Abstract
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.
Highlights
Chemotaxis is the behaviour of cells to bias the direction of their motility in reaction to perceived chemical gradients[1,2]
We speculated that pH-changes as a result of flagellar motor rotation differences in chemotactic cells might be observable from pHluorin emission ratio changes. pHluorin was expressed in the cytoplasm or the periplasm of motile E. coli and its fluorescence emission ratio was quantified as a function of external pH
We followed interactions between CheY~P and CheZ as a proxy of ligand binding to the chemoreceptors in E. coli using bimolecular fluorescence complementation (BiFC) with split-eGFPs
Summary
Chemotaxis is the behaviour of cells to bias the direction of their motility in reaction to perceived chemical gradients[1,2]. Assays can be based on Föster resonance energy transfer (FRET) measurements of dynamic interactions between fluorescently-labeled protein partners in the chemotaxis signaling pathway in cells exposed to rapidly fluctuating chemical environments[17,18]. The dynamic interaction between CheY~P and its phosphatase CheZ or FliM has been measured by FRET17,18, and by direct observation of single molecules to motor complexes[28], and can be calibrated as a function of the attractant concentration[17]. In order to find potential alternative readouts for chemotactic behaviour of cells that might at some point enable development of biosensor assays, we decided to probe parts of the chemotactic signaling pathway and flagellar motor activity using fluorescence markers. Our hypothesis was that we might localize CheY~P/CheZ interactions in chemotactic cells from reconstituted split-GFP, which might change in number, position or intensity www.nature.com/scientificreports depending on cells being attracted or repelled. Microscope assays were established to measured pHluorin emission ratio changes in populations of cells under conditions of active chemoattraction
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