During sporulation the development of the pH i in the mother cell and the nascent fore-spore may also give insight in the level of independence of the two cells. To gain further insight in the putative pH i dynamics of these differentiation processes, we studied the pH i of the mother cell and fore-spore independently. We reasoned that in analogy to eukaryotes, pH i could be a global regulator, as well as an indicator of the metabolic and energetic state of the cell. (2007) have shown that this too, is a carefully orchestrated process. Germination is less well understood, but Keijser et al. The best described mode of differentiation of this Gram-positive prokaryote is sporulation, with the pathways controlling sporulation understood in great molecular detail ( Eichenberger et al., 2004 Steil et al., 2005 Wang et al., 2006). subtilis is generally considered to be the bacterial model organism for cellular differentiation. In prokaryotic organisms, the relationships between pH i and growth and development have not been studied extensively ( Padan and Schuldiner, 1987).īecause of its various well-described differentiation modes, B. In multicellular eukaryotes pH i is thought to be important during growth and differentiation ( Cruciat et al., 2010). Gene expression as a response to glucose starvation was found to be mediated by changes in the pH i, through the protonation state-dependent binding of a transcription factor to membrane-associated phosphatidic acid ( Young et al., 2010). In the model eukaryote Saccharomyces cerevisiae, pH i was found to be a signal controlling growth ( Orij et al., 2011). Furthermore, in many organisms proton gradients are required for the greater part of ATP synthesis while uptake systems often depend on the proton gradient over the cell membrane ( Krulwich et al., 1998, 2011 Slonczewski et al., 2009). Many intracellular enzymes show optimal activity and stability in a narrow pH range near neutrality. The internal pH (pH i) of living cells plays a fundamental role in many chemical reactions. Such effects were absent when acetic was added at identical concentrations. The presence of sorbic acid in the germination medium inhibited a rise in the intracellular pH of germinating spores and inhibited germination. Upon full germination the pH i rose dependent on the medium to 7.0–7.4. Dormant spores were characterized by an pH i of 6.0 ± 0.3. Our results show strong, compartment-specific expression of IpHluorin that allowed accurate pH i measurements of live cultures during exponential growth, early and late sporulation, spore germination, and during subsequent spore outgrowth. subtilis that are specifically active during vegetative growth on glucose (P ptsG) or during sporulation (P spoIIA, P spoIIID, and P sspE). The new version, which showed an approximate 40% increase in fluorescence intensity, was expressed from developmental phase-specific, native promoters of B. 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 internal pH (pH i) of a living cell is one of its most important physiological parameters. Department of Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands.
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