31P-NMR and 13C-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 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.

Cd(II), Pb(II) and Zn(II) ions regulate expression of the metal-transporting P-type ATPase ZntA in Escherichia coli.

ZntA is a cation-translocating ATPase which exports from Escherichia coli Cd(II) and Pb(II), as well as Zn(II). The metal-dependent ATP hydrolysis activity of purified ZntA was recently characterised and showed a specificity for Cd(II), Pb(II) and Zn(II). zntA expression has been reported to be up-regulated primarily by Zn(II), mediated by the regulatory protein ZntR, belonging to the MerR transcriptional regulator family. In contrast to previous claims, we now show, using a Phi(zntA-lacZ) monolysogen, that Cd(II) is the most effective inducer of zntA, which is also induced significantly by Pb(II). The Cd(II)- and Pb(II)-dependent transcriptional up-regulation of zntA is also mediated by ZntR.

Evidence for the transport of zinc(II) ions via the pit inorganic phosphate transport system in Escherichia coli.

A locus involved in zinc(II) uptake in Escherichia coli K-12 was identified through the generation of a zinc(II)-resistant mutant by transposon (Tn10dCam) mutagenesis. The mutation was located within the pitA gene, which encodes the low-affinity inorganic phosphate transport system (Pit). The pitA mutant accumulated reduced amounts of zinc(II) when exposed to 0.5-2.0 mM ZnSO(4) during growth in Luria-Bertani medium.

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.

Transport of glucose by a phosphoenolpyruvate:mannose phosphotransferase system in Pasteurella multocida.

Pasteurella multocida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIAGlc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida growth on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.