Summary: Novel recombinant plasmids derived from the killer plasmid k2 have been constructed to study plasmid biology and gene function. recombination between native resident k2 and suitable disruption vectors, employing the KlTRP1 gene fused to a plasmid promoter as selection marker, yielded ORF2 and ORF6 deletion plasmids at high frequencies. As judged from Southern hybridization and plasmid restriction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6, respectively, carry deletions in their putative DNA (ORF2) and RNA (ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for over 350 generations of growth did not favour maintenance of hybrids over wild-type k2. Thus, neither rk2/2 nor rk2/6 was fully functional and able to displace parental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy number relative to k2 with rk2/2 more drastically affected than rk2/6 implying a direct involvement of the ORF2 product in plasmid replication and an indirect maintenance function for the ORF6 gene product.


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