Control of GTP Homeostasis by (p)ppGpp

描述（由申请方提供）：核苷酸（p）ppGpp对于胁迫抗性和抗生素耐受性至关重要。在革兰氏阴性细菌大肠杆菌中，（p）ppGpp与RNA聚合酶相互作用，在应激时重编程转录。然而，在厚壁菌门（Firmicutes）（由病原体（葡萄球菌属、肠球菌属、李斯特菌属、炭疽芽孢杆菌）和有益微生物组成的主要类别的革兰氏阳性细菌）中，（p）ppGpp如何介导应激抗性仍然知之甚少。该提议的中心假设是，在厚壁菌门中，（p）ppGpp调节GTP水平以调节细胞代谢对外部环境的多效性，从而维持体内平衡并促进抗生素耐受性。我们最近的工作与细菌枯草芽孢杆菌，一个广泛研究的，遗传上适合的模型厚壁菌门，提供了强有力的支持这一假设。将基因组学和代谢组学方法与遗传学和生物化学相结合，我们确定了GTP生物合成途径中的酶为（p）ppGpp作用的主要靶点，并揭示了一种对细胞活力至关重要的新型GTP-（p）ppGpp反馈回路。因此，我们的发现提供了一个以前未认识到的观点（p）ppGpp的合成和功能在B。枯草杆菌。我们的目标是建立在这个基础上，通过（p）ppGpp-GTP在B的压力管理开发一个全面的模型。我们的数据预测将适用于厚壁菌门和超越。具体目标是：1）表征（p）ppGpp如何调节其靶标GTP和HprT; 2）阐明（p）ppGpp如何控制GTP反馈和糖酵解;和3）定义（p）ppGpp如何有助于抗生素耐受性。

DESCRIPTION (provided by applicant): The nucleotide (p)ppGpp is critical for stress resistance and antibiotic tolerance. In the Gram-negative bacterium Escherichia coli, (p)ppGpp interacts with RNA polymerase to reprogram transcription upon stress. However, in Firmicutes, a major class of Gram- positive bacteria comprised of pathogens (Staphylococcus, Enterococcus, Listeria, Bacillus anthracis) and beneficial microbes, how (p)ppGpp mediates stress resistance remains poorly understood. The central hypothesis of this proposal is that, in Firmicutes, (p)ppGpp regulates GTP levels to adjust pleiotropic aspects of cellular metabolism to the external environment, thus maintaining homeostasis and facilitating antibiotic tolerance. Our recent work with the bacterium Bacillus subtilis, a widely studied, genetically amenable model for Firmicutes, lends strong support for this hypothesis. Integrating genomic and metabolomic approaches with genetics and biochemistry, we identified enzymes in the GTP biosynthesis pathway as major targets for (p)ppGpp action, and unmasked a novel GTP-(p)ppGpp feedback loop that is critical for cellular viability. Our discoveries thus provide a previously unrecognized view of (p)ppGpp synthesis and function in B. subtilis. We aim to build on this foundation to develop a comprehensive model of stress-management via (p)ppGpp-GTP in B. subtilis that our data predicts will be applicable to Firmicutes and beyond. The specific aims are: 1) Characterize how (p)ppGpp regulates its targets Gmk and HprT; 2) Elucidate how (p)ppGpp controls GTP feedback and glycolysis; and 3) Define how (p)ppGpp contributes to antibiotic tolerance.