AUTHOR=van Beilen Johan W., Brul Stanley 

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

JOURNAL=Frontiers in Microbiology

VOLUME=4

YEAR=2013

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2013.00157

DOI=10.3389/fmicb.2013.00157

ISSN=1664-302X

ABSTRACT=<p>The internal pH (pH<sub>i</sub>) of a living cell is one of its most important physiological parameters. To monitor the pH inside <italic>Bacillus subtilis</italic> 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 <italic>comGA</italic>. The new version, which showed an approximate 40% increase in fluorescence intensity, was expressed from developmental phase-specific, native promoters of <italic>B. subtilis</italic> that are specifically active during vegetative growth on glucose (P<sub>ptsG</sub>) or during sporulation (P<sub>spoIIA</sub>, P<sub>spoIIID</sub>, and P<sub>sspE</sub>). Our results show strong, compartment-specific expression of IpHluorin that allowed accurate pH<sub>i</sub> measurements of live cultures during exponential growth, early and late sporulation, spore germination, and during subsequent spore outgrowth. Dormant spores were characterized by an pH<sub>i</sub> of 6.0 ± 0.3. Upon full germination the pH<sub>i</sub> 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.</p>