Functional exploration of a deep Mycobacterium tuberculosis phosphoproteome

摘要 磷信号为细菌适应提供了主要的通道。两组分系统(TCS) 长期以来一直被视为细菌中典型的磷信号系统，但越来越多的O- Ser/Thr激酶介导的磷酸化被认为是一种相关的细菌磷信号 机制也是如此。我们现在证明，事实上，结核分枝杆菌(Mtb)有一种膨胀的， 分布的、协作的O-磷酸化系统，其规模和复杂性通常仅与 与真核生物的关系。通过使用全面的STPK功能丧失和功能获得突变小组和定量 质谱仪，我们发现70%的Mtb蛋白在Ser/Thr/Tyr上被磷酸化，鉴定出数千个 丝氨酸/苏氨酸蛋白激酶底物的表达，并表明丝氨酸/苏氨酸蛋白激酶共同控制着 ~30%的结核分枝杆菌基因。在这里，我们将测试Ser/Thr激酶之间新的和广泛的调节联系 并检测O-磷酸化在转录调控中的作用 各种因素。作为我们对Mtb磷酸化的详尽分析的结果，我们还获得了第一个证据 Mtb中的Arg磷酸化--革兰氏阳性细菌外的第一个细菌Arg磷酸化蛋白质组。我们会 确定相关的磷酸酶并测试pArg作为ClpP-的降解标签的想法 介导的蛋白质分解。

ABSTRACT Phosphosignaling provides the major conduit for bacterial adaptation. The two component systems (TCSs) have long been viewed as the canonical phosphosignaling systems in bacteria, but increasingly, O- phosphorylation mediated by Ser/Thr kinases is recognized as a relevant bacterial phosphosignaling mechanism as well. We now show that in fact, Mycobacterium tuberculosis (Mtb) has an expansive, distributed, and cooperative O-phosphorylation system of a size and complexity that is typically only associated with eukaryotes. By using a comprehensive STPK loss- and gain-of-function mutant panel and quantitative mass spectrometry, we show that >70% of Mtb proteins are phosphorylated on Ser/Thr/Tyr, identify thousands of individual Ser/Thr kinase substrates, and show that the Ser/Thr kinases collectively control the expression of ~30% of Mtb genes. Here, we will test a new and extensive regulatory connection between the Ser/Thr kinases and the His kinases of the TCSs and test the role of O-phosphorylation on the regulation of transcription factors. As a result of our exhaustive analysis of Mtb phosphorylation, we also obtained the first evidence of Arg phosphorylation in Mtb- the first bacterial Arg phosphoproteome outside of gram-positive bacteria. We will identify the relevant phosphoenzymes and test the idea that pArg functions as a degradation tag for ClpP- mediated proteolysis.