Recently, an additional rare amino acid pyrrolysine (Pyl), was identified, which expanded the canonical genetic code to 22 amino acids . Pyl is inserted in response to UAG codon in several methanogenic archaea . Although the mechanism of Pyl biosynthesis and incorporation into protein is not fully understood, the presence of a tRNApyl gene (pylT) with the CUA anticodon and of class II aminoacyl-tRNA synthetase gene (pylS) argued for cotranslational incorporation of Pyl . In Desulfitobacterium hafniense, a single bacterium, in which a Pyl-containing protein was found, PylS consists of two proteins: PylSn and PylSc .