西瓜噬酸菌Aac-5AAA ATPases基因moxR和ruvB功能研究

AAA ATPases(ATPase associated with diverse cellular activities)是一种与细胞活性相关的酶,广泛存在于所有生物中,同时具有蛋白酶和分子伴侣活性。为了研究AAA ATPases相关基因缺失对西瓜噬酸菌(Acidovorax citrulli)的影响,本研究通过同源重组双交换的方法构建西瓜噬酸菌moxR基因以及ruv B基因全缺失突变菌株及互补菌株,测定了突变菌株的致病性、烟草过敏反应、运动性、生长能力、生物膜形成等;并利用实时荧光定量PCR方法测定了AAA ATPases与Ⅲ型分泌系统相关基因(hrc N和hrp E)、毒性蛋白相关基因(trb C和vir B)、鞭毛相关基因(fliR和fli M)、菌毛相关基因(pin C-1和pin C-2)以及AAA ATPases亚家族Ruv B相关基因(ruv A和ruv C)的关系。结果表明,moxR基因以及ruv B基因全缺失突变菌株的致病力降低、运动性减弱、生长能力下降、生物膜形成能力增强、烟草过敏性反应减弱、突变菌株中重要的致病基因hrc N和hrp E表达量显著下调,表明AAA ATPases相关基因moxR、ruv B在西瓜噬酸菌的致病力上发挥重要作用。

Functional analysis of AAA ATPases genes (moxR and ruvB) in Acidovorax citrulli

AAA ATPases found in all kingdoms of living organisms participate in diverse cellular processes including protein degradation and chaperon activity. In order to study the roles of AAA ATPases related genes in Acidovorax citrulli, the causal agent of bacterial fruit blotch, we generated the gene deletion mutant strains ΔmoxR, ΔruvB and their gene complemented strains by homologous recombination approach. The influence of AAA ATPases on A. citrulli was illustrated by testing the virulence, hypersensitive response (HR), twitching motility, biofilm formation and growth rate of these strains. The different expression levels of genes related to type III secretion system (hrcN and hrpE), toxic protein (trbC and virB), flagellum (fliR and fliM), pilus (pinC-1 and pinC-2) and ruvB related genes (ruvA and ruvC) were also analyzed in these strains by qPCR. Our results indicated that the genes (moxR and ruvB) deletion mutants caused significant reduction in virulence, twitching, growth ability, HR but increase in biofilm formation. The expression of hrcN and hrpE decreased in the mutant strains compared to that in the wild type. These findings demonstrated that moxR and ruvB play important roles in pathogenicity of A. citrulli.