School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia.
J Antimicrob Chemother. 2021 May 12;76(6):1428-1432. doi: 10.1093/jac/dkab055.
IS26 plays a major role in the dissemination of antibiotic resistance determinants in Gram-negative bacteria.
To determine whether insertion sequence IS26 is able to move alone (simple transposition) or if it exclusively forms cointegrates.
A two-step PCR using outward-facing primers was used to search for circular IS26 molecules. Gibson assembly was used to clone a synthetic IS26 containing a catA1 chloramphenicol resistance gene downstream of the tnp26 transposase gene into pUC19. IS activity in a recA-Escherichia coli containing the non-conjugative pUC19-derived IS26::catA1 construct and the conjugative plasmid R388 was detected using a standard mating-out assay. Transconjugants were screened for resistance.
Circular IS26 molecules that would form with a copy-out route were not detected by PCR. The synthetic IS26::catA1 construct formed CmRTpR transconjugants (where CmR and TpR stand for chloramphenicol resistant and trimethoprim resistant, respectively), representing an R388 derivative carrying the catA1 gene at a frequency of 5.6 × 10-7 CmRTpR transconjugants per TpR transconjugant, which is comparable to the copy-in activity of the unaltered IS26. To test for simple transposition of IS26::catA1 (without the plasmid backbone), 1200 CmRTpR colonies were screened and all were resistant to ampicillin, indicating that the pUC19 backbone was present. Hence, IS26::catA1 had only formed cointegrates.
IS26 is unable to move alone and cointegrates are the exclusive end products of the reactions mediated by the IS26 transposase Tnp26. Consequently, when describing the formation of complex resistance regions, simple 'transposition' of a single IS26 should not be invoked.
IS26 在革兰氏阴性菌中抗生素耐药决定因素的传播中起着重要作用。
确定插入序列 IS26 是否能够单独移动（简单转位）或它是否仅形成共整合体。
使用向外引物的两步 PCR 用于搜索环形 IS26 分子。使用 Gibson 组装将包含 tnp26 转座酶基因下游的 catA1 氯霉素抗性基因的合成 IS26 克隆到 pUC19 中。在含有非共轭 pUC19 衍生的 IS26::catA1 构建体和共轭质粒 R388 的 recA-大肠杆菌中检测 IS 活性，使用标准交配试验。筛选转导体对抗性。
PCR 未检测到会形成复制出途径的环形 IS26 分子。合成的 IS26::catA1 构建体形成 CmRTpR 转导体（其中 CmR 和 TpR 分别代表氯霉素抗性和甲氧苄啶抗性），代表携带 catA1 基因的 R388 衍生物，其频率为 5.6×10-7 CmRTpR 转导体每 TpR 转导体，这与未改变的 IS26 的复制活性相当。为了测试 IS26::catA1 的简单转位（没有质粒骨架），筛选了 1200 个 CmRTpR 菌落，所有菌落均对氨苄青霉素有抗性，表明存在 pUC19 骨架。因此，IS26::catA1 仅形成共整合体。
IS26 不能单独移动，共整合体是 IS26 转座酶 Tnp26 介导的反应的唯一终产物。因此，在描述复杂抗性区域的形成时，不应援引单个 IS26 的简单“转位”。
