In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis
In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis
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DOI：
10.1128/jb.186.18.6124-6132.2004
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发表时间：
2004-09-01
影响因子：
3.2
通讯作者：
Price, CW
中科院分区：
文献类型：
--
作者：
Kim, TJ;Gaidenko, TA;Price, CW
细菌暴露在不同的生长限制压力下，会诱导一组共同的蛋白质的合成，这些蛋白质对未来可能致命的压力提供了广泛的保护。在枯草芽孢杆菌及其近亲中，这种一般的应激反应是由Sigma(B)转录因子控制的。环境和能量胁迫的信号通过多组分网络激活sigma(B)，该网络通过伙伴转换机制发挥作用，在该机制中，蛋白质之间的相互作用由丝氨酸和苏氨酸磷酸化控制。在这里，我们测试了当前模型对该网络的环境信号分支的中心预测。我们使用等电聚焦和免疫印迹实验来确定RsbRA和RsbS调节因子在体内的磷酸化状态，这两种调节因子协同作用负调控RsbU环境信号磷酸酶。正如模型预测的那样，RsbRA和RsbS的磷酸化形式与非磷酸化形式的比率都随着盐或乙醇胁迫的反应而增加。然而，这两个调控因子在稳态和应激条件下的磷酸化程度上有很大的不同，RsbRA总是更高度修饰。突变分析表明，环境信号所需的RSBT激酶也是RsbRA和RsbS在体内磷酸化所必需的。此外，RsbRA的T171a改变阻断了环境信号，也阻止了RsbRA在体内的磷酸化，阻碍了RsbS的磷酸化。这些体内结果证实了先前的遗传分析，并将RsbRA和RsbS的磷酸化形式与环境应激信号的主动传递联系起来。
Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a common set of proteins which provide broad protection against future, potentially lethal stresses. Among Bacillus subtilis and its relatives, this general stress response is controlled by the sigma(B) transcription factor. Signals of environmental and energy stress activate sigma(B) through a multicomponent network that functions via a partner switching mechanism, in which protein-protein interactions are governed by serine and threonine phosphorylation. Here, we tested a central prediction of the current model for the environmental signaling branch of this network. We used isoelectric focusing and immunoblotting experiments to determine the in vivo phosphorylation states of the RsbRA and RsbS regulators, which act in concert to negatively control the RsbU environmental signaling phosphatase. As predicted by the model, the ratio of the phosphorylated to unphosphorylated forms of both RsbRA and RsbS increased in response to salt or ethanol stress. However, these two regulators differed substantially with regard to the extent of their phosphorylation under both steady-state and stress conditions, with RsbRA always the more highly modified. Mutant analysis showed that the RsbT kinase, which is required for environmental signaling, was also required for the in vivo phosphorylation of RsbRA and RsbS. Moreover, the T171A alteration of RsbRA, which blocks environmental signaling, also blocked in vivo phosphorylation of RsbRA and impeded phosphorylation of RsbS. These in vivo results corroborate previous genetic analyses and link the phosphorylated forms of RsbRA and RsbS to the active transmission of environmental stress signals.