Department of Fundamental Microbiology, University of Lausanne, 1015, Lausanne, Switzerland.
Sci Rep. 2019 Mar 7;9(1):3845. doi: 10.1038/s41598-019-40655-x.
Bacterial chemotaxis signaling may be interesting for the development of rapid biosensor assays, but is difficult to quantify. Here we explore two potential fluorescent readouts of chemotactically active Escherichia coli cells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of stable or unstable 'split'-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically active E. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise for biosensor assays based on bacterial chemotaxis activity.
细菌趋化性信号转导可能对快速生物传感器测定法的发展很有意义，但却难以定量。在这里，我们探索了两种可能的荧光读出方法来检测趋化性活跃的大肠杆菌细胞。在第一种方法中，我们分别将非荧光的稳定或不稳定的“分裂型”绿色荧光蛋白的非荧光部分与趋化信号蛋白 CheY 和 CheZ 融合，以此来探测它们之间的相互作用。野生型趋化性细胞但不是缺乏 CheA 激酶的突变体产生可区分的荧光焦点，其中三分之二定位在细胞极与化学感受器一起，三分之一定位在运动复合物。基于稳定的分裂型 eGFP 的荧光焦点在暴露于吸引剂或排斥剂的细胞中显示出小的波动，但基于不稳定的 ASV 标记的 eGFP 的荧光焦点在焦点强度变化和每个细胞中的焦点数量方面表现出更高的动态行为。对于第二种读出方法，我们在趋化性活跃的大肠杆菌的细胞质和周质中表达了 pH 敏感荧光蛋白 pHluorin。pHluorin 荧光作为 pH 函数的校准表明，在接近化学引诱剂的细胞中，细胞质 pH 暂时增加，而周质 pH 降低。因此，这两种读出方法都有望应用于基于细菌趋化性活动的生物传感器测定法。
