Protein expression-independent response of intensity-based pH-sensitive fluorophores in Escherichia coli

Abstract

Fluorescent proteins that modulate their emission intensities when protonated serve as excellent probes of the cytosolic pH. Since the total fluorescence output fluctuates significantly due to variations in the fluorophore levels in cells, eliminating the dependence of the signal on protein concentration is crucial. This is typically accomplished with the aid of ratiometric fluorescent proteins such as pHluorin. However, pHluorin is excited by blue light, which can complicate pH measurements by adversely impacting bacterial physiology. Here, we characterized the response of intensity-based, pH-sensitive fluorescent proteins that excite at longer wavelengths where the blue light effect is diminished. The pH-response was interpreted in terms of an analytical model that assumed two emission states for each fluorophore: a low intensity protonated state and a high intensity deprotonated state. The model suggested a scaling to eliminate the dependence of the signal on the expression levels as well as on the illumination and photon-detection settings. Experiments successfully confirmed the scaling predictions. Thus, the internal pH can be readily determined with intensity-based fluorophores with appropriate calibrations irrespective of the fluorophore concentration and the signal acquisition setup. The framework developed in this work improves the robustness of intensity-based fluorophores for internal pH measurements in E. coli, potentially extending their applications.

Heterogeneous Distribution of Proton Motive Force in Nonheritable Antibiotic Resistance

“…Hence, we measured the membrane potential in the R-A-exp population and compared it with that of the No-exp cells. We did this with a fluorescence microscopy assay detailed in our recent work ( 42 ). We employed a cationic reporter dye, thioflavin T (ThT), which is taken up by cells due to their negatively charged membranes ( 43 ); dissipation of the membrane charge leads to the release of the dye from the cell.…”

“…Volumes of 50 μL of cell suspensions were placed on 12-mm-diameter coverslips that had been treated with 0.01% poly- l -lysine for 5 min and then washed in copious amounts of MB. The coverslips were placed in a perfusion chamber that allowed a constant flow of MB-ThT solution from a reservoir ( 28 , 42 ). A Nikon Ti-E microscope with a 60× water immersion objective was used to image the cells.…”