Ann Zahle Andersen

Research Article: The metabolic pH response in Lactococcus lactis: An integrative experimental and modelling approach

Lactococcus lactis is characterised by its ability to convert sugar almost exclusively into lactic acid. This organic acid lowers extracellular pH, thus inhibiting growth of competing bacteria. Although L. lactis is able to survive at low pH, glycolysis is strongly affected at pH values below 5, showing reduced rate of glucose consumption. Therefore, in order to deepen our knowledge on central metabolism of L. lactis in natural or industrial environments, an existing full scale kinetic model of glucose metabolism was extended to simulate the impact of lowering extracellular pH in non-growing cells of L. lactis MG1363. Validation of the model was performed using (13)C NMR, (31)P NMR, and nicotinamide adenine dinucleotide hydride auto-fluorescence data of living cells metabolizing glucose at different pH values. The changes in the rate of glycolysis as well as in the dynamics of intracellular metabolites (NADH, nucleotide triphosphates and fructose-1,6-bisphosphate) observed during glucose pulse experiments were reproduced by model simulations. The model allowed investigation of key enzymes at sub-optimum extracellular pH, simulating their response to changing conditions in the complex network, as opposed to in vitro enzyme studies. The model predicts that a major cause of the decrease in the glycolytic rate, upon lowering the extracellular pH, is the lower pool of phosphoenolpyruvate available to fuel glucose uptake via the phosphoenolpyruvate-dependent transport system.

On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae

We employed the fluorescent cyanine dye DiOC2(3) to measure membrane potential in semi‐anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found that the mitochondrial membrane potential was oscillating, and that these oscillations displayed the same frequency and duration as the NADH oscillations. It was confirmed that DiOC2(3) localizes itself in the mitochondrial membrane and thus reports qualitative changes solely in mitochondrial membrane potential. Our studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolarization of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane potential oscillations and delocalized DiOC2(3), while glycolysis continued to oscillate unaffected. Copyright