Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium.
Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium.
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DOI：
10.1371/journal.pone.0172630
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Gerlach RG
中科院分区：
文献类型：
--
作者：
Hoffmann S;Schmidt C;Walter S;Bender JK;Gerlach RG
定点无瘢痕突变是现代发病机制研究的重要工具。我们描述了一种优化的两步方案，用于肠道沙门氏菌鼠伤寒血清型的基因组编辑，以在单个菌株中实现多个连续的诱变步骤。该系统基于λ Red重组酶催化的可选择抗生素抗性标记的整合，然后替换该盒。通过表达大范围核酸酶I-SceI来选择无标记突变体，所述大范围核酸酶I-SceI在仍然携带抗性基因座的细菌中诱导双链断裂。我们的新双功能质粒pWRG 730允许λ Red重组酶的热诱导表达和I-SceI的tet诱导生产。甲基接受趋化蛋白（MCP）是一种跨膜化学感受器，可感受包括氨基酸、糖、离子和氧在内的大量环境信号。基于MCP的感觉输入，趋化性是沙门氏菌毒力的关键组成部分。为了确定单个MCP的贡献，我们顺序删除了七个MCP基因。通过PCR验证单个突变，并通过全基因组测序确认最终七个MCP突变体WRG 279的遗传完整性。在HeLa细胞感染模型中对连续的MCP突变体进行了功能测试，该模型揭示了非趋化性突变体和缺乏MCP Chem（Tar）的菌株的侵袭率增加。WRG 279的表型逆转与质粒为基础的表达Chem。互补的WRG 279突变体也表现出部分恢复趋化性在半固体琼脂上的群集试验。我们优化的无瘢痕删除方案能够有效和精确地操纵沙门氏菌基因组。如全基因组测序所证明的，可以实现多个后续诱变步骤而不引入不需要的突变。7个MCP基因的顺序缺失揭示了在S.鼠伤寒沙门氏菌与宿主细胞，可能会提供新的见解沙门氏菌宿主细胞的机制。
Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give new insights into mechanisms of Salmonella host cell sensing.