Identification of the histidine protein kinase KinB in Pseudomonas aeruginosa and its phosphorylation of the alginate regulator AlgB
Identification of the histidine protein kinase KinB in Pseudomonas aeruginosa and its phosphorylation of the alginate regulator AlgB
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DOI：
10.1074/jbc.272.29.17952
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Ohman, DE
中科院分区：
文献类型：
--
作者：
Ma, S;Wozniak, DJ;Ohman, DE
藻酸盐胞外多糖是铜绿假单胞菌引起的慢性肺部感染中的重要毒力因子。藻酸盐合成的两种正激活剂algB和algR是双组分调节系统的反应调节剂超家族的成员。AlgB属于响应调节剂的NtrC亚家族，并且是高水平生产藻酸盐所必需的。在这项研究中，在algB的下游发现了一个编码66 kh α多肽的开放阅读框，命名为kinB。KinB的序列与双组分调节系统的组氨酸蛋白激酶成员同源。对携带kinB-lacZ蛋白融合体的铜绿假单胞菌菌株的Western印迹分析和对KinB-phoA融合体的研究表明，KinB定位于内膜并且具有NH 2-末端周质结构域。产生并纯化了含有KinB的COOH末端的KinB衍生物。在[γ-P-32]ATP的存在下，观察到纯化的COOH末端KinB蛋白在体外进行进行性自磷酸化。此外，KinB的磷酸化标记可迅速转移到纯化的AlgB上，组氨酸蛋白激酶保守残基的取代可消除KinB的自磷酸化，这些结果为KinB编码AlgB同源组氨酸蛋白激酶提供了证据。
The exopolysaccharide alginate is an important virulence factor in chronic lung infections caused by the bacterium Pseudomonas aeruginosa. Two positive activators for alginate synthesis, algB and algR, are members of a superfamily of response regulators of the two-component regulatory system. AlgB belongs to the NtrC subfamily of response regulators and is required for high-level production of alginate. In this study, an open reading frame encoding a polypeptide of 66 kh)a, designated kinB, was identified immediately downstream of algB. The sequence of KinB is homologous to the histidine protein kinase members of two-component regulatory systems. Western blot analysis of a P. aeruginosa strain carrying a kinB-lacZ protein fusion and studies of KinB-phoA fusions indicate that KinB localizes to the inner membrane and has a NH2-terminal periplasmic domain, A KinB derivative containing the COOH terminus of KinB was generated and purified. In the presence of [gamma-P-32]ATP, the purified COOH-terminal KinB protein was observed to undergo progressive autophosphorylation in vitro. Moreover, the phosphoryl label of KinB could be rapidly transferred to purified AlgB, Substitutions of the residues conserved among histidine protein kinases abolished KinB autophosphorylation, These results provide evidence that KinB encodes the AlgB cognate histidine protein kinase.