In situ 31P nuclear magnetic resonance for observation of polyphosphate and catabolite responses of chemostat-cultivated Saccharomyces cerevisiae after alkalinization

Abstract

The proposed pH buffering and phosphagenic functions of polyphosphate were investigated by subjecting chemostat-cultivated Saccharomyces cerevisiae to alkalinization (NaOH addition) and anaerobiosis. The subsequent changes in intracellular phosphate-containing species were observed in situ by nuclear magnetic resonance (NMR) spectroscopy by using the NMR cultivator we developed. For the alkalinization experiments, changes in catabolite secretion were also measured in parallel experiments. Additionally, a range of potential neutralization capacity was investigated: a dilute culture and concentrated cultures with low or high polyphosphate content. The concentrated cultures displayed increased cytosolic pH and rapid polyphosphate degradation to small chains. The pH changes and extent of polyphosphate degradation depended inversely on initial polyphosphate content. The dilute culture restored extracellular pH rapidly and secreted acetate. The concentrated culture with low polyphosphate reserves also secreted acetate. In contrast to the alkalinization-induced polyphosphate dynamics, anaerobiosis resulted in the complete hydrolysis of polyphosphate to P(i), as opposed to small chains, and reduced cytosolic pH. The results and calculations suggest that the bulk of NMR-observable polyphosphate (vacuolar) degradation to short polymers conceivably contributes to neutralizing added alkalinity. In other circumstances, such as anaerobiosis, degradation serves other functions, such as phosphorylation potential regulation.