline as a carbon source. Utilization of these compounds as nitrogen sources showed similar but not identical patterns. Fifteen fusions were subcloned together with adjacent DNA. Sequence analysis and further genetic analysis indicated that insertions of the first group are localized in the occ region of the Ti plasmid. Insertions of the second group were localized to a gene encoding ornithine cyclodeaminase. This gene is very similar to, but distinct from, a homolog located on the Ti plasmid. This gene is located immediately downstream from a gene encoding an arginase. Genetic experiments indicated that this arginase gene is essential for octopine and arginine catabolism. Insertions of the third group was localized to a gene whose product is required for degradation of proline. We therefore have identified all steps required for the catabolism of octopine to glutamate.