微生物学

The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively low-throughput such that collecting enough data to fully characterize populations of cells is challenging. Flow cytometry avoids this drawback, and is a powerful tool that allows for rapid, high-throughput measurement of fluorescent readouts in individual cells. When combined with pH-sensitive fluorescent reporters, it can be used to characterize the intracellular pH of large populations of cells at the single-cell level. We adapted microscopy and flow-cytometry based methods to measure the intracellular pH of yeast. Cells can be grown under near-native conditions up until the point of measurement, and the protocol can be adapted to single-point or dynamic (time-resolved) measurements during changing environmental conditions.

The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H⁺ and Na⁺ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na⁺ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na⁺ concentration quantitatively.

The characteristics of Ca²⁺ and H⁺ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H⁺ concentration and increase in cytoplasmic Ca²⁺ concentration. The higher aquaporin activities can protect cells against osmotic stresses by altering water flow into and out of the cells. In order to confirm the contribution of aquaporins to the cell tolerance to different osmotic stresses, net Ca²⁺ and H⁺ fluxes are measured using the noninvasive micro-test technique (NMT). NMT provides the real-time in situ detection of net ion transport across membranes. Here, we describe the protocol of in situ detection of net Ca²⁺ and H⁺ fluxes across transformed Pichia pastoris cells in response to glycerol and polyethylene glycol 6000 (PEG6000) treatments. The transformed yeast cells are loaded onto a coverslide pre-processed in the poly-L-lysine solution (0.1% w/v aqueous solution). After cell immobilization, microelectrodes are positioned above a monolayer of attached cell population. Micro-volts differences are measured at two excursion points manipulated by a computer. Micro-volts differences could be converted into ion fluxes using the ASET 2.0 and iFluxes 1.0 Software. The method is expected to promote the application of NMT in microbiology. We are very grateful to Younger USA (Xuyue Beijing) NMT Service Center for their critical reading of the manuscript.

DiOC₂ (Novo et al., 2000) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts towards red emission as the dye molecules self associate at the higher cytosolic concentrations caused by larger membrane potentials. Proton ionophores such as CCCP destroy membrane potential by eliminating the proton gradient. The magnitude of membrane potentials varies with different bacterial species. For many gram-positive species, including Staphylococcus aureus and Micrococcus luteus, the red:green ratio tends to vary with the intensity of the proton gradient while in many gram-negative bacteria such as Escherichia coli and Salmonella choleraesuis, the response of the dye does not appear to be proportional to proton gradient intensity. Mycobacterium tuberculosis itself is a difficult organism to work with because of its rigid cell wall.