Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli
Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli
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DOI：
10.1128/jb.182.12.3544-3552.2000
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发表时间：
2000-06-01
影响因子：
3.2
通讯作者：
Bourret, RB
中科院分区：
文献类型：
--
作者：
Boesch, KC;Silversmith, RE;Bourret, RB
大肠杆菌CheZ蛋白刺激CheY的去磷酸化，CheY是趋化性信号转导途径中的反应调节剂，其机制未知。CheZ的遗传分析落后于生物化学和生物物理表征。为了确定推定的区域的功能的重要性，在CheZ，我们进行随机诱变和分离107 nonchemotactic CheZ突变体。错义突变聚集在cheZ的六个区域，而无义突变和移码突变在整个基因中均匀分布。基因内互补实验表明，当含有截短的CheZ（1-189）肽和CheZA 65 V、CheZL 90 S或CheZD 143 G的基因的相容质粒都存在时，群集活性恢复，这意味着在CheZ二聚体的每条链中存在至少两个独立的功能结构域。六个突变的CheZ蛋白质，每个功能丧失的错义突变簇一个，被纯化和生化鉴定。111个测试的突变体蛋白质在其去磷酸化CheY-P的能力上有缺陷，其活性范围为野生型CheZ的0.45 - 16%。CheZ的磷酸酶活性与在CheY磷酸供体乙酰磷酸存在下与CheY形成大的化学交联复合物的能力之间存在良好的相关性。考虑到遗传和生物化学数据，这组CheZ突变体中最严重的功能障碍似乎集中在位于CheZ蛋白的拟议大N-末端结构域中的区域。
The Escherichia coli CheZ protein stimulates dephosphorylation of CheY; a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically.;lll of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.