Mode of Replicon Fusion Mediated by the Duplicated Insertion Sequence IS21 in Escherichia coli

摘要

The insertion sequence IS 21 (2.1 kb) originating from the broad-host-range IncP plasmid R68 transposes infrequently; by contrast, the IS 21 tandem repeat found on the derivative R68.45 is highly active in transpositional mobilization of other replicons in a variety of Gram-negative bacteria. The mobilized plasmids are joined to R68.45 by single IS 21 copies in direct orientation.—The formation of IS 21 tandem duplications was observed in cointegrates between R68.45 and pBR325::IS 21 and also in an RP1::IS 21 plasmid derivative in which a segment located between two directly repeated copies of IS 21 was deleted spontaneously. We speculate that IS 21 tandem repeats can arise when the termini of two IS 21 elements are specifically joined in a transposition or deletion event.—A resistance gene flanked by two IS 21 elements in direct orientation did not behave as a transposon. The Ω fragment carrying transcription and translation stop signals was inserted into various sites of the IS 21 tandem repeat; in this way it could be shown that the left IS 21 element (which is next to the kanamycin resistance gene in R68.45) was 100 times more active in cointegrate formation than was the righthand element.—Cointegrates between the conjugative plasmid R751 and pBR325 derivatives carrying IS 21 and IS 21 ::Ω in tandem contained a single IS 21 at one replicon junction and a single IS 21 ::Ω at the other. In the IS 21 duplications the inner IS 21 ends were preferentially recognized (presumably by IS 21 transposase), whereas the outer termini were not required for cointegrate formation. Based on these findings a conservative (simple) pathway of transposition is proposed for R68.45 and other plasmids with an IS 21 tandem repeat. In this model R68.45 is pictured as a large transposon whose ends are joined together to form a circular molecule which is capable of autonomous replication.