³¹P‐NMR and ¹³C‐NMR studies of mannose metabolism in Plesiomonas shigelloides

Abstract

The metabolism of mannose was examined in resting cells in vivo using 13 C-NMR and 31 P-NMR spectroscopy, in cell-free extracts in vitro using 31 P-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 13 C 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 P i were estimated by in vivo 31 P-NMR spectroscopy. The intracellular concentrations of phosphoenolpyruvate and P i 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 P i 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.Keywords: mannose; metabolism; microbial diversity; NMR; Plesiomonas shigelloides.The genus Plesiomonas appears to occupy a position between the families Enterobacteriaceae and Vibrionaceae in the gamma group of Proteobacteria [1]. Plesiomonas shigelloides is a facultatively anaerobic chemo-organotrophic Gram-negative bacterium. It has been implicated as the causative agent of gastroenteritis as well as of extraintestinal infections, primaly septicemia and meningitis [2]. To understand the ability of P. shigelloides to survive in its environment, it is essential to know the mechanisms by which nutrients are taken up and metabolized.In many Gram-negative bacteria, the specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates active transport and concomitant phosphorylation of sugars and hexitols [3,4]. The PTS uses phosphoenolpyruvate as the phosphoryl donor in a transfer chain that involves two general energy-coupling enzymes, Enzyme I and HPr, and the sugarspecific membrane-bound Enzyme II complex across the cell membrane.In order to study the metabolic diversity in Gram-negative bacteria, we have investigated sugar metabolism in P. shigelloides. In the present study, we show that mannose is transferred into the cells by a PTS producing mannose 6-phosphate. Curiously, when P. shigelloides was grown in nutrient broth supplemented with mannose, a lower growth yield than on nutrient broth was observed. Mannose and glucose catabolism were investigated in vivo, in resting cells using 13 C-NMR spectroscopy and in vitro, in cell-free extracts using 31 P-NMR spectroscopy a...

Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase‐encoding gene, MAN1, and its impact on pathogenicity

“…Furthermore, excess mannose in the environment appears to cause toxicity in the C. neoformans man1 cell, such that the cell seems unable to grow at levels above 50 mM mannose. A similar observation of mannose toxicity has been seen in Plesiomonas shigelloides (Rager et al ., 2000), Escherichia coli and honeybees, in which a non‐functional PMI enzyme leads to a marked accumulation of mannose‐6‐phosphate and an increase in intracellular ATP, which becomes lethal to the cell (de la Fuente et al ., 1986). Whether C. neoformans possesses a similar mechanism of toxicity is unclear.…”

<i>Escherichia coli</i> Response to Exogenous Pyrophosphate and Analogs

“…The cells were resuspended in a buffer containing 50 mM MES, 100 mM PIPES, 60 mM NaCl pH 7.4 and lyzed by addition of cold perchloric acid (0.6 M final concentration) as described previously [Rager et al, 2000]. After centrifugation, the supernatant was adjusted to pH 8.5 by adding NaOH.…”

Broad identification of bacterial type from pus by 1H MR spectroscopy

“…Proton magnetic resonance spectroscopy ( 1 H MRS) is a noninvasive and nondestructive technique and is widely utilized to study the metabolic pathways in healthy and diseased tissues. A number of MR spectroscopic studies have been performed on the various microorganisms [5–8]. The identification of bacteria is based on the manifestation of characteristic signals in the spectra of different bacteria that are contributed to by constituents of the cell wall [9].…”