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In previous studies we determined the nucleotide sequence of the gene cluster containing lys20, hacA (lys4A), hacB (lys4B), orfE, orfF, rimK, argC, and argB of Thermus thermophilus, an extremely thermophilic bacterium. In this study, we characterized the role of each gene in the cluster by gene disruption and examined auxotrophy in the disruptants. All disruptants except for the orfE disruption showed a lysine auxotrophic phenotype. This was surprising because this cluster consists of genes coding for unrelated proteins based on their names, which had been tentatively designated by homology analysis. Although the newly found pathway contains alpha-aminoadipic acid as a lysine biosynthetic intermediate, this pathway is not the same as the eukaryotic one. When each of the gene products was phylogenetically analyzed, we found that genes evolutionarily-related to the lysine biosynthetic genes in T. thermophilus were all present in a hyperthermophilic and anaerobic archaeon, Pyrococcus horikoshii, and formed a gene cluster in a manner similar to that in T. thermophilus. Furthermore, this gene cluster was analogous in part to the present leucine and arginine biosyntheses pathways. This lysine biosynthesis cluster is assumed to be one of the origins of lysine biosynthesis and could therefore become a key to the evolution of amino acid biosynthesis.