BarTn7: Optimizing Bacterial Lineage Tracking at Sub-Species Resolution for Population Dynamics in Ecological and Evolutionary Studies

Communities of bacteria undergo population bottlenecks which are crucial to their population, ecological, and evolutionary dynamics. However, conventional amplicon sequencing cannot distinguish such demographic shifts between very closely related lineages. Here we describe BarTn7, an optimized method for bacterial lineage tracking through chromosomal integration of phenotypically neutral DNA barcodes with transposon Tn7. By combining conventional conjugative plasmids into a single vector and leveraging a parts-based strategy to optimize delivery to different recipients, BarTn7 increases barcoding efficiency and enables the systematic application of this tool to diverse bacteria. We tested BarTn7 in multiple bacterial species and confirmed a lack of lineage-specific growth effects. We then used BarTn7 to measure the colonization of Panicum virgatum roots under differing phosphate concentrations. Lineage tracking enabled discrimination between unique colonization events and the proliferation of existing bacteria during root colonization, the ratio of which differed between three plant-associated bacterial species. The strategy further allowed the measurement of phosphate-dependent ingress rates of the root endosphere by Paraburkholderia phytofirmans PsJN. We then demonstrated the effectiveness of BarTn7 to detect adaptive mutation(s) and facilitate identification of mutant lineages. Additionally, we demonstrated that BarTn7 is more accurate than conventional 16S rRNA gene sequencing at measuring community composition of a 5-member synthetic bacterial community (SynCom) and compares favorably to shotgun metagenomics. Our results illustrate the utility of BarTn7 as a simple, cost-efficient, and broadly applicable method of measuring bacterial population dynamics at sub-species resolution.