Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli
Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli
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DOI：
10.1186/s12866-019-1561-0
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发表时间：
2019-08-22
期刊：
影响因子：
4.2
通讯作者：
Kell, Douglas B.
中科院分区：
文献类型：
--
作者：
Jindal, Srijan;Yang, Lei;Kell, Douglas B.
背景人们普遍认为，大多数外源性物质通过磷脂双分子层扩散穿过生物膜，蛋白质转运蛋白的使用只是偶尔的辅助手段。根据另一种观点，磷脂双层转运可以忽略不计，几种不同的转运蛋白可能参与单个分子类型的摄取。我们在这里认识到，基因敲除集合的可用性允许一个评估所有潜在的转运蛋白的贡献，和基于荧光的流式细胞术提供了一个方便的高通量测定单个细胞中的外源性摄取。结果我们使用高通量流式细胞术评估单个基因敲除大肠杆菌菌株摄取两种膜渗透性阳离子荧光染料的能力，即碳菁diS-C3（5）和DNA染料SYBR绿色。个别菌株表现出大范围的吸收分布。不同菌株之间的碳菁的模式稳态摄取的范围是36倍。ATP合酶α-和β-亚基的敲除极大地抑制了摄取，这意味着大多数摄取是ATP驱动的，而不是由膜电位驱动的。几十个转运蛋白改变了超过50%的稳态摄取的染料相对于野生型，在任何方向（增加或减少）;敲除已知的流入和流出转运蛋白的行为如预期的那样，给予信任的一般策略。许多具有最低摄取的敲除是未知功能的转运蛋白基因（“y-基因”）。类似地，“ASKA”集合中的几种过表达变体具有预期的相反效果。SYBR绿色获得了类似的结果（范围约为69倍）。虽然它也含有苯并噻唑基序，但当在各种菌株中进行比较时，其摄取与碳菁的摄取之间的相关性可以忽略不计（尽管膜电位在每种情况下可能是相同的）。总之，我们得出结论，这些染料的吸收可能是由大量的转运蛋白的pupillion广泛和目前未知的特异性催化，和非常大的范围之间的“最低”和“最高”的摄取水平，即使在敲除的只是单一的基因，强烈暗示磷脂双层运输确实可以忽略不计。这项工作也投下了严重的怀疑后，使用这些染料作为定量染色代表生物能参数或数量的细胞DNA在未固定的细胞（体内）。相比之下，它开辟了它们作为高通量筛选中的转运蛋白测定底物的潜在用途。
Background It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient 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 of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely 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 alpha- and beta-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by 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 either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to 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 results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane 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 of putatively 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 bilayer transport is indeed negligible. This work also 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.