Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus
Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus
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DOI：
10.1128/jb.00403-10
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发表时间：
2010-09-01
影响因子：
3.2
通讯作者：
Yang, Zhaomin
中科院分区：
文献类型：
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作者：
Black, Wesley P.;Schubot, Florian D.;Yang, Zhaomin
由IV型皮利提供动力的黄色粘球菌社会滑行运动需要细胞表面上存在胞外多糖（EPS）。Dif化学感受系统对于EPS产生的调节是必不可少的。先前证明DifA（甲基接受趋化蛋白[MCP]样）、DifC（CheW样）和DifE（CheA样）刺激EPS产生，而DifD（CheY样）和DifG（CheC样）抑制EPS产生。发现DifD在EPS调节中不起DifE下游的作用，因为difD difE双突变体表型模仿difE单突变体。已经提出DifA、DifC和DifE形成三元信号传导复合物，其通过DifE的激酶活性正向调节EPS产生。DifD被认为是磷酸化DifE（DifE类似于P）的磷酸盐库，而DifG将作为磷酸化DifD（DifD类似于P）的磷酸酶增强DifD的功能。在这里，我们报告在体外磷酸化研究与所有的Dif化学感受蛋白，表达和纯化大肠杆菌。DifE被证明是一种自身激酶。与DifA-DifC-DifE复合物的形成一致，发现DifA和DifC一起而不是单独地影响DifE自身磷酸化。发现DifD不直接抑制DifE自磷酸化，其接受来自自磷酸化的DifE的磷酸。虽然DifD类似于P在体外具有异常长的去磷酸化半衰期，但DifG有效地将DifD类似于P作为磷酸酶去磷酸化。这些结果支持这样的模型，其中DifE与DifA和DifC复合以通过下游靶标的磷酸化来调节EPS产生，而DifD和DifG协同作用以类似于P将磷酸盐从DifE转移。
Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE similar to P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD similar to P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD similar to P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD similar to P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE similar to P.