Deep mutational scan of ErmDL in the absence or presence of erythromycin or telithromycin

Antibiotic-dependent translational arrest on the ermD leader (ermDL) sequence is known to induce the expression of the ermD (erythromycin resistance methyltransferase) gene, thereby conferring resistance to MLS (macrolide, lincosamide, streptogramin B) antibiotics. This process depends both on the sequence of the nascent ErmDL peptide and the presence of macrolide antibiotics, such as erythromycin or telithromycin. In order to identify the sequence determinants within ErmDL that lead to translational arrest, we used high-throughput inverse toeprinting following in vitro translation in the presence or absence of erythromycin or telithromycin to perform a deep mutational scan of ermDL. Our data indicate that ErmDL has two distinct arrest motifs: a previously identified R/K-X-R/K motif (R/K indicates a positively charged amino acid and X any amino acid) which is strictly needed for macrolides that do not have a cladinose moiety, such as telithromycin, and an N-terminal motif that can compensate for the absence of this motif during erythromycin-dependent arrest.