Control of GTP Homeostasis by (p)ppGpp

说明(申请人提供)：核苷酸(P)ppGpp对抗逆性和抗生素耐受性至关重要。在革兰氏阴性菌大肠杆菌中，(P)ppGpp与RNA聚合酶相互作用，在应激条件下重新编程转录。然而，在由病原菌(葡萄球菌、肠球菌、李斯特菌、炭疽芽孢杆菌)和有益微生物组成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。这一建议的中心假设是，在Firmicmins中，(P)ppGpp调节GTP水平，以调节细胞代谢的多效性方面以适应外部环境，从而维持体内平衡并促进抗生素耐受。我们最近对枯草芽孢杆菌的研究，这是一种被广泛研究的、在遗传上适用于非米米库特的模型，为这一假设提供了强有力的支持。将基因组和代谢学方法与遗传学和生物化学相结合，我们确定GTP生物合成途径中的酶是(P)ppGpp作用的主要靶点，并揭示了一个对细胞活力至关重要的新的GTP-(P)ppGpp反馈环。因此，我们的发现为枯草杆菌中(P)ppGpp的合成和功能提供了一个以前未被认识到的观点。我们的目标是在此基础上，通过(P)ppGpp-GTP在枯草杆菌中开发一个全面的压力管理模型，我们的数据预测将适用于Firmicmin和更远的地方。具体目标是：1)确定(P)ppGpp如何调节其靶标GMK和HPRT；2)阐明(P)ppGpp如何控制GTP反馈和糖酵解；以及3)确定(P)ppGpp如何促进抗生素耐药性。

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.