Quantification of Intracellular pH.

Intracellular pH was determined using the pH-sensitive fluorescent dye BCECF-AM. In brief, the fluorescence of this dye depends on the environmental pH, with high pH environments showing increased levels of fluorescence. It is a ratiometric dye, meaning that we utilize the ratio of fluorescence emission (525 nm) when excited at two different wavelengths (470 nm and 440 nm). Because the relative intensities in these two channels depend on both the imaging conditions (light intensity, emission filters, exposure time, etc.) and the pH, the conversion from the ratio of intensities to pH units needs to be calibrated utilizing the same approach as the experimental collection. Here, we used the ionophore nigericin as a way to collapse the pH gradient between the inside of control cells and their external buffered environment. This allowed us to measure intensities ratios and build a conversion function from intensities to pH while the BCECF dye was still in the presence of cellular components.

Cultures of each experimental strain, including an additional WT culture used for calibration of ratiometric fluorescence calibration (control), were grown overnight in LB broth. Due to the significant autofluorescence exhibited by LB broth, it could not be utilized for this microscopy-based approach. To address this issue, we thoroughly washed and resuspended cells from the overnight cultures in Ringer’s buffer, which is specifically formulated to minimize osmotic stress and commonly used to wash cells prior to staining and visualization on a microscope (114, 115). Specifically, a 1 mL aliquot from each culture was washed two times via centrifugation and resuspended in 500 µL of unbuffered Ringer’s buffer (1/4X) (Oxoid). We stained cells for 30 min at 37 °C with 2 µM BCECF-AM (Invitrogen). In addition to BCECF-AM staining, we added 5 µL of nigericin (Sigma Aldrich) to the tube containing the WT ladder standard to allow intracellular pH to equilibrate with the external environment. Following staining, we performed two additional washes via centrifugation using 500 µL of unbuffered 1/4X Ringers’ solution (Oxoid). 100 µL aliquots of each experimental strain and the ladder sample were transferred into five separate tubes. We centrifuged each tube for 1 min at 13,000×g and resuspended the pellets in one of seven 1/4X buffered Ringers’ solutions (pH 6.92, 7.25, 7.72, 8.33, 8.69, 9.06, and 9.59). We prepared buffered Ringers’ solutions using 50 mM of 1,3-Bis[tris(hydroxymethyl)amino]propane (Acros Organics) and adjusted the pH to the appropriate level using 1 M HCl. Once resuspended into pH-adjusted solutions, we imaged cells after approximately 30 min.

For each condition, 7 µL of cells was sandwiched between a 22 mm square #1.5 coverslip and a standard glass slide, providing sufficient fluid volume for cells to remain in solution but in approximately the same focal plane of the microscope. Cells were imaged on a Nikon Ti inverted microscope (Nikon Instruments, Melville, NY) equipped with an Andor Zyla sCMOS camera (Oxford Instruments, Abingdon, Oxfordshire), Spectra/Aura light engine (Lumencor, Beaverton, OR), and an Apo TIRF 60X Oil DIC N2 NA 1.49 objective (Nikon Instruments, Melville, NY). The BCECF fluorescence intensity was imaged in two channels (1: Ex 470 nm, Em 525/50 nm, and 2: Ex 440 nm, Em 525/50 nm). We used a semiautomated MATLAB script (MathWorks, Natwick, MA) to segment cells and quantify the fluorescence intensity. In brief, the process of segmentation identifies which pixels in the image correspond to individual objects or regions. Within each of these regions, the total intensity from each fluorescence channel was computed. Regions around these cells were used as proxies for the local background intensity and subtracted from the total intensity in each region. From these background subtracted intensities, the intensity ratio was calculated. To assist in the automation of this process, we utilized standard approaches in image processing which are described below. Images underwent a spatial bandpass filter (spatial frequencies between 220 nm and 1,100 nm) to remove salt and pepper noise and background intensity. Objects were then segmented as individual regions if above a certain intensity threshold. Because cells were plated at a sufficiently low density to ensure single objects, a relative threshold for each image was set as the intensity such that approximately the top 1,000 pixel values were above the threshold (quantile of 0.9998). To ensure that the entire region of each cell was included, and to deal with the fact that these two imaging channels have a small spatial shift from one channel to the other, these regions were then dilated by disks of radius 2 µm. Background regions were defined by dilating these regions by a further 2 µm, and removing the objects from these masks, yielding regions that are near, but not overlapping with, individual objects. The intensity for each object in each channel was determined as the background-subtracted sum of all the pixels in each region. Lists of relative intensities of these objects were then imported into RStudio. Samples treated with nigericin were used to determine the coefficients A and B in a fit of pH_obs = A*log(intensityRatio)+B.

To statistically determine differences in intracellular pH across the WT and engineered mutant strains, we fit the relationship between intracellular pH and environmental pH to a logarithmic function [Intracellular pH ~ a*log(Environmental pH)+k] using a nonlinear least squares method in the R package “nlm.” The fit was calculated across the measured interval of pH 7 to 9.5, and CI were calculated via uncertainty propagation with the function predictNLS which calculates CI for the fitted values of nonlinear models by using first-/second-order Taylor expansion and Monte Carlo simulation.