The molecular circuitry of toxin-antitoxin systems and their contribution to microbial dormancy.

在实验室中，细菌通常在营养丰富的环境中以最大的生长速度进行研究。事实上，微生物生命的典型特征是间断的、次优的生长条件。为了在营养不良、压力大的条件下长时间生存，许多细菌进入生长停滞状态，直到恢复到更有利的条件。我们的研究计划旨在定义这些转变背后的分子电路，使用高度易处理的模型细菌，嗜肺军团菌。L.嗜肺菌通常在淡水变形虫内复制，但可以在非复制的细胞外状态中一次花费数月。细菌调节其生长的一种机制是通过毒素-抗毒素系统。尽管它们的名字，这些“毒素”并不传递给其他细胞，而是用来限制编码它们的细胞的复制。毒素-抗毒素系统通常作为操纵子内的双基因模块存在，所述操纵子编码能够沿着相应抗毒素抑制生长的蛋白质毒素。它们在细菌中普遍存在，并与噬菌体防御、生物膜形成和休眠有关，但它们在细胞生理学中的大部分功能仍然未知。大多数物种中的大量系统使它们的实验询问复杂化：例如，大多数埃希氏菌和沙门氏菌物种中有30多个系统，分枝杆菌中有近90个系统。与此相反，L.嗜肺菌仅具有7种预测的毒素-抗毒素系统，但具有明确定义的在非复制状态下长时间存活的需要。简而言之，我们的研究计划旨在建立L。pneumophila是系统研究毒素-抗毒素系统的理想模式生物。我们将确定它们的分子靶点和作用机制。我们将系统地研究这些系统对L. pneumophila环境持久性、细胞内复制和基因组稳定性。在这个项目中，我们的目标是：1。将每个毒素-抗毒素系统置于微生物细胞的遗传网络中。 2.广泛表征没有所有毒素-抗毒素系统的细菌。 3.通过系统的互补研究确定单个毒素-抗毒素系统对特定表型的贡献。 毒素-抗毒素系统领域充满了未解之谜、争议和希望。具体来说，虽然有越来越多的文献的分子机制的基础上毒素抗毒素系统，这种系统背后的生物学仍然不清楚。利用一种独特的易处理和非常适合的模式微生物，我们的计划旨在正面解决这一知识缺口。最近对数十种军团菌进行了测序-每种都有相应的不同毒素-抗毒素系统供探索-我们提出的计划为可想象的未来提供了一条明确的前进道路。

In the lab, bacteria are often studied in nutrient-rich environments under maximal growth rates. The reality is that microbial life is typically characterized by punctuated, suboptimal conditions of growth. In order to survive extended periods of time under nutrient-poor, stressful conditions, many bacteria enter a growth-arrested state until there is a return to more favourable conditions. Our research program seeks to define the molecular circuitry behind these transitions, using a highly tractable model bacterium, Legionella pneumophila. L. pneumophila typically replicates inside freshwater amoebae, but can spend months at a time in a non-replicative, extracellular state. One mechanism by which bacteria regulate their growth is through toxin-antitoxin systems. Despite their name, these "toxins" are not delivered to other cells, but rather serve to limit the replication of the cells that encode for them. Toxin-antitoxin systems typically exist as two-gene modules within an operon that encode a protein toxin capable of inhibiting growth along with a corresponding antitoxin. They are ubiquitous in bacteria and have been implicated in phage defense, biofilm formation, and dormancy, yet much of their function in cellular physiology is still unknown. The large number of systems in most species complicates their experimental interrogation: for instance, there are over 30 systems in most Escherichia and Salmonella species, and nearly 90 in Mycobacterium. In contrast, L. pneumophila has only 7 predicted toxin-antitoxin systems yet has a well-defined need to survive for extended periods in a non-replicative state. Put simply, our research program seeks to establish L. pneumophila as a consummate model organism for the systematic study of toxin-antitoxin systems. We will define their molecular targets and their mechanisms of action. We will systematically examine the contributions of these systems to L. pneumophila environmental persistence, intracellular replication, and genome stability. Within this program, our objectives are to: 1. Place each toxin-antitoxin system within the genetic network of the microbial cell. 2. Extensively characterize bacteria devoid of all toxin-antitoxin systems. 3. Determine the contribution of individual toxin-antitoxin systems to specific phenotypes through systematic complementation studies. The field of toxin-antitoxin systems is full of unlocked mysteries, controversies, and promise. Specifically, while there is a growing body of literature on the molecular mechanisms underpinning toxin-antitoxin systems, the underlying biology behind such systems remains poorly defined. Leveraging a uniquely tractable and well-suited model microbe, our program seeks to address this knowledge-gap head-on. With dozens of additional Legionella species recently sequenced - each with a corresponding diverse set of toxin-antitoxin systems to explore - the program we propose has a clear path forward for the conceivable future.