Characterization of a Methanosarcina acetivorans mutant unable to translate UAG as pyrrolysine

Anirban Mahapatra, Asmita Patel, Jitesh A. Soares, Ross C. Larue, Jun Kai Zhang, William W. Metcalf, Joseph A. Krzycki

Research output: Contribution to journalArticlepeer-review


The methyltransferases initiating methanogenesis from trimethylamine, dimethylamine and monomethylamine possess a novel residue, pyrrolysine. Pyrrolysine is the 22nd amino acid, because it is encoded by a single amber (UAG) codon in methylamine methyltransferase transcripts. A dedicated tRNA CUA for pyrrolysine, tRNAPyl, is charged by a pyrrolysyl-tRNA synthetase with pyrrolysine. As the first step towards the genetic analysis of UAG translation as pyrrolysine, a 761 base-pair genomic segment in Methanosarcina acetivorans containing the pylT gene (encoding tRNAPyl) was deleted and replaced by a puromycin resistance cassette. The ΔppylT mutant lacks detectable tRNAPyl, but grows as wild-type on methanol or acetate. Unlike wild-type, the ΔppylT strain cannot grow on any methylamine, nor use monomethylamine as sole nitrogen source. Wild-type cells, but not ΔppylT, have monomethylamine methyltransferase activity during growth on methanol. Immunoblot analysis indicated monomethylamine methyltransferase was absent in ΔppylT. The phenotype of ΔppylT reveals the deficiency in methylamine metabolism expected of a Methanosarcina species unable to decode UAG codons as pyrrolysine, but also that loss of pylT does not compromise growth on other substrates. These results indicate that in-depth genetic analysis of UAG translation as pyrrolysine is feasible, as deletion of pylT is conditionally lethal depending on growth substrate.

Original languageEnglish (US)
Pages (from-to)56-66
Number of pages11
JournalMolecular Microbiology
Issue number1
StatePublished - Jan 2006

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology


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