Interactions between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a H-1 and C-13 nuclear magnetic resonance and enzymatic study

The effect of the presence of ammonia on [1-C-13]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by C-13 and H-1 nuclear magnetic resonance (NMR), Ammonia halved the level of glyc ogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-ac etate ratio. The 13C enrichment of succinate and acetate was precisely quan tified by C-13-filtered spin-echo difference H-1-NMR spectroscopy. The pres ence of ammonia did not modify the C-13 enrichment of succinate C-2 (withou t ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic di lution of metabolites due to utilization of endogenous glycogen was not aff ected. In contrast, the presence of ammonia markedly decreased the C-13 enr ichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of th e succinate synthesis pathway, The reversal of glycolysis was unaffected by the presence of ammonia as shown by C-13-NMR analysis, Study of cell extra cts showed that the main pathways of ammonia assimilation in F, succinogene s were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthet ase activity was not detected, Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation i n the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase a ctivities were evidenced by spectrophotometry and H-1 NMR. When cells were incubated in vivo with [1-C-13]glucose, only C-13-labeled aspartate, glutam ate, alanine, and valine were detected. Their labelings were consistent wit h the proposed amino acid synthesis pathway and with the reversal of the su ccinate synthesis pathway.