Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability

Abstract

Plasmid R68.45 is useful in promoting the transfer of chromosomal markers in bacteria of many genera. In donors harboring R68.45, chromosome-mobilizing ability (Cma) may be lost without the loss of other plasmid markers. Cma can be somewhat stabilized by maintenance of the donors in the presence of kanamycin (Km). Variants of R68.45 were isolated from 4 bacterial species of 3 genera. Plasmid variants isolated included those without Cma, without transfer function (Tra) and Cma, or without Tra, Cma and Km resistance. In Erwinia carotovora ssp. atroceptica EA153, the loss of plasmid markers is dependent on the culture medium on which the donors are maintained. Restriction endonuclease analyses of the variant plasmids revealed that most are deletion mutants of R68.45. In all cases when the uncertainty in the ends of the deletions was not too great, one end of the deletion was shown to originate within or near the direct DNA duplication in R68.45 which is required for Cma and which maps close to the Km resistance determinant. The types of deletions observed are consistent with what might be expected for deletions generated by tandemly repeated insertion sequences. The DNA duplication is the source of much of the instability observed in R68.45. Data are presented for E. carotovora ssp. atroceptica EA153 which suggest that another region of R68.45 may also play a role in its stability in this species.