Figure 3. The target of amsacrine was identified by exploiting synthetic lethality.
(a) The USA300 wild-type strain (1) and tn::1050 strain (2) were grown on TSA or amsacrine (m-AMSA), o-AMSA, or the topoisomerase inhibitors ciprofloxacin and etoposide. The strains exhibit opposite sensitivities to the AMSA analogs as compared to the topoisomerase inhibitors. (b) Synthetic lethality between 1050 and the target of amsacrine (denoted by red line, top) was exploited in a selection on amsacrine for resistant mutants. A Δ1050 strain (tn::1050) was plated on 10 μg/ml amsacrine. Class I mutants with alterations in the target of amsacrine (left) were distinguished from class II mutants containing extragenic suppressors (right, denoted by blue “gene X”) by testing for growth in a combination of tunicamycin (1 μg/ml) and amsacrine (10 μg/ml). Extragenic suppressors preserve the synthetic lethality between WTAs and the target of amsacrine, while mutations in the target of amsacrine that prevent inhibition disrupt this interaction.