School of Life and Environmental Sciences, The University of Sydney, NSW, Australia
School of Life and Environmental Sciences, The University of Sydney, NSW, Australia.
mSphere. 2021 Jan 27;6(1):e01321-20. doi: 10.1128/mSphere.01321-20.
IS 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 IS and some close relatives, IS, IS, and a natural hybrid, IS, which are found predominantly in spp., to interact was examined. IS consists of 175 bp from IS at the left end, with the remainder from IS 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 -negative strain, IS, IS, and IS 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 IS However, using mixed pairs, targeted cointegration was detected only when IS was paired with the IS/ 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 IS paired with IS::, an artificially constructed IS derivative that includes the 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. The IS family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS, ISIS, and IS, 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 IS family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS and naturally occurring close relatives of IS that are found near resistance genes in and are widespread in species.
IS 通过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）和一个靶标物的复制插入机制，另一种是涉及两个位于不同 DNA 分子中的 IS 的靶向保守机制。在这项研究中，我们研究了 IS 及其一些近亲，如 IS、IS 和一个自然杂种 IS 的相互作用能力。IS 由 IS 的左末端的 175bp 组成，其余部分来自 IS。这些 IS 都具有相同的 14bp 末端反向重复序列，并且 Tnp26、Tnp1006 和 Tnp1008 转座酶之间的配对同一性为 83.7%至 93.1%，应该能够识别彼此的末端。在一个-阴性菌株中，IS、IS 和 IS 都通过复制插入途径和靶向保守途径形成共整合体，尽管靶向反应的频率至少比 IS 低 10 倍。然而，使用混合配对时，仅当 IS 与 IS/杂种配对时才检测到靶向共整合，该杂种也编码 Tnp1008，并且形成的靶向共整合体均源自在 DNA 序列相同的末端发生的反应。使用与 IS 配对的 IS::进行反应时，也会在具有扩展 DNA 同一性的末端发生反应，IS::是人工构建的包含基因的 IS 衍生物。因此，需要具有相同的转座酶和反应末端的相同 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，然后迁移并解决形成的 Holliday 连接。IS 家族包括那些通常与多重耐药革兰氏阴性和革兰氏阳性细菌中的抗生素抗性基因相关的 IS。IS 在革兰氏阴性物种中最为普遍，并且可以产生与多重耐药病原体中存在的抗生素抗性基因交错排列的直接定向 IS 簇。这种能力依赖于 IS 家族成员的新型双重机制能力。然而，最近发现的由 IS、ISIS 和 IS 介导的共整合体形成的靶向保守模式的机制与任何以前研究过的 IS 运动机制都不同，其机制尚不清楚。一个重要的问题是，IS 和转座酶的哪些特征对于允许 IS 家族成员进行靶向保守反应至关重要。在这项研究中，通过涉及 IS 和自然发生的近亲的混合伴侣杂交来解决这个问题，这些近亲在和中发现的抗性基因附近，并且在物种中广泛存在。
