BONCAT-Live for isolation and cultivation of active environmental bacteria

In diverse environments, microbes drive a myriad of processes, from geochemical and nutrient cycling to interspecies interactions, including associations with plants and animals. Their physiological state is dynamic and impacted by abiotic and biotic conditions, responding to environmental fluctuations by changes in cellular metabolism, according to their genetic potential. Molecular, cellular, and genomic approaches can identify and measure microbial responses and adaptation to environmental changes in native communities. However, isolating individual microbial cells that respond to specific changes for cultivation has been difficult. To address this, we implemented a novel bacterial isolation approach (BONCAT-Live) by integrating bio-orthogonal non-canonical amino acid tagging (BONCAT) in diverse native communities, with isolation and cultivation of cells responding to specific stimuli, at different time scales. In frozen Arctic permafrost samples, we identified and isolated dormant bacteria that become active after thawing under native or nutrient-enriched conditions. From the <i>Populus</i> tree rhizosphere, we isolated strains that thrive under high concentrations of root exudates that act as defense compounds and nutrients. In the human microbiome, we identified and isolated bacteria that rapidly proliferated when exposed to metabolites provided by the host or other co-occurring microbes. Further characterization of isolated bacterial strains will provide opportunities for in-depth determination of how these microbes adapt to changes in their environments, individually and as part of model communities.IMPORTANCEDynamic microbial activity transforms environments and impacts health and disease in associations with plants and animals, including humans. Identifying the contribution of individual microbes to those processes in real time has not been generally compatible with their selective cultivation. BONCAT-Live tracks which bacteria in environmental samples are translationally active and couples it with single-cell isolation and cultivation. By studying the response of individual community members to specific natural or induced physical or chemical changes in the environment and culturing those organisms, BONCAT-Live enables further insights into microbial metabolic strategies, community dynamics, and environmental adaptations.