根癌农杆菌Ⅵ型分泌系统关键组分ClpⅤ型ATPase基因表达及活性位点分析  

Biochemical Characterization of ClpⅤ Type ATPase,the Key Component of Type Ⅵ Secretion System in Agrobacterium tumefaciens C58

作　　者：蔺晓丽[1,2] 徐胜娟[3] 刘应保[3] 郭建春[2] 王瑶[3] 

机构地区：[1]海南大学农学院,海口570228 [2]中国热带农业科学院生物技术研究所热带作物生物技术国家重点实验室,海口571101 [3]西北农林科技大学生命科学学院,杨凌712100

出　　处：《基因组学与应用生物学》2011年第2期136-144,共9页Genomics and Applied Biology

基　　金：国家自然科学基金(30570020和30770026);西北农林科技大学人才专项资金(Z111021007)共同资助

摘　　要：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。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.

关 键 词：根癌农杆菌 ClpⅤ型ATPase基因 酶学活性 突变分析 

分 类 号：S432.4[农业科学—植物病理学]