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进行最大生长速度的研究。现实情况是，微生物生命的典型特征是断断续续的、次优的生长条件。为了在营养不良、压力大的条件下生存很长一段时间，许多细菌进入生长停滞状态，直到恢复到更有利的条件。我们的研究项目试图使用一种高度易驯化的模式细菌--嗜肺军团菌来定义这些转变背后的分子电路。嗜肺乳杆菌通常在淡水阿米巴体内复制，但一次可以在非复制的细胞外状态下呆上几个月。 细菌调节生长的一种机制是通过毒素-抗毒素系统。尽管他们的名字，这些“毒素”不会传递给其他细胞，而是用来限制细胞的复制，为他们编码。毒素-抗毒素系统通常以操纵子中的两个基因模块的形式存在，这些操纵子编码一种能够抑制生长的蛋白质毒素和相应的抗毒素。它们普遍存在于细菌中，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许多功能仍不清楚。大多数物种的大量系统使他们的实验询问变得复杂：例如，大多数大肠杆菌和沙门氏菌物种中有30多个系统，而分枝杆菌中有近90个系统。相比之下，嗜肺乳杆菌只有7个预测的毒素-抗毒素系统，但有一个明确的需求，需要在非复制状态下长期生存。 简而言之，我们的研究计划旨在将嗜肺乳杆菌建立为系统研究毒素-抗毒素系统的完美模式生物。我们将定义它们的分子靶标和它们的作用机制。我们将系统地研究这些系统对嗜肺乳杆菌环境持久性、细胞内复制和基因组稳定性的贡献。 在这项计划中，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.广泛鉴定缺乏所有毒素-抗毒素系统的细菌。 3.通过系统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开的谜团、争议和希望。具体地说，虽然有越来越多的文献关于支撑毒素-抗毒素系统的分子机制，但这种系统背后的潜在生物学仍然定义不清。利用一种独特的易驯化和适合的模式微生物，我们的计划试图正面解决这一知识差距。随着最近对数十种额外的军团菌进行测序--每个军团菌都有相应的不同的毒素-抗毒素系统来探索，我们提出的计划为可预见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.