Catch me if you can: Capturing extracellular DNA transformation in mixed cultures via Hi-C sequencing

Environmental microorganisms evolve constantly under various stressors using different adaptive mechanisms, including horizontal gene transfer. Microorganisms benefit from transferring genetic information that code for antibiotic resistance via mobile genetic elements (plasmids). Due to the complexity of natural microbial ecosystems, quantitative data on the transfer of genetic information in microbial communities remain unclear. Two 1-L chemostats (one control and one test) were inoculated with activated sludge, fed with synthetic wastewater, and operated for 45 days at a hydraulic retention time of 1 day to study the transformation capacity of a rolling-circle plasmid encoding GFP and kanamycin resistance genes, at increasing concentrations of kanamycin (0.01-2.5-50-100 mg L ⁻¹ ) representing environmental, wastewater, lab-selection, and gut or untreated pharmaceutical wastewater discharge environments. The plasmid DNA was spiked daily at 5 µg L ⁻¹ in the test chemostat. The evolution of the microbial community composition was analyzed by 16S rRNA gene amplicon sequencing and metagenomics, and the presence of the plasmid by quantitative PCR. We used Hi-C sequencing to identify natural transformant microorganisms under steady-state conditions with low (2.5 mg L ⁻¹ ) and high (50 mg L ⁻¹ ) concentrations of kanamycin. Both chemostats selected for the same 6 predominant families of Spirosomaceae, Comamonadaceae, Rhodocyclaceae, Rhizobiaceae, Microbacteriaceae , and Chitinophagaceae , while biomass formation in the presence of kanamycin was higher with the plasmid. Hence, the antibiotic exerted the main pressure on microbial selection, while the plasmid helped these populations better resist the antibiotic treatment and grow. The kanamycin resistance gene increased in both reactors (log 7 gene copies g VSS ⁻¹ ). When higher antibiotic concentrations were applied, the GFP/16S ratio was increased, highlighting plasmids accumulation in the test reactor over time. The plasmid transformed mainly inside populations of Bosea sp ., Runella spp ., and Microbacterium sp .. This study made one significant step forward by demonstrating that microorganisms in enrichments from activated sludge biomasses can acquire exogenous synthetic plasmids by transformation. Graphical abstract