We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.

We have further characterized the transposon Tn5-facilitated chromosomal gene transfer system developed for Agrobactenium tumefaciens 15955. Using a strain whose chromosome contained Tn5, we compared the chromosome-mobilizing ability of plasmid pDP37 (containing Tn5) with that of its parent plasmid R68.45. For R68.45, we observed nonpolar transmission from multiple origins. For pDP37 we found polarized transmission from a single origin near ilv. When we examined the transmission gradients of a number of pDP37-containing donor strains each differing at the site of the chromosomal insertion we found just two classes. One set of donors transmitted markers with a gradient of Ilv+ > Rif r > His+ > Met+, whereas the second set transferred His+ > Rift > Ilv+ > Met+. Using representatives from each transmission class of donor strains, we conducted matings to measure the degree of linkage between pairs of adjacent donor markers. From this information we developed a map of the A. tumefaciens 15955 chromosome. Attempts to isolate R-prime plasmids or Hfr-like donors were unsuccessful.