A Metagenome-Based Methodology to Track Genomically Recoded Strains and Assess Their Effects on Indigenous Microbes.

Assessing the effects of the release of biologically contained microorganisms into the environment represents a challenging task as it requires both the tracking of escape events as well as the changes that result in the indigenous microbes, which cannot be effectively determined based on conventional culture-based methodologies. Toward closing this gap, we set up closed, laboratory mesocosms with water from a nearby recreational-use freshwater reservoir that were subsequently spiked with the Escherichia coli strain DEP to simulate an accidental spill of a synthetic organism into the environment. Strain DEP is a chloramphenicol-resistant synthetic auxotroph harboring three redesigned genes encoding nonstandard amino acid (nsAA)-dependent gene products for l-4,4'-biphenylalanine (BipA) dependence. Shotgun metagenome sequencing of the mesocosms revealed a sharp decline in the relative abundance of strain DEP over time, with minimal impact on the indigenous freshwater microbial communities as evidenced by the recovery of these communities to the preperturbation state after 2 days of incubation. Further, there were no observations of transfer of the nsAA-dependent genes to the indigenous populations at the limit of detection of our metagenome sequencing effort or based on culturing on BipA-supplemented media. Collectively, our results show that this particular strain DEP may not pose a serious environmental threat if accidentally released into the environment due to low competitiveness against the indigenous freshwater microbes and the lack of escape mutants. Notably, this work establishes a holistic approach to assess biocontainment efficacy that should be applicable to additional genetically modified organisms.