Link between phosphate starvation and glycogen metabolism in Corynebacterium glutamicum, revealed by metabolomics

Abstract

In this study, we analyzed the influence of phosphate (P(i)) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under P(i) limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under P(i) excess, the glycogen level in P(i)-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under P(i) excess, did so under P(i) limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under P(i) limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited P(i) concentrations.

FIG. 1.

Influence of growth with different P_i concentrations on the metabolome of C. glutamicum. (A) Growth of C. glutamicum ATCC 13032 in CGXII minimal medium with 222 mM glucose and different concentration of P_i. Cells were precultured twice in CGXII glucose medium with 0.13 mM potassium P_i and then transferred to CGXII medium containing 0.13 mM, 0.26 mM, 0.65 mM, or 13 mM inorganic P_i. The experiment was performed in triplicate, and mean values and standard deviations are shown. After 24 h, samples of all cultures were taken and used for metabolite analysis by GC-TOF MS. The 2,517 mass fragments detected in all samples were used for PLS-DA, representing one symbol of the score plots. (B) PLS-DA score plot of the metabolome samples taken after 24 h of growth with different P_i concentrations. t[1] and t[2] represent vectors for the most significant components of the matrix x of mass ion abundances. The plot shows a directionality of the metabolite pattern from P_i excess to limitation. (C) PLS-DA score plot of the samples taken after 8, 12, and 24 h from cultures grown in CGXII glucose medium with either 13 mM P_i or 0.13 mM P_i.

FIG. 2.

Growth (OD₆₀₀, circles), carbon source consumption (triangles), and cellular glycogen pools (squares) are shown for C. glutamicum cultivated in CGXII minimal medium with 222 mM glucose (A and C) or with 300 mM potassium acetate (B and D) either under P_i excess or P_i limitation. The inoculum was precultivated twice in the same medium under P_i limitation. Panel A also shows lactate formation (rhombic symbols), which did not occur during growth on acetate (B). Mean values and standard deviations of triplicate cultures are shown.

FIG. 3.

In vivo ³¹P-NMR spectrum (A) and measurements to determine the cytoplasmic concentrations of intracellular P_i, phosphate monoesters (PME), NDP-glucose, and polyphosphate (polyP) in cells cultivated for 8 h (a) and 24 h (b) in CGXII glucose minimal medium with 13 mM P_i (black bars) or for 8 h (c) and 24 h (d) in CGXII glucose minimal medium with 0.13 mM P_i (white bars) (B). Signals representing intracellular P_i (1), phosphate monoesters (2), NDP-glucose (3), and polyphosphate (4) are marked in the ³¹P-NMR spectra, which were recorded using a Varian Inova 400 MHz spectrometer operating at a ³¹P frequency of 161.985 MHz, as described in Materials and Methods. The experiment was performed twice with comparable results.

FIG. 4.

Growth of (A) and glucose consumption by (B) of C. glutamicum wild type (black and red) and the ΔsugR mutant (green and blue) in CGXII minimal medium with 4% (wt/vol) glucose and either 13 mM P_i (black and green) or 0.13 mM P_i (red and blue). Mean values and standard deviations of triplicate cultures are shown.

FIG. 5.

Simulated phenotypes under growth optimization of the genome-scale metabolic network model. In silico solutions of growth rates and glycogen (GLN) formation under variation of P_i uptake in combination with either glucose (GLC) uptake (A and C) or acetate (ACE) uptake (B and D) form three-dimensional surfaces in each case. For comparison, measured growth rates for P_i-limited and -excess cultures, including experimental errors, are mapped as light gray rectangles.