A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum

The four genes required for selenocysteine incorporation were isolated from the gram-positive. amino acid-fermenting anaerobe Eubacterium acidaminophi lum which expresses various selenoproteins of different functions. The sel genes were located In an unique organization on a continuous fragment of ge nomic DNA in the order selDl (selenophosphate synthetase 1), selA (selenocy steine synthase). selB (selenocysteine-specific elongation factor), and sel C (selenocysteine-specific tRNA). A second gene copy, encoding selenophosph ate synthetase 2 (selD2), was present on a separate fragment of genomic DNA . SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, se lD1 and selD2, contained an In-frame UGA codon encoding selenocysteine, whi ch corresponds to Cys-17 of Escherichia coli SelD. The function of selA, se lB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzy l viologen-dependent formats dehydrogenase activity in these strains after anaerobic growth in the presence of formate, selA and selC from E. acidamin ophilum were functional and complemented the respective mutant strains to 8 3% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these condit ions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not suff icient to restore formate dehydrogenase activity.