Genetic screen identifies cell wall enzyme is key for freshwater survival of Francisella tularensis

Human infection with Francisella tularensis, a potentially lethal bacterial pathogen, typically occurs after exposure to contaminated water, soil, food, or an infected animal. While F. tularensis can persist in environmental sources over long periods of time, the genetic requirements that permit its long-term viability are not understood. To address this question, we developed a laboratory model for persistence of F. tularensis in fresh water, finding that viable cells could be recovered for 3 - 8 weeks after incubation at 4{degrees}C. Using this model, we took an unbiased, transposon insertion sequencing approach to identify genes critical for this persistence of F. tularensis cells. We found that mutants in mpl, a gene encoding murein peptide ligase, are defective for persistence in fresh water. Previous studies had identified mpl as critical for intramacrophage survival. Murein peptide ligase plays a role in peptidoglycan recycling, suggesting that F. tularensis uses this enzyme to maintain cell wall integrity during hypoosmotic and intramacrophage stress conditions. Our results highlight the importance of understanding how bacterial cell envelopes have evolved and adapted to maintain their integrity in a variety of stress conditions.