Characterization and mapping of the agrocinopine-agrocin 84 locus on the nopaline Ti plasmid pTiC58

Abstract

Overlapping segments of pTiC58 inserted into cosmid vectors were used to characterize the agrocinopine-agrocin 84 locus from the nopaline/agrocinopine A and B Agrobacterium tumefaciens strain C58. All of the clones conferring agrocin 84 sensitivity on agrobacteria also conferred uptake of agrocin 84 and agrocinopines A and B. Transposon Tn3-HoHo1 insertion mutations of one such clone were generated that simultaneously abolished agrocin 84 sensitivity and transport of agrocinopines A and B and agrocin 84. Such insertions were found to cluster within a 4.4-kilobase region. Analysis of beta-galactosidase activity in these insertion mutants suggested a single transcriptional unit regulated at the transcriptional level by agrocinopines A and B. The smallest DNA fragment subcloned from the region to confer all three activities was 8.5 kilobases long. This subclone was still properly regulated, indicating that the regulatory gene is closely linked to the locus. The data are consistent with a single operon encoding catabolism of agrocinopines A and B and conferring sensitivity to agrocin 84. Based on these results, we support the locus name acc, for agrocinopine catabolism.