地毯草黄单胞菌双组分系统VgrS-VgrR基因敲除及表型筛选

Gene deletion and phenotype observation of two-component system VgrS-VgrR in Xanthomonas axonopodis pv.manihotis

【目的】研究地毯草黄单胞菌双组分系统VgrS-VgrR与致病性的关系,为木薯细菌性病害的高效防控提供分子生物学证据。【方法】采用同源重组方法构建vgrS和vgrR的插入失活突变体,用可移动的cosmid载体p HM1构建互补菌株。检测突变体的致病性、细菌游动性、胞外酶、胞外多糖的变化,观察细菌对H_2O_2和金属离子胁迫的反应。【结果】相比野生型菌株,vgrS和vgrR突变体接种寄主植物木薯后致病力显著降低,突变体的游动性减少、蛋白酶活性减弱、H_2O_2耐受性降低,在高浓度金属离子Fe^(2+)、Fe^(3+)、Cu^(2+)、Ni^(2+)、Zn^(2+)、Co^(2+)的胁迫条件下菌体生长显著减弱。然而,vgrS和vgrR突变体的胞外多糖含量显著升高,分别是野生型的2.14和1.89倍。【结论】阐明了VgrS-VgrR系统在细菌致病过程中发挥的重要作用,为鉴定VgrS-VgrR调控机制提供线索,为药物筛选提供靶向目标。

[Objective]In this study,we investigated the relationship between the VgrS-VgrR two-component system(TCS)of Xanthomonas axonopodis pv.manihotis(Xam)and the bacterial pathogenicity.The study can provide molecular biological evidence for the effective prevention and control of cassava bacterial blight.[Methods]We constructed the insertion-inactivation mutants of vgrS and vgrR by homologous recombination,and constructed the complementary strains of each mutant by using the mobile cosmid vector pHM1.The study measured the pathogenicity of each mutant,its swimming and the changing in extracellular enzyme and extracellular polysaccharide production.We also observed the bacterial response to hydrogen peroxide and metal ion stress.[Results]Compared with wild-type strain,both vgrS and vgrR mutants exhibited significantly reduced pathogenicity after inoculation of the host plant cassava.Both mutants exhibited decreased swimming,reduced protease activity and hydrogen peroxide tolerance.Under the stress conditions of high concentrations of metal ions Fe^(2+),Fe^(3+),Cu^(2+),Ni^(2+),Zn^(2+)and Cu^(2+),the mutants’growth were also significantly decreased.However,the extracellular polysaccharide content of vgrS and vgrR mutants increased dramatically,which was 2.14 and 1.89 times that of the wild type,respectively.[Conclusion]The results clarified the important role of the VgrS-VgrR TCS in the pathogenesis of Xam,and provided clues for further research on the regulation mechanism of VgrS-VgrR and drug screening targeting Xam.