Multinuclear NMR spectroscopy of the cellular slime mold Polysphondylium pallidum. Monitoring of the encystment and excystment processes.

Abstract

Polysphondylium pallidum microcysts and amoebae have been investigated by 31P- and natural-abundance proton-decoupled 13C-NMR spectroscopy. Microcysts have been found to contain as prominent metabolites a phosphomonoester, inositol hexakisphosphate (1.4 mM), two phosphodiesters [glycerophosphocholine (5.5 mM) and glycerophosphoethanolamine (2.6 mM)], as well as nucleoside triphosphates (3 mM) and polyphosphates (greater than 10 mM), the polyamines 1,3-diamino-propane (3.5 mM), putrescine (16 mM) and spermidine (3 mM) and the sugar trehalose (31 mM). In vivo 31P-NMR has shown that the level of nucleoside triphosphates in microcysts was maintained metabolically and that the pH of their cytosol, deduced from the chemical shift of cytosolic Pi was 7.2. The absence of trehalose, glycerophosphocholine and glycerophosphoethanolamine in P. pallidum amoebae was the most remarkable difference from microcysts. Microcyst germination (excystment), induced by reduction of the ionic strength of the microcyst bathing medium, was monitored non-invasively by 31P- and 13C-NMR spectroscopy. The major modifications observed during excystment were the progressive disappearance of trehalose used as energy source, of glycerophosphocholine and glycerophosphoethanolamine used as membrane phospholipid precursors, and, finally, the appearance of NMR-visible polyphosphates and of cellobiose. As a mirror situation, P. pallidum amoebae responded to a high-ionic-strength stress by production of trehalose, glycerophosphocholine, and glycerophosphoethanolamine, and induction of an encystment process.