Hu Lizhi, Kong Wei, Yang Dezhi, Han Qiangqiang, Guo Lin, Shi Yixin
The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
School of Life Sciences, Arizona State University, Tempe, AZ, United States.
Front Microbiol. 2019 Jul 3;10:1515. doi: 10.3389/fmicb.2019.01515. eCollection 2019.
Histone-like nucleoid structuring protein (H-NS) in enterobacteria plays an important role in facilitating chromosome organization and functions as a crucial transcriptional regulator for global gene regulation. Here, we presented an observation that H-NS of serovar Typhimurium could undergo protein phosphorylation at threonine 13 residue (T13). Analysis of the H-NS wild-type protein and its T13E phosphomimetic substitute suggested that T13 phosphorylation lead to alterations of H-NS structure, thus reducing its dimerization to weaken its DNA binding affinity. Proteomic analysis revealed that H-NS phosphorylation exerts regulatory effects on a wide range of genetic loci including the PhoP/PhoQ-regulated genes. In this study, we investigated an effect of T13 phosphorylation of H-NS that rendered transcription upregulation of the PhoP/PhoQ-activated genes. A lower promoter binding of the T13 phosphorylated H-NS protein was correlated with a stronger interaction of the PhoP protein, i.e., a transcription activator and also a competitor of H-NS, to the PhoP/PhoQ-dependent promoters. Unlike depletion of H-NS which dramatically activated the PhoP/PhoQ-dependent transcription even in a PhoP/PhoQ-repressing condition, mimicking of H-NS phosphorylation caused a moderate upregulation. Wild-type H-NS protein produced heterogeneously could rescue the phenotype of mutant and fully restored the PhoP/PhoQ-dependent transcription enhanced by T13 phosphorylation of H-NS to wild-type levels. Therefore, our findings uncover a strategy in typhimurium to fine-tune the regulatory activity of H-NS through specific protein phosphorylation and highlight a regulatory mechanism for the PhoP/PhoQ-dependent transcription via this post-translational modification.
肠道细菌中的类组蛋白核仁结构蛋白（H-NS）在促进染色体组织方面发挥着重要作用，并作为全局基因调控的关键转录调节因子。在此，我们展示了一项观察结果，即鼠伤寒血清型的H-NS可在苏氨酸13残基（T13）处发生蛋白质磷酸化。对H-NS野生型蛋白及其T13E磷酸模拟替代物的分析表明，T13磷酸化导致H-NS结构改变，从而减少其二聚化以削弱其DNA结合亲和力。蛋白质组学分析显示，H-NS磷酸化对包括PhoP/PhoQ调控基因在内的广泛遗传位点发挥调节作用。在本研究中，我们研究了H-NS的T13磷酸化对PhoP/PhoQ激活基因转录上调的影响。T13磷酸化的H-NS蛋白与启动子的较低结合与PhoP蛋白（一种转录激活因子，也是H-NS的竞争者）与PhoP/PhoQ依赖性启动子的更强相互作用相关。与H-NS缺失不同，即使在PhoP/PhoQ抑制条件下，H-NS缺失也会显著激活PhoP/PhoQ依赖性转录，模拟H-NS磷酸化会导致适度上调。异源产生的野生型H-NS蛋白可以挽救突变体的表型，并将由H-NS的T13磷酸化增强的PhoP/PhoQ依赖性转录完全恢复到野生型水平。因此，我们的研究结果揭示了鼠伤寒中的一种策略，即通过特定的蛋白质磷酸化来微调H-NS的调节活性，并突出了通过这种翻译后修饰对PhoP/PhoQ依赖性转录的调节机制。
