Objectives: The FusB protein from Staphylococcus aureus is an elongation factor G (EF-G)–binding protein that mediates resistance to the antibiotic fusidic acid (FA), and represents the major cause of resistance to FA in clinical strains. However, several lines of evidence suggest that resistance to FA is probably not the original, native role of this protein. To gain insight into the cellular role(s) of FusB, including resistance to FA, we examined the effect of FusB on the individual reactions that form part of the functional cycle of EF-G.
Methods: In vitro translocation assays were performed using a heterologous translation system comprising 70S ribosomes from E. coli and recombinant (polyhistidine-tagged) S. aureus EF-G. A series of established biochemical and rapid kinetic experiments were performed to assess EF-G function, both in the presence and absence of FusB and FA.
Results: FusB exhibited no gross effects on translation or the EF-G-mediated translocation reaction, either in single- or multiple-turnover experiments. FusB increased the affinity of EF-G for its energy substrate, GTP, but not for GDP. No effect was seen on EF-G-mediated GTP hydrolysis. FusB caused a dose-dependent increase in the rate of dissociation of post-translocation complexes (EF-G•GDP•ribosome), and both prevented formation and promoted dissociation of EF-G•GDP•ribosome•FA complexes.
Conclusions: The ability of FusB to both block formation and cause dissociation of EF-G•GDP•ribosome•FA complexes provides a biochemical explanation for FusB-mediated resistance to FA. Since FusB increased affinity of EF-G for GTP, and lowered the affinity of EF-G for GDP•ribosome complexes, it seems likely that the native role of FusB is as an accessory translation factor that optimises the function of EF-G with respect to GTP when cellular GTP:GDP ratios are suboptimal (e.g. during starvation).