IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes.
IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes.
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DOI：
10.1128/msphere.00038-16
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发表时间：
2016-03
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
在革兰氏阴性菌中，IS26通过形成携带许多不同抗性基因的转座子将抗生素抗性基因募集到移动的基因库中。除了复制转座，IS26最近被证明使用一种新的保守运动机制，其中传入的IS26靶向预先存在的一个。在这里，我们已经证明了如何IS26绑定的I类转座子可以产生从易位单位（TU）只含有一个IS26和一个抗性基因通过保守反应。TU被并入到现有的IS26旁边，产生I类转座子，并且如果靶向的IS26在转座子中，则产物类似于共享中心IS26的两个转座子，这是在一些抗性区域中观察到的构型，并且当转座子串联复制时。虽然同源重组也可以合并TU，但Tnp26更有效。这提供了对IS26如何构建转座子并将额外的转座子带入抗性区域的深入了解。IS26转座酶Tnp26通过复制途径催化IS26移动到新位点并使相邻DNA缺失或倒位。分子内缺失反应产生由DNA片段和单个IS26组成的环状分子，我们称之为可转位单位或TU。最近，Tnp26被证明可以催化不同质粒中IS26的两个预先存在的拷贝之间的另外的分子间保守反应。在这里，我们研究了同源重组和Tnp26催化的反应从TU转座子的产生的相对贡献。在体外产生含有aphA1a卡那霉素和新霉素抗性基因或泰特（D）四环素抗性决定簇的环状TU，并转化到携带R388：：IS26的大肠杆菌recA细胞中。在R388：：IS26中并入IS26旁边的TU形成具有直接定向的插入序列（IS）的转座子。引入第二个TU产生了包含aphA1a基因和泰特（D）决定簇的区域，但只有三个IS26拷贝。整合反应，这需要一个预先存在的IS26，是精确和保守的，是50倍更有效时，两个IS26拷贝可以产生一个积极的Tnp26。当两个IS通过tnp26中的移码失活时，在大肠杆菌recA细胞中未检测到TU掺入，但在大肠杆菌recA+细胞中确实发生了TU掺入。然而，Tnp催化的反应比RecA依赖的同源重组效率高100倍。Tnp26以复制或保守模式发挥功能的能力可能解释了在革兰氏阴性菌中发现的抗性区域中IS26结合的转座子的突出性。在革兰氏阴性菌中，IS26通过形成携带许多不同抗性基因的转座子，将抗生素抗性基因招募到移动的基因库中。除了复制转座，IS26最近被证明使用一种新的保守运动机制，其中传入的IS26靶向预先存在的一个。在这里，我们已经证明了如何IS26绑定的I类转座子可以产生从易位单位（TU）只含有一个IS26和一个抗性基因通过保守反应。TU被并入到现有的IS26旁边，产生I类转座子，并且如果靶向的IS26在转座子中，则产物类似于共享中心IS26的两个转座子，这是在一些抗性区域中观察到的构型，并且当转座子串联复制时。虽然同源重组也可以合并TU，但Tnp26更有效。这提供了对IS26如何构建转座子并将额外的转座子带入抗性区域的深入了解。
In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions. The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA+ cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions.