Diversity of Tn4001Transposition Products: the Flanking IS256Elements Can Form Tandem Dimers and IS Circles

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Abstract
We show that both flanking IS256elements carried by transposon Tn4001are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256belonging to one Tn4001copy attacks an IS256end carried by the second Tn4001copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis inEscherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such asStreptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.