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

Abstract

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, tRNA(Pyl), 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 tRNA(Pyl)) was deleted and replaced by a puromycin resistance cassette. The DeltappylT mutant lacks detectable tRNA(Pyl), but grows as wild-type on methanol or acetate. Unlike wild-type, the DeltappylT strain cannot grow on any methylamine, nor use monomethylamine as sole nitrogen source. Wild-type cells, but not DeltappylT, have monomethylamine methyltransferase activity during growth on methanol. Immunoblot analysis indicated monomethylamine methyltransferase was absent in DeltappylT. The phenotype of DeltappylT 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.