Jindal, Srijan, Yang, Lei, Day, Philip J and Kell, Douglas B 
ORCID: 0000-0001-5838-7963
(2019)
Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli.
BMC MICROBIOLOGY, 19 (1).
195-.
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Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli.pdf - Published version Download (1MB) | Preview |
Abstract
Background:It is widely believed that most xenobiotics cross biomembranes by diffusing through thephospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternativeview, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptakeof an individual molecular type. We recognise here that the availability of gene knockout collections allows one toassess the contributions of all potential transporters, and flow cytometry based on fluorescence provides aconvenient high-throughput assay for xenobiotic uptake in individual cells.Results:We used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNAdye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-stateuptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthaseα- andβ-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by amembrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% withrespect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and effluxtransporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the mostreduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants inthe‘ASKA’collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the rangebeing approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlationbetween its uptake and that of the carbocyanine when compared across the various strains (although themembrane potential is presumably the same in each case).Conclusions:Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters ofputatively broad and presently unknown specificity, and that the very large range between the‘lowest’and the‘highest’levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayertransport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stainsfor 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | Drug transporters, Keio, Carbocyanine, SYBR Green, Membrane energisation |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 03 Oct 2019 08:32 |
| Last Modified: | 19 Jan 2023 00:24 |
| DOI: | 10.1186/s12866-019-1561-0 |
| Open Access URL: | https://doi.org/10.1186/s12866-019-1561-0 |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3056826 |
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