Abstract
Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.
Highlights
Fusidic acid-resistant elongation factor-G (EF-G) mutants of Staphylococcus aureus show fitness loss and compensation
Comparison of the S. aureus EF-G Mutants in Ribosomal Complex Formation with Fusidic acid (FA)—In the presence of 70S ribosomes, EF-G hydrolyzes GTP and EF-G1⁄7GDP can be trapped on the 70S ribosome with 0.5 mM FA, resulting in a 70S EF-G1⁄7GDP-FA complex
Only 5– 8% of complex was formed with F88L and F88L/M16I mutants, comparable with the background without 70S or EF-G or FA (Ͼ10%) (Fig. 1A)
Summary
Fusidic acid-resistant EF-G mutants of Staphylococcus aureus show fitness loss and compensation. Results: Slower translocation and ribosome recycling from restricted conformational change, plus increased tRNA drop-off, cause fitness loss in F88L, which are recovered in F88L/M16I, leading to fitness compensation. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. The switch II region contains a set of highly conserved residues, including a Phe at its tip, which is Phe-88 in S. aureus and Phe-90 in Thermus thermophilus This residue is thought to be important for transmitting the conformational changes of switch II between the GTP and GDP forms of EF-G [19], direct evidence has been lacking so far. Our study shows how the cross-talk between different residues in EF-G influences its function and provides insights into the molecular mechanism of FA resistance, fitness loss, and fitness compensation in S. aureus
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
