Accumulation of mutations in both gyrB and parE genes is associated with high-level resistance to novobiocin in Staphylococcus aureus

Abstract

Coumarin-resistant mutants of Staphylococcus aureus were isolated by three-step selection with novobiocin at different concentrations. Sequencing analysis of the gyrB and parE genes of the first-, second-, and third-step mutants revealed that successive point mutations first occurred specifically in the gyrB gene, followed by a point mutation in the parE gene and then an additional point mutation in the gyrB gene. These findings demonstrate that DNA gyrase is the primary target and that topoisomerase IV is the secondary target for novobiocin and that the accumulation of point mutations in both the gyrB and the parE genes is associated with high-level resistance to novobiocin in S. aureus. Moreover, our results show that the amino acid substitutions (Asp-89 to Gly and Ser-128 to Leu) found in GyrB are associated with resistance to novobiocin but not to coumermycin A1, suggesting that the interactions of novobiocin and coumermycin A1 with GyrB differ at the molecular level.

FIG. 1.

Relationships between S. aureus RN4220 and its resistant mutants isolated by stepwise exposure to novobiocin and between phenotype and genotype in first-, second-, and third-step mutants. G1, group 1; G2, group 2; G3, group 3; G4, group 4; G5, group 5; G6, group 6; G7, group 7. NOV^s and COU^s, mutant susceptibility to NOV and COU, respectively, did not change compared to that of the parent strain; NOV^r and COU^r, mutant susceptibility to NOV and COU, respectively, decreased compared to that of the parent strain; NOV, novobiocin; COU, coumermycin A1; superscript S, D, or T on gyrB and parE, the gene had a single, a double, or a triple point mutation, respectively.

FIG. 2.

Alignment of GyrB and ParE N-terminal amino acid sequences. Triangles, residues from E. coli GyrB region involved in ATP binding (26); asterisks and dots under the amino acid sequences, identical and similar residues in all four proteins, respectively; dashes, gaps introduced to maximize similarities; boxes, amino acid sequences that underwent changes in the novobiocin-resistant mutants; Ec, E. coli; Sa, S. aureus.