IS Family Members IS and IS Also Form Cointegrates by Copy-In and Targeted Conservative Routes.

IS has been shown to form cointegrates both by a copy-in mechanism involving one insertion sequence (IS) and a target and by a targeted conservative mechanism involving two ISs. IS is the flagship of a group of 65 bacterial ISs in the recently redefined IS/IS family. Here, whether other family members can also use two mechanisms was examined using members of the IS/IS and IS isoform groups, which are associated with antibiotic resistance genes in staphylococci and enterococci, respectively. Transposases Tnp257 and Tnp1216 have 39% and 47% amino acid identities, respectively, with Tnp26 and are 62% identical to one another. Using a novel transposition assay, pUC-based plasmids carrying these ISs integrated into the chromosome of a temperature-sensitive strain grown at the restrictive temperature. In the cointegrates, the plasmid carrying IS was flanked by various 8-bp target site duplications, consistent with random target selection. However, in a mating-out assay, only the targeted conservative reaction was detectable at a low frequency in a -negative strain, indicating that IS is at least 100-fold less active than IS For IS, in mating-out assays, both copy-in and targeted conservative cointegrate formation were detectable at frequencies similar to those observed for IS Duplication of various 8-bp target sites was detected for the copy-in route. For both IS and IS, when both of the plasmids carried an IS, the targeted conservative route occurred at a significantly higher frequency than the copy-in route, and only cointegrates formed by the conservative route were detected. IS differs from other studied ISs in the reactions that it can undertake. The differences make IS uniquely suited to its key role in the recruitment and spread of antibiotic resistance genes in Gram-negative bacteria. However, whether other ISs in the IS/IS family can perform the same reactions is not known. IS/IS and IS isoforms found associated with antibiotic resistance genes in the Gram-positive bacteria staphylococci, enterococci, streptococci, and clostridia are related to IS However, the way that they move had not been investigated, limiting interpretation of their role in resistance gene dissemination and in the formation of cointegrates and complex resistance regions in staphylococci and enterococci. Here, they are shown to share the broad catalytic capabilities of IS, demonstrating that it is likely that all members of the redefined IS/IS family of bacterial ISs likewise are able to use both the copy-in and conservative routes.