Genetic analysis of selenocysteine biosynthesis in the archaeon Methanococcus maripaludis

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Abstract
In Archaea selenocysteine (Sec) is synthesized in three steps. First seryl-tRNA synthetase acylates tRNASec with serine to generate Ser-tRNASec. Then phosphoseryl-tRNASec kinase (PSTK) forms Sep-tRNASec, which is converted to Sec-tRNASec by Sep-tRNA:Sec-tRNA synthase (SepSecS) in the presence of selenophosphate produced by selenophosphate synthetase (SelD). A complete in vivo analysis of the archaeal Sec biosynthesis pathway is still unavailable, and the existence of a redundant pathway or of a rescue mechanism based on the conversion of Sep-tRNASec to Cys-tRNASec during selenium starvation, cannot be excluded. Here we present a mutational analysis of Sec biosynthesis in Methanococcus maripaludis strain Mm900. Sec formation is abolished upon individually deleting the genes encoding SelD, PSTK or SepSecS; the resulting mutant strains could no longer grow on formate while growth with H2 + CO2 remained unaffected. However, deletion of the PSTK and SepSecS genes was not possible unless the selenium-free [NiFe]-hydrogenases Frc and Vhc were expressed. This required the prior deletion of either the gene encoding SelD or that of HrsM, a LysR-type regulator suppressing transcription of the frc and vhc operons in the presence of selenium. These results show that M. maripaludis Mm900 is facultatively selenium-dependent with a single pathway of Sec-tRNASec formation.

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