Targeted Conservative Cointegrate Formation Mediated by IS26 Family Members Requires Sequence Identity at the Reacting End.
Targeted Conservative Cointegrate Formation Mediated by IS26 Family Members Requires Sequence Identity at the Reacting End.
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DOI：
10.1128/msphere.01321-20
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发表时间：
2021-01-27
期刊：
影响因子：
4.8
通讯作者：
Hall RM
中科院分区：
文献类型：
--
作者：
Harmer CJ;Hall RM
IS 26家族包括通常发现与多重耐药革兰氏阴性和革兰氏阳性细菌中的抗生素耐药基因相关的IS。IS 26在革兰氏阴性物种中最普遍，并且可以产生散布有在多重耐药病原体中看到的直接定向的IS 26的抗生素耐药基因簇。IS 26使用两种不同的途径形成共整合体，一种是涉及一个插入序列（IS）和一个靶的拷贝机制，另一种是涉及不同DNA分子中的两个IS的靶向保守机制。在这项研究中，IS 26和一些近亲，IS 1006，IS 1008，以及一个自然杂交种，IS 1006/IS 1008，主要存在于不动杆菌属，进行了检查。IS 1006/1008由IS 1006左端的175 bp组成，其余部分来自IS 1008。这些IS都具有相同的14 bp末端反向重复序列，并且Tnp 26、Tnp 1006和Tnp 1008转座酶具有83.7%至93.1%的成对同一性，应该能够识别彼此的末端。在recA阴性大肠杆菌菌株中，IS 1006、IS 1008和IS 1006/1008各自通过拷贝入途径和通过靶向保守途径形成共整合体，尽管靶向反应的频率比IS 26低至少10倍。然而，使用混合对，只有当IS 1008与IS 1006/1008杂交体（其也编码Tnp 1008）配对时才检测到靶向共整合，并且所形成的靶向共整合体都产生于在DNA序列相同的末端发生的反应。使用与IS 26：：catA 1（一种人工构建的包含catA 1基因的IS 26衍生物）配对的IS 26，反应也发生在末端，具有延伸的DNA同一性。因此，在反应末端需要相同的转座酶和相同的DNA序列。这些特征表明，靶向保守途径通过单个转座酶催化的链转移进行，然后迁移并解析形成的霍利迪连接。重要信息IS 26家族包括在多重耐药革兰氏阴性菌和革兰氏阳性菌中常见的与抗生素耐药基因相关的IS。IS 26在革兰氏阴性物种中最普遍，并且可以产生散布有在多重耐药病原体中看到的直接定向的IS 26的抗生素耐药基因簇。这种能力依赖于IS 26家族成员的新型双重机制能力。然而，最近发现的由IS 26、IS 257/IS 431和IS 1216介导的共整合体形成的靶向保守模式的潜在机制，与任何先前研究的IS运动机制不同，尚未得到很好的理解。一个重要的问题是IS和转座酶的哪些特征是允许IS 26家族成员进行靶向保守反应的关键。在这项研究中，这个问题得到了解决，使用混合伴侣杂交涉及IS 26和IS 26的天然近亲，发现附近的耐药基因在鲍曼不动杆菌和广泛的不动杆菌属物种。
The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. IS26 forms cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid, IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end, with the remainder from IS1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other’s ends. In a recA-negative Escherichia coli strain, IS1006, IS1008, and IS1006/1008 each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26. However, using mixed pairs, targeted cointegration was detected only when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed. IMPORTANCE The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS26, IS257/IS431, and IS1216, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS26 and naturally occurring close relatives of IS26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species.