EDGE FGT: Genome-editing tools for keystone freshwater heterotrophs

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Microbial activity drives carbon emission and burial in freshwater environments, which cover only 4% of Earth's surface but both emit and store disproportionally large amounts of carbon. Up to 60% of the bacteria in fresh waters belong to a specific group of Actinobacteria, and this group likely controls conversion of organic matter to CO2 and buried biomass in these ecosystems. Although these bacteria have fewer than 2000 genes, they thrive in bogs, algae-choked lakes, pristine alpine ponds, rivers, estuaries, and reservoirs. To understand how bacteria with so few genes are able to dominate in such a diverse range of environments, we propose to develop genome-editing tools in two easy-to-work-with species of freshwater Actinobacteria. The work proposed here, and the research that builds on it, will enable scientists working on freshwater carbon cycling, bacterial-algal interactions, heterotrophy, and light capture to identify the genes in freshwater Actinobacteria that contribute to these processes. That work, in turn, will provide experimental data linking genes to ecosystem functions. Laboratory research on the physiology and metabolism of freshwater clades of Actinobacteria has been hampered by having only a few cultivated species, none with available genome editing tools. To understand how such an apparently homogeneous group of bacteria contributes to nutrient cycling in such a diverse range of ecological settings, we propose to develop systems for targeted and random mutagenesis in Rhodoluna lacicola and Aurantimicrobium sp. strain MWH-Mo1, two easily cultivated, representative species of freshwater Actinobacteria. Targeted mutagenesis will enable not just inactivation of specific genes, but also insertion of genes for heterologous expression from the chromosome. Random transposon-based mutagenesis will enable construction of comprehensive mutant libraries that can be screened for fitness under a variety of different environmentally relevant stresses. These tools will catalyze research that connects simple genomes to complex phenotypes, phenotypes to biochemical networks in microbial communities, and microbial community metabolism to ecosystem carbon budgets.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.