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.

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

Malfatti F;
2014

Abstract

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11368/2959837
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