Factors determining frequency of plasmid cointegration mediated by insertion sequence IS 1

We demonstrate that mutants with deletions at either end of the insertion sequence IS 1 lose the ability to mediate cointegration of two plasmids, whereas mutants with deletions or an insertion within IS 1 can mediate cointegration at a reduced frequency. These results, together with the nucleotide sequence analysis of the IS 1 mutants, indicate that the two ends of IS 1 ( insL and insR ) and two genes ( insA and insB ) that are encoded by IS 1 are required for cointegration. Using a plasmid carrying two copies of IS 1 , we found that the individual IS 1s mediate cointegration at different characteristic frequencies, and that each of two parts of plasmid DNA segments flanked by the two IS 1s is a transposon, mediating plasmid cointegration at a unique frequency. When one IS 1 was replaced with a mutant IS 1 , the remaining wild-type IS 1 complemented the cointegration ability of the mutant IS 1 as well as a resulting mutant transposon that was then flanked by a wild-type IS 1 and a mutant IS 1 . The efficiency of this complementation reflected the characteristic ability of an individual IS 1 present on the plasmid to promote cointegration. The results suggest that the IS 1 -encoded proteins are produced in different amounts, depending on the location of IS 1 in the plasmid, and that these amounts determine the efficiency of complementation of the cointegration ability of a mutant IS 1 as well as a mutant transposon. However, the location of an individual IS 1 itself can also determine the frequency of cointegration in the presence of a given amount of the IS 1 proteins. On the basis of the observation that the cointegration ability of a mutant IS 1 is less efficiently complemented than is the ability of a mutant transposon, we also suggest that the IS 1 -encoded proteins can function in trans , but act preferentially on the IS 1 or transposon sequence from which they are produced in promoting cointegration.