A General Workflow for Characterization of Nernstian Dyes and Their Effects on Bacterial Physiology

Abstract

The electrical membrane potential (V m) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes. Because V m also plays a role in signal transduction, measuring it is of great interest. Over the years, a variety of techniques have been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favorite strategy, and their cytoplasmic accumulation depends on V m according to the Nernst equation. However, a careful calibration of Nernstian probes that takes into account the tradeoffs between the ease with which the signal from the dye is observed and the dyes' interactions with cellular physiology is rarely performed. Here, we use a mathematical model to understand such tradeoffs and apply the results to assess the applicability of the Thioflavin T dye as a V m sensor in Escherichia coli. We identify the conditions in which the dye turns from a V m probe into an actuator and, based on the model and experimental results, propose a general workflow for the characterization of Nernstian dye candidates.

The Microbiologist’s Guide to Membrane Potential Dynamics

“…Accordingly, membrane potential can be determined by measuring the ratio between the fluorescence intensities inside and outside the cell. Lipophilic fluorescent cationic dyes, such as tetramethylrhodamine, methyl ester (TMRM), have been used to probe bacterial membrane potential [11,12,59,64,90,91]. Nernstian dyes present a particularly powerful and convenient tool for microbiological investigations, although their use for quantitative biophysical investigations requires more careful calibrations [91,92].…”

“…Lipophilic fluorescent cationic dyes, such as tetramethylrhodamine, methyl ester (TMRM), have been used to probe bacterial membrane potential [11,12,59,64,90,91]. Nernstian dyes present a particularly powerful and convenient tool for microbiological investigations, although their use for quantitative biophysical investigations requires more careful calibrations [91,92]. A potential drawback of Nernstian dyes is that their permeabilities tend to be low with Gram-negative bacteria, which requires pretreatments of cells with EDTA to chelate divalent cations [90].…”

“…Low permeability would also mean that they are not suitable for capturing fast dynamics. Another point to consider is that, when used at high concentrations, Nernstian dyes can become invasive to cellular membrane potential [12,91]. Membrane-bound dyes for measuring membrane potential, such as aminonaphthylethenylpyridinium (ANEP) dyes, are a preferable choice for capturing fast dynamics, although they suffer from a weaker signal-to-noise ratio.…”

Environmental conditions define the energetics of bacterial dormancy and its antibiotic susceptibility

“…Average values for each time point were obtained from 8 to 10 experiments for a total of 494 cells in M63 with glucose (media), 605 cells in the media with chloramphenicol and 594 cells in media with no glucose. Measurements are performed every 90 s. Due to our flow slide design [Mancini et al, 2020] the switch between growth medium used during slide preparation and dormancy-inducing medium occurs 2 to 5 min from the beginning of the recording. b, Example motor speed trace (proportional to the PMF) from a single cell in the three conditions shown in a, with the addition of the trace where the growth medium has been supplemented with 10 mM indole (yellow).…”

“…11. As in Mancini et al [2020], the plasmid was constructed by PCR amplification of the backbone from plasmid pWR20 Pilizota and Shaevitz [2012] and the sequence containing Q7*. Later steps included purification 9/15 of the PCR products followed by restriction with the restriction enzymes AvrII and NotI (NEB, UK) and ligation with the T4 DNA ligase (Promega, UK).…”