Compartment-specific pH monitoring in Bacillus subtilis using fluorescent sensor proteins: a tool to analyze the antibacterial effect of weak organic acids

Abstract

The internal pH (pHi) of a living cell is one of its most important physiological parameters. To monitor the pH inside Bacillus subtilis during various stages of its life cycle, we constructed an improved version (IpHluorin) of the ratiometric, pH-sensitive fluorescent protein pHluorin by extending it at the 5′ end with the first 24 bp of comGA. The new version, which showed an approximate 40% increase in fluorescence intensity, was expressed from developmental phase-specific, native promoters of B. subtilis that are specifically active during vegetative growth on glucose (PptsG) or during sporulation (PspoIIA, PspoIIID, and PsspE). Our results show strong, compartment-specific expression of IpHluorin that allowed accurate pHi measurements of live cultures during exponential growth, early and late sporulation, spore germination, and during subsequent spore outgrowth. Dormant spores were characterized by an pHi of 6.0 ± 0.3. Upon full germination the pHi rose dependent on the medium to 7.0–7.4. The presence of sorbic acid in the germination medium inhibited a rise in the intracellular pH of germinating spores and inhibited germination. Such effects were absent when acetic was added at identical concentrations.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells

“…Growth rate and average colony pH i calculations with Multichannel-SporeTracker were always performed from the time point at which a clearly detectable surface increase, i.e., growth, had resumed. pH i was then generally ϳ7.5, values seen previously by van Beilen et al in liquid populations (4,20).…”

“…B. subtilis PptsG-IpHluorin cells were grown to exponential phase in MOPS medium to pH 7.4 containing spectinomycin (50 g/ml). At an OD 600 of 0.4, the cells were centrifuged (1,073 ϫ g for 10 min) and resuspended in phosphate-citrate buffers (0.1 M citrate and 0.2 M K 2 HPO 4 ), with pH values ranging from 5.5 to 8.5, as described previously by us (20). The cells were then permeabilized with the potassium ionophore valinomycin (1 M) and the protonophore nigericin (1 M) (13).…”

“…This treatment allows for the equilibration of the pH i with the externally set pH. Subsequently, cells in the phosphate-citrate buffer of different pH (5.5 to 8.5) were transferred to agarose pads with the ionophores at 1 M and of corresponding buffered pH values in closed aircontaining chambers (20). For each pH, fluorescence images of ϳ200 cells were analyzed with Multichannel-SporeTracker to construct a calibration curve (Fig.…”

“…To monitor the pH i of exponentially growing B. subtilis cells for an extended period of time, the B. subtilis PptsG-IpHluorin (trp2C amyE3= spcR PptsG-IpHluorin amyE5=) construct was used (20). This construct consists of the pHluorin gene (16), which was inserted after the first 24 bp of comGA adjacent to the promoter PptsG, resulting in IpHluorin.…”

“…pHluorin, a ratiometric and pH-sensitive green fluorescent protein (GFP) variant (16), allows direct, fast, and localized pH i measurements. It has been successfully used in S. cerevisiae (17,18) and more recently in B. subtilis (19)(20)(21) to probe cellular responses to various growth conditions, glucose pulses, respiratory chain inhibitors, and a few other treatments. A specific advantage of a fluorescence microscopy-based method is that it can provide information with (sub)cellular resolution (22,23).…”