Bacteriophage PB2, a temperate phage that lysogenizes Agrobacterium tumefaciens, the organism responsible for the induction of crown gall tumors, will be analyzed in order to determine its efficacy as a vector for the insertion of genes into the Agrobacterium T-DNA. Analysis will include both a physical component an a genetic component. The physical analysis will include the identification and cloning of the DNA restriction fragments containing the phage and chromosomal att sites utilized by the phage for integration into the bacterial chromosome during lysogeny. To accomplish this, a number of independently isolated PB2 lysogens will be analyzed via Southern blots and PB2 DNA probes to identify the phage fragment carrying the phage att site. This fragment will be isolated and cloned into a plasmid vector, after which it will be used as a probe to identify the att site located on the Agrobacterium chromosome. Several alternative methods will be utilized for this identification, after which the fragment containing the site will be isolated and cloned. The genetic analysis will include the isolation and physical mapping of both nonsense and deletion PB2 mutants in order to determine which regions of the phage may be removable for the production of a phage replacement vector similar to those produced from lambda. The result of this work will serve as a basis for future work on agrophages and as the basis for the development of a PB2 vector which will allow genes to be rapidly inserted into the T-DNA portion of the Agrobacterium tumefaciens Ti plasmid.
