Abstract：
The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.
摘要：
脑膜炎奈瑟氏球菌的有效天然转化允许快速构建细菌突变体,其中目的基因被抗生素抗性盒中断或替换。然而,这被证明是一把双刃剑，即，尽管有助于这种重要的人类病原体的遗传表征，它限制了构建无抗生素抗性标记的无标记突变体的策略的开发。此外,在脑膜炎奈瑟球菌中也缺乏有效的补充或标记工具。在这项研究中,我们通过开发新的有效工具来构建无标记突变体（使用双重反选择策略），显着扩展了脑膜炎球菌遗传工具箱，遗传互补（使用整合载体），和细胞标记(使用自标记蛋白标签)。这个扩展的工具箱为更深入的脑膜炎奈瑟氏球菌遗传表征铺平了道路，也可能对其他奈瑟氏球菌物种有用。重要的脑膜炎奈瑟菌和淋病奈瑟菌是两种重要的人类病原体。专注于这些细菌的研究需要基因工程，这是由他们经历转变的自然能力所促进的。然而,突变工程的易用性导致奈瑟氏菌社区忽视了更复杂的基因编辑工具的开发，特别是对于脑膜炎奈瑟菌。在这项研究中,通过开发用于无标记突变体构建的新颖有效的工具，我们显着扩展了脑膜炎球菌遗传工具箱，遗传互补，和细胞标记。这个扩展的工具箱为更深入的脑膜炎奈瑟氏球菌遗传表征铺平了道路，也可能对其他奈瑟氏球菌物种有用。
