Abstract 1544 Dual routes for B. anthracis asparaginyl-tRNA formation

In Bacillus anthracis, there are two distinct routes for attaching asparagine (Asn) to its cognate transfer RNA (tRNAAsn), an essential step in protein synthesis. In the direct route, asparaginyl-tRNA synthetase (AsnRS) directly ligates Asn to tRNAAsn. For the indirect route a two-step pathway is employed where a non-discriminating-AspRS (ND-AspRS) ligates aspartate (Asp) to tRNAAsn which is them amidated by GatCAB, an amidotransferase to synthesize Asn on tRNAAsn. The ND-AspRS, GatCAB, and tRNAAsn associate to form the transamidosome. B. anthracis encodes two distinct AspRSs with one of bacterial origins and the other acquired through horizontal gene transfer from archaea, while also encoding an AsnRS to directly ligate Asn to tRNAAsn. The archaeal AspRS serves as the non-discriminating enzyme for tRNA-dependent asparagine biosynthesis. We have successfully overproduced in E. coli and purified the enzymes for both pathways to further characterize the two routes under various physiologically relevant conditions to elucidate why B. anthracis acquired an archaeal aspartyl-tRNA synthetase and encodes two distinct pathways for asparaginyl-tRNA synthesis. The work was supported by funding from the National Science Foundation (NSF- 1615770).