Construction and Use of Transposon MycoTetOP2 for Isolation of Conditional Mycobacteria Mutants
Construction and Use of Transposon MycoTetOP2 for Isolation of Conditional Mycobacteria Mutants
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DOI：
10.3389/fmicb.2019.03091
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发表时间：
2020-01
影响因子：
5.2
通讯作者：
Sarah D. Riggs-Shute;J. Falkinham;Zhaomin Yang
中科院分区：
文献类型：
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作者：
Sarah D. Riggs-Shute;J. Falkinham;Zhaomin Yang
分枝杆菌在其生物学的许多方面是独特的。通过正向遗传分析来识别对其生长至关重要的基因的遗传工具的发展，为促进我们对其细胞、生理和生化过程的理解带来了巨大的希望。在这里，我们报告了一种新的转座子，MycoTetOP 2，以帮助识别这些基因的直接转座子诱变的发展。这种基于mariner的转座子含有嵌套的无水四环素（ATc）诱导型启动子，以驱动转录从其两端向外。此外，它还包括大肠杆菌R6Kγ来源，以便于识别插入位点。将MycoTetOP 2置于分枝杆菌中具有温度敏感性DNA复制起点的穿梭质粒中。这允许携带质粒的分枝杆菌在允许的温度下繁殖。然后可以使所得的细胞群体经受温度变化以选择转座子突变体。这种转座子及其传递系统，一旦构建，在快速生长的模型耻垢分枝杆菌和13个突变株与ATC依赖性生长进行了测试。在这些突变体中的插入位点的鉴定导致了9个独特的遗传位点，这些基因对M.耻垢病和结核分枝杆菌。这些结果表明，MycoTetOP 2及其递送载体为通过正向遗传学研究分枝杆菌提供了有价值的工具。
Mycobacteria are unique in many aspects of their biology. The development of genetic tools to identify genes critical for their growth by forward genetic analysis holds great promises to advance our understanding of their cellular, physiological and biochemical processes. Here we report the development of a novel transposon, MycoTetOP2, to aid the identification of such genes by direct transposon mutagenesis. This mariner-based transposon contains nested anhydrotetracycline (ATc)-inducible promoters to drive transcription outward from both of its ends. In addition, it includes the Escherichia coli R6Kγ origin to facilitate the identification of insertion sites. MycoTetOP2 was placed in a shuttle plasmid with a temperature-sensitive DNA replication origin in mycobacteria. This allows propagation of mycobacteria harboring the plasmid at a permissive temperature. The resulting population of cells can then be subjected to a temperature shift to select for transposon mutants. This transposon and its delivery system, once constructed, were tested in the fast-growing model Mycobacterium smegmatis and 13 mutants with ATc-dependent growth were isolated. The identification of the insertion sites in these mutants led to nine unique genetic loci with genes critical for essential processes in both M. smegmatis and Mycobacterium tuberculosis. These results demonstrate that MycoTetOP2 and its delivery vector provide valuable tools for the studies of mycobacteria by forward genetics.