Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes byEscherichia coli

Abstract

AbstractWe used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar findings were made with SYBR Green (the range being some 69-fold), though despite it too containing a benzimidazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of possibly broad and presently unknown specificity. This casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.