Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis

Abstract.

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H₂/CO₂, alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-¹³C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by ¹³C-NMR. Carbons derived from pyruvate contained 14.5% ¹³C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3–5% ¹³C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less ¹³C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO₂ exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO₂ exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.