Cloning of the Glu-tRNA ^Gln amidotransferase (gatCAB) gene from Staphylococcus aureus

In Gram(+) bacteria and organelles in higher eukaryotes, Gln-tRNA^Gln utilized for protein biosynthesis is formed by a tRNA-dependent amino acid transformation using mischarged Glu-tRNA^Gln as the intermediate. In this study, the gatCAB gene encoding Glu-tRNA^Gln amidotransferase (Glu-AdT) of Staphylococcus aureus was cloned and its nucleotide sequence was determined. The S. aureus gatCAB gene was organized in an operon structure consisting of three open reading frames (gatC, gatA, and gatB), similar to that of Bacillus subtilis. The gene sequences for the A and B subunits of Glu-tRNA^Gln amidotransferase showed significant homology (77 and 87% homology with amino acid sequence) with the gatA and gatB genes of B. subtilis, yet the C subunit (gatC) showed a relatively lower homology with the B. subtilis gatC gene and other orthologues. The cloned S. aureus Gln-tRNA^Gln amidotransferase gene was highly expressed in Escherichia coli, and the resulting crude enzyme could convert misacylated Glu-tRNA^Gln into Gln-tRNA^Gln in vitro.