Abstract: A protein with canonical lysyl-tRNA synthetase activity was purified from Methanococcus maripaludis, cloned, and sequenced. The predicted amino acid sequence of the enzyme indicated a novel class I polypeptide structurally unrelated to class II lysyl-tRNA synthetase reported in eubacteria, eukaryotes, and the Crenarchaeote Sulfobus solfataricus. A similar class I polypeptide was isolated from Borrelia burgdorferi, the causative agent of Lyme disease, and an open reading frame encoding a class I-type lysyl-tRNA synthetase was identified in the genome of Treponema pallidum, the causative agent of syphilis. The B. burdorferi gene encoding tRNALysl was cloned and used to make tRNA in vitro. The fundamental difference between pathogen and host in an essential enzyme suggests that class I-type lysyl-tRNA synthetase provides a target for the development of medical and veterinary therapeutics and diagnostics for Borrelia and other microorganism infections.