Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro.

unlabelledPyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr(+) strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr(+) strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri. IMPORTANCE: The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens.