Futile cycling of glycogen in Fibrobacter succinogenes as shown by in situ 1H-NMR and 13C-NMR investigation

Abstract

Glycogen was synthesized during all the growth phases in the rumen anaerobic cellulolytic bacterium Fibrobacter succinogenes. Glycogen synthesis and degradation were monitored using in situ 13C and 1H-NMR spectroscopy in resting cells of F. succinogenes. The cells were incubated at 37 degrees C under anaerobic conditions with [1-13C]glucose and [2-13C]glucose. 1H-NMR spectra were used to quantify enrichment by 13C of metabolism products. Glucose was utilized for energy requirements of the bacterium, essentially via the Embden-Meyerhof pathway, leading to the synthesis of succinate and acetate, while glycogen was stored. From [1-13C]glucose, labeling occurred on C2 of succinate and acetate, and on both C1 and C6 of glycogen, the labeling on C1 being predominant. The C6-labeling of glycogen may be explained by scrambling and reversal of the glycolytic pathway at the triose-phosphate and fructose 1,6-bisphosphate level. When the bacteria were incubated first with [1-13C]glucose, then washed and incubated with [2-13C]glucose, the pattern of 13C labeling in the products of the metabolism, as shown by 13C and 1H-NMR spectra, indicated that glycogen was degraded at the same time as it was being stored, suggesting futile cycling of glycogen. The hydrolysis of previously stored glycogen can provide, in the presence of glucose, up to 30% of the carbon source for the bacteria.