The critical roles of (p)ppGpp in homeostasis and antibiotic tolerance in Gram positive bacteria

项目摘要 细菌经常遇到压力，包括营养饥饿，温度变化， 抗生素攻击，这很容易使他们的细胞内环境陷入混乱。生存和 为了适应，细菌发展了多种应激反应来调节细胞内过程 相应地虽然控制应激反应的转录网络已经被广泛研究， 表征，我们的知识除了转录调控之外还有重大空白。主题 我的研究是阐明压力信号机制，这种机制是通过快速变化来传递的， 浓度的“alarmones”-信号核苷酸，有助于提醒细胞 及时强调。我的实验室在表征保守的 alarmone（p）ppGpp.（p）ppGpp由应力诱导，并介导深刻的、多效性的 几乎所有细菌的生理变化，使健身，生存和进化。我们确定 多个嘌呤合成酶、复制酶和转录阻遏物， 在革兰氏阳性芽孢杆菌属物种中由（p）ppGpp直接调节。这些规定进一步 发现在许多病原体中是保守的，并且对于体内平衡，抗饥饿性， 抗生素持久性和基因组完整性。目前，我们还在研究（p）ppGpp如何 调节不同的细菌生活方式之间的转换：快速生长和生物膜形成。 此外，我们还检测到其他核苷酸alarmones，包括AppppA，pGpp，ppApp和c-di- AMP是由温度和细胞壁应力诱导形成的， 强大的保护网络。我们未来的研究将回答以下基本问题： 不同的压力是否会引发不同的警报素，细菌又是如何合成它们的？ 什么是不同的警报素的直接相互作用的目标，以及它们如何促进细菌 健身和影响细菌的发展，如生物膜的形成和孢子形成？怎么 细菌整合来自不同警报素的多种线索，以快速和适当地适应 不同的环境？我们将联合收割机代谢组学、转录组学和蛋白质组学与 生物化学和细胞生物学方法来回答这些问题。我们得到了一份 从病原体芽孢杆菌的蛋白质组进行的系统筛选中获得的报警素靶点 炭疽病我们将在相关的非致病性细菌枯草芽孢杆菌中研究这些过程 我们有丰富的经验。B。枯草芽孢杆菌生长迅速， 操纵我们在芽孢杆菌中表征的核苷酸信号传导机制适用于 其他不易处理的致病菌，并可用于开发抗菌策略 通过针对他们的压力反应。

Project Summary Bacteria frequently encounter stresses including nutrient starvation, temperature changes, and antibiotic assault, which could easily throw their intracellular environment into chaos. To survive and to adapt, bacteria developed diverse stress responses to regulate intracellular processes accordingly. While the transcriptional networks governing stress responses have been extensively characterized, there are major gaps in our knowledge beyond transcription regulation. The theme of my research is to elucidate stress signaling mechanisms that are transmitted by rapid changes in concentration of ‘alarmones’ – signaling nucleotides which are instrumental for alerting cells about stresses in a timely manner. My laboratory has extensive experience in characterizing the conserved alarmone (p)ppGpp. (p)ppGpp is induced by stresses and mediates profound, pleiotropic physiological changes in almost all bacteria to allow fitness, survival, and evolution. We identified multiple purine synthesis enzymes, a replication enzyme and a transcription repressor that are directly regulated by (p)ppGpp in Gram-positive Bacillus species. These regulations were further found to be conserved in many pathogens and are critical for homeostasis, starvation resistance, antibiotic persistence, and genome integrity. Currently, we are also investigating how (p)ppGpp regulates the switch between distinct bacterial lifestyles: planktonic growth and biofilm formation. Additionally, we detected other nucleotide alarmones including AppppA, pGpp, ppApp, and c-di- AMP, which are induced by different stresses including temperature and cell wall stress, to form a robust protective network. Our future research will answer the following fundamental questions: How are the different alarmones triggered by different stresses, and how do bacteria synthesize them? What are the direct interaction targets of different alarmones, and how do they promote bacterial fitness and influence bacterial development such as biofilm formation and sporulation? How do bacteria integrate multiple cues from different alarmones for rapid and appropriate adaptation to diverse environments? We combine metabolomics, transcriptomics, and proteomics with biochemical and cell biological approaches to answer these questions. We obtained a list of alarmone targets from systematic screens performed with the proteome of the pathogen Bacillus anthracis. We will study these processes in the related non-pathogenic bacterium Bacillus subtilis for which we have extensive experience. B. subtilis grows fast and is highly amenable to genetic manipulation. The nucleotide signaling mechanisms we characterize in Bacillus are applicable to other, less tractable, pathogenic bacteria, and can be used for developing antimicrobial strategies by targeting their stress responses.