CAJ | 학술논문

细菌Ⅵ型分泌系统（type Ⅵ secretion system,T6SS）是近年发现的一种分布广泛且与细菌致病性密切相关的新型分泌系统,ClpⅤ型ATPase是不同细菌T6SS中均含有的关键保守组分,据推测可能为底物蛋白的分泌提供动力,但对其生化功能尚未进行深入鉴定。在本研究中,我们克隆了根癌农杆菌（Agrobacterium tumefaciens）C58中的ClpⅤ型ATPase基因atu4344并在大肠杆菌中表达,酶学检测证实了其ATPase活性,定点突变分析发现K232/608和E299/675突变体均丧失ATPase活性,表明Walker A模体和Walker B模体与其活性密切相关。这是首次对植物病原细菌T6SS中的ClpⅤ型ATPase进行生化鉴定和突变分析研究。
세균Ⅵ형분비계통（type Ⅵ secretion system,T6SS）시근년발현적일충분포엄범차여세균치병성밀절상관적신형분비계통,ClpⅤ형ATPase시불동세균T6SS중균함유적관건보수조분,거추측가능위저물단백적분비제공동력,단대기생화공능상미진행심입감정。재본연구중,아문극륭료근암농간균（Agrobacterium tumefaciens）C58중적ClpⅤ형ATPase기인atu4344병재대장간균중표체,매학검측증실료기ATPase활성,정점돌변분석발현K232/608화E299/675돌변체균상실ATPase활성,표명Walker A모체화Walker B모체여기활성밀절상관。저시수차대식물병원세균T6SS중적ClpⅤ형ATPase진행생화감정화돌변분석연구。
Type Ⅵ secretion system （T6SS） is a new discovered secretion system which widely distributed in many Gram-negative bacterial pathogens.ClpⅤ,one of the key components of T6SS containing a conserved AAA＋ domain （ATPase associated with diverse cellular activities）,has been presumed to be the ATPase which drives effector protein secretion through hydrolyzing ATP.However,the functional and biochemical roles of ClpⅤ remain largely uncharacterized.In this study,the clpⅤ gene （atu4344） of Agrobacterium tumefaciens C58 was cloned and heterologously expressed in E.coli.By using the coupled-enzyme assay and site-directed mutagenesis,it was confirmed that the wild-type ClpⅤ exhibited ATPase activity,but neither the K232/608 nor E299/675 mutants were active,which demonstrated that both the Walker A and Walker B motif are essential for ClpⅤ activity in vitro.This was the first biochemical identification and mutation analysis on ClpⅤ-type ATPase in T6SS of plant pathogenic bacteria.