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

The metabolism of mannose was examined in resting cells in vivo using C-13- NMR and P-31-NMR spectroscopy, in cell-free extracts in vitro using P-31-NM R spectroscopy, and by enzyme assays. Plesiomonas shigelloides was shown to transport mannose by a phosphoenolpyruvate-dependent phosphotransferase sy stem producing mannose 6-phosphate. However, a toxic effect was observed wh en P. shigelloides was grown in the presence of mannose. Investigation of mannose metabolism using in vivo C-13 NMR showed mannose 6 -phosphate accumulation without further metabolism. In contrast, glucose wa s quickly metabolized under the same conditions to lactate, ethanol, acetat e and succinate. Extracts of P. shigelloides exhibited no mannose-6-phospha te isomerase activity whereas the key enzyme of the Embden-Meyerhof pathway (6-phosphofructokinase) was found. This result explains the mannose 6-phos phate accumulation observed in cells grown on mannose. The levels of phosph oenolpyruvate and P-i were estimated by in vivo P-31-NMR spectroscopy. The intracellular concentrations of phosphoenalpyruvate and P-i were relatively constant in both starved cells and mannose-metabolizing cells. In glucose- metabolizing cells, the phosphoenolpyruvate concentration was lower, and ab out 80% of the P-i was used during the first 10 min. It thus appears that t he toxic effect of mannose on growth is not due to energy depletion but pro bably to a toxic effect of mannose 6-phosphate.