Individual cell DNA synthesis within natural marine bacterial assemblages as detected by 'click' chemistry

Individual cell growth rates enhance our understanding of microbial roles in regulating organic matter flux in marine and other aquatic systems. We devised a protocol to microscopically detect and quantify bacteria undergoing replication in seawater using the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), which becomes incorporated into bacterial DNA and is detected with a 'click' chemistry reaction in <1 h. Distinct EdU localization patterns were observed within individual labeled cells, e.g. some displayed 2 or more distinct EdU loci within a single DAPI-stained region, which likely indicated poleward migration of nascent DNA during the early phase of replication. Cell labeling ranged from 4.4 to 49%, comparable with cell labeling in parallel incubations for H-3-thymidine microautoradiography. Meanwhile, EdU signal intensities in cells ranged >3 orders of magnitude, wherein the most intensely labeled cells comprised most of a sample's sum community EdU signal, e.g. 26% of cells comprised 80% of the sum signal. This ability to rapidly detect and quantify signals in labeled DNA is an important step toward a robust approach for the determination of single-cell growth rates in natural assemblages and for linking growth rates with microscale biogeochemical dynamics.