31P-NMR and 13C-NMR studies of mannose metabolism in Plesiomonas shigelloides. Toxic effect of mannose on growth.

Abstract

The metabolism of mannose was examined in resting cells in vivo using 13C-NMR and 31P-NMR spectroscopy, in cell-free extracts in vitro using 31P-NMR spectroscopy, and by enzyme assays. Plesiomonas shigelloides was shown to transport mannose by a phosphoenolpyruvate-dependent phosphotransferase system producing mannose 6-phosphate. However, a toxic effect was observed when P. shigelloides was grown in the presence of mannose. Investigation of mannose metabolism using in vivo 13C NMR showed mannose 6-phosphate accumulation without further metabolism. In contrast, glucose was quickly metabolized under the same conditions to lactate, ethanol, acetate and succinate. Extracts of P. shigelloides exhibited no mannose-6-phosphate isomerase activity whereas the key enzyme of the Embden-Meyerhof pathway (6-phosphofructokinase) was found. This result explains the mannose 6-phosphate accumulation observed in cells grown on mannose. The levels of phosphoenolpyruvate and Pi were estimated by in vivo 31P-NMR spectroscopy. The intracellular concentrations of phosphoenolpyruvate and Pi were relatively constant in both starved cells and mannose-metabolizing cells. In glucose-metabolizing cells, the phosphoenolpyruvate concentration was lower, and about 80% of the Pi was used during the first 10 min. It thus appears that the toxic effect of mannose on growth is not due to energy depletion but probably to a toxic effect of mannose 6-phosphate.