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首页> 外文期刊>Helicobacter >Xer-cise in Helicobacter pylori: One-step Transformation for the Construction of Markerless Gene Deletions
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Xer-cise in Helicobacter pylori: One-step Transformation for the Construction of Markerless Gene Deletions

机译：幽门螺杆菌的Xer-cise：一步构建无标记基因缺失的构建。

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Background: Xer-cise is an efficient selectable marker removal technique that was first applied in Bacillus subtilis and Escherichia coli for the construction of markerless gene deletions. Xer-cise marker excision takes advantage of the presence of site-specific Xer recombination in most bacterial species for the resolution of chromosome dimers at the dif site during replication. The identification and functional characterization of the difH/XerH recombination system enabled the development of Xer-cise in Helicobacter pylori. Methods: Markerless deletions were obtained by a single natural transformation step of the Xer-cise cassette containing rpsL and cat genes, for streptomycin susceptibility and chloramphenicol resistance respectively, flanked by difH sites and neighboring homologous sequences of the target gene. Insertion/deletion recombinant H. pylori were first selected on chloramphenicol- containing medium followed by selection on streptomycin-containing medium for clones that underwent XerH mediated excision of the rpsL-cat cassette, resulting in a markerless deletion. Results: XerH-mediated removal of the antibiotic marker was successfully applied in three different H. pylori strains to obtain markerless gene deletions at very high efficiencies. An unmarked triple deletion mutant was also constructed by sequential deletion of ureA, vacA and HP0366 and removal of the selectable marker at each step. The triple mutant had no growth defect suggesting that multiple difH sites per chromosome can be tolerated without affecting bacterial fitness. Conclusion: Xer-cise eliminates the need for multiple passages on non selective plates and subsequent screening of clones for loss of the antibiotic cassette by replica plating.
机译：背景：Xer-cise是一种有效的选择性标记去除技术，最早应用于枯草芽孢杆菌和大肠杆菌中，用于构建无标记基因缺失。 Xer-cise标记切除可利用大多数细菌物种中特定位点Xer重组的存在来解决复制过程中dif位点的染色体二聚体。 difH / XerH重组系统的鉴定和功能表征使得幽门螺杆菌中Xer-cise的开发成为可能。方法：通过含有rpsL和cat基因的Xer-cise盒的单个自然转化步骤获得无标记缺失，其链霉素敏感性和氯霉素抗性分别位于目标基因的difH位点和邻近同源序列旁。首先在含氯霉素的培养基上选择插入/缺失重组幽门螺杆菌，然后在含链霉素的培养基上选择克隆，这些克隆经过XerH介导的rpsL-cat盒切除，无标记缺失。结果：XerH介导的抗生素标记去除成功应用于三种不同的幽门螺杆菌菌株，以非常高的效率获得无标记的基因缺失。还通过依次删除ureA，vacA和HP0366并在每个步骤去除选择标记来构建未标记的三重缺失突变体。该三重突变体没有生长缺陷，表明每个染色体可以容忍多个difH位点，而不会影响细菌的适应性。结论：Xer-cise消除了在非选择性平板上进行多次传代以及随后通过复制平板进行克隆筛选而筛选出的抗生素盒丢失的需求。

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