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

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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.