Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome

We constructed several versatile sets of vectors that can be used to introd uce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 c hromosome without affecting T-DNA transfer. One set contains a fragment con taining the lacI(q) and lacZ genes and a multiple cloning site from pBluesc riptII SK(+) inserted into a PstI site between the pgl and picA genes on an incP alpha plasmid, The resulting plasmid contains eight unique restrictio n endonuclease sites and the ability to use blue-white screening for the pr esence of an insert. A second plasmid also contains a beta -lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resist ance marker for the selection of genes integrated into the chromosome follo wing double homologous recombination (homogenotization). A third plasmid co ntains, in addition to the lacZ, lacI(q), and P-lactamase genes within the pcl/picA locus, a sacRB gene cassette within the vector to counterselect ag ainst the presence of the vector within A. tumefaciens, To test this system , we introduced a wild-type virD2 gene into the A. tumefaciens chromosome a t the pgl/picA locus. When a Ti plasmid harboring a deletion of virD2 was i n this strain, the integrated virD2 gene complemented the virD2 deletion an d the resulting transformation phenotype was identical to that resulting fr om A. tumefaciens strains harboring a wild-type virD2 gene located on a rep licating plasmid.