The insertion sequence IS21 of R68.45 and the molecular basis for mobilization of the bacterial chromosome

The IncP-1 plasmid mutant R68.45, which is able to mobilize the chromosomes of many Gram-negative bacteria, was shown to carry a 2.10-kb insertion sequence designated IS21. This sequence transposed to the small multicopy plasmid pED815 at a high frequency (2 × 10^?3) and in two pED815::IS21 derivatives inactivated the tetracycline-resistance and replication functions, respectively. We propose that the chromosome-mobilizing ability of R68.45 is due to the formation of an R68.45-chromosome cointegrate during transposition of IS21. This would account for its high efficiency and the absence of a fixed chromosomal origin of transfer in Pseudomonas aeruginosa PAO, and its ability to function in a variety of bacterial hosts. R68.45 is formed from R68 by duplication of a 2.1-kb DNA segment including a distinctive cluster of seven restriction endonuclease sites. The two copies of the duplicated segment are probably contiguous and so might have arisen by a transition type of mechanism. IS21 is similar in length to the duplicated segment and includes the same set of seven cleavage sites located at similar distances from the two termini. However, the single copy of the duplicated segment in R68 transposed at an undetectably low frequency (<6 × 10^?8); either the duplicated segment and IS21, although overlapping, are not identical, or they are identical but the transposition system is nonfunctional in R68. Our further investigations of R68.45 and of several independently isolated chromosome-mobilizing derivatives of R68 demonstrated that these were indistinguishable from each other and that they did not include any P. aeruginosa PAO DNA. Furthermore, we searched without success for sequences corresponding to IS21, and to the Escherichia coli K-12 insertion sequences IS1, IS2, and IS3, on the chromosomes of P. aeruginosa PAO and PAT and P. putida PPN, and on several Pseudomonas plasmids. The contribution of homology to low-frequency chromosomal mobilization by these plasmids is discussed.