Monitoring of inorganic polyphosphate dynamics in Corynebacterium glutamicum using a novel oxygen sparger for real time 31P in vivo NMR

Abstract

For the first time in intact bacterial cells, the dynamics of the build-up of soluble cytosolic inorganic polyphosphate (polyP) during aeration, and its breakdown during anaerobiosis have been observed with a time resolution of 50 s. Under conditions of 60-80% saturation with pure oxygen, the accumulation of high levels of intracellular polyP was detected when inorganic phosphate (Pi) and glucose or acetate were added to Corynebacterium glutamicum cell suspensions (3 ml, ~40 mg dw/ml). The maximum levels of polyP reached were estimated to 600 mM P units in the cytosol or ~3% phosphorus [w/w] in the cell dry weight. C. glutamicum polyP was apparently of high molecular weight (containing probably a few hundred units) as inferred from signal distribution, but a temporary average polyP chain length of about n = 40 could be estimated at the initial stages of polyP formation. After each addition of glucose or acetate, oxygen levels followed a steep decline to ~20% and then an increase to the previous level. In contrast, polyP levels rose after the addition of substrate, and declined again, while the oxygen level recovered. When the oxygen supply was completely switched off, the polyP signal declined immediately, with concomitant re-appearance of phosphomonoester signals (sugar phosphates and related compounds). Both processes, the increase of polyP during aeration and supply with substrate and Pi, and the decrease during anaerobiosis, occurred within minutes. Only within these relatively brief windows of time between successive feedings with substrate or between aeration and anaerobiosis, high levels of polyP could be observed. Thus, our findings indicate that polyP occurs not only as the long known granular storage material in some Corynebacteria, such as C. diphtheriae or C. imitans, but that formation and breakdown of soluble polyP in C. glutamicum is a very dynamic process that may play a decisive role in C. glutamicum and in other strains of this genus. These investigations were made possible by combining nuclear magnetic resonance (NMR) techniques with novel methods of oxygen sparging and online substrate distribution. The sparger was custom made from titanium to fit into 10 mm o.d. NMR tubes. Both the size and the spacing of the holes in the sparger were calculated for optimum distribution of oxygen at 30 deg C through 3 ml of C. glutamicum cell suspensions. The experiments were carried out using in vivo 31P NMR, and monitoring of oxygen was performed with a miniature oxygen optode in real time. Glucose or acetate and/or phosphate stock solutions could be added in SitU. 31P NMR analyses of intracellular phosphorus metabolites were sampled with a time resolution of 50 s. The sparger unit, including optode and supply lines, could be easily switched from one sample to another after completion of an experiment. lt is suggested to use these analytical tools to investigate other bacterial strains and even cell extracts, shedding further light on the novel roles of polyP in living Cells