Class I and class II lysyl-tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.

Methanosarcina spp. begin methanogenesis from methylamines with methyltransferases made via the translation of UAG as pyrrolysine. In vitro evidence indicates two possible routes to pyrrolysyl-tRNA^Pyl. PylS ligates pyrrolysine to tRNA^Pyl. Alternatively, class I and class II lysyl-tRNA synthetases (LysRS1 and LysRS2) together form lysyl-tRNA ^Pyl, a potential intermediate to pyrrolysyl-tRNA^Pyl. The unusual possession of both LysRS1 and LysRS2 by Methanosarcina spp. may also reflect differences in catalytic properties. Here we assessed the in vivo relevance of these hypotheses. The lysK and mtmB transcripts, encoding LysRS1 and monomethylamine methyltransferase, were detectable in Methanosarcina barkeri during early log growth on trimethylamine, but not methanol. In contrast, lysS transcript encoding LysRS2 was detectable during log phase with either substrate. Methanosarcina acetivorans strains bearing deletions of lysK or lysS grew normally on methanol and methylamines with wild-type levels of monomethylamine methyltransferase and aminoacyl-tRNA^Pyl. The lysK and lysS genes could not replace pylS in a recombinant system employing tRNA ^Pyl for UAG suppression. The results support an association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine. However, decreased lysyl-tRNA^Lys in the lysS mutant provides a possible rationale for stable transfer of the bacterial lysS gene to methanoarchaea.