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Bacterial DNA type II topoisomerases (DNA gyrase and topoisomerase IV) catalyze the modification of the topological state of DNA in cells. Topoisomerase IV appears to have a particular role in decatenation of daughter chromosomes (18). These enzymes are composed of two subunits which form A₂B₂ tetramers composed of GyrA and GyrB subunits in the case of DNA gyrase and GrlA (ParC in Escherichia coli) and GrlB (ParE in E. coli) subunits in the case of topoisomerase IV in Staphylococcus aureus. For both enzymes, the A subunit is responsible for DNA breakage and reunion (10), while the B subunit catalyzes the hydrolysis of ATP (5). DNA gyrase is the target of several antibiotics. The fluoroquinolones, such as ciprofloxacin and norfloxacin, inhibit the DNA breakage-reunion cycle by binding to the gyrase-DNA complex. The coumarins, such as novobiocin and coumermycin, act by inhibiting ATP hydrolysis mediated by the GyrB subunit of DNA gyrase. Topoisomerase IV is also the target of several fluoroquinolones (6, 8). The action of coumarins on topoisomerase IV has been demonstrated by showing the inhibition of the activity of the purified enzyme by novobiocin (2, 12) and by showing the increased coumarin susceptibility of a mutant with an altered GrlB subunit (Asn470Asp) (7). No mutations in GrlB homologous to those in the GyrB subunit that cause coumarin resistance have been reported.

The novel phenotype of quinolone resistance and coumarin hypersusceptibility of the GrlB (Asn470Asp) mutant was postulated to be due to a novel mechanism, possibly associated with altered catalytic function (7). We showed in the study described here that certain GrlA mutations also exhibit this phenotype. This class of GrlA mutations, in contrast to those localized in the common quinolone resistance-determining region, is closer to the active site of enzyme breakage of DNA, suggesting possible effects on enzyme function. The dominance of these mutations was also studied and was found to be similar to that of the GrlB (Asn470Asp) mutation.

In vitro mutants from strain MT5. The strains and plasmids used in this study are described in Table 1. Strain MT5 carries the nov-142 locus (gyrB [Ile102Ser and Arg144Ile]), which is responsible for high-level resistance to coumarins (7). Some mutants of MT5 selected with ciprofloxacin or norfloxacin showed mutations in GrlA at position 80 or 116 (Table 2) (11, 14). For both kinds of mutants an increase in the level of fluoroquinolone resistance was observed, but only mutants carrying the mutation at position 116 exhibited a slight but reproducible decrease in the level of novobiocin resistance (twofold) (Table 2). In order to study the effects of these mutations on novobiocin susceptibility, cloning of the different grlA genes was done.

Cloning of grlA genes. The grlA genes of strain ISP794 (wild-type), MT5224c4 (grlA [Phe-80]), MT5224c2 (grlA [Pro-116]), and MT111 (grlA [Glu-116]) were amplified by PCR with Vent DNA polymerase and primers containing a BamHI site as described previously (7). The PCR product was cloned into the BamHI site of pGEM3-zf(+). In order to verify that no mutation was introduced by the polymerase, the sequences of the entire grlA gene for each allele were determined, and no changes were found. The grlA genes were then subcloned into the BamHI site of pSK950, a shuttle vector carrying the thermosensitive replicon of plasmid pE194 from S. aureus. Plasmids pSKISA (grlA⁺), pSKC2A (grlA [Pro-116]), pSKC4A (grlA [Phe-80]), and pSKMTA (grlA [Glu-116]) were obtained (Table 1). pSKC4A (grlA [Phe-80]) was introduced into wild-type strain S. aureus RN4220 (r), and pSKISA (grlA⁺) was introduced into S. aureus EN20 (a derivative of RN4220 with the grlA [Phe-80] mutation on the chromosome) (11). MICs were measured with Mueller-Hinton agar supplemented with serial twofold increasing concentrations of drugs, and the cells were grown at 30°C. Unexpectedly, for cells carrying pSKISA and pSKC4A the MICs were similar in comparison to the MICs for cells containing the vector plasmid alone (data not shown), suggesting that the grlA genes were not expressed. The region that was amplified by PCR included the putative promoter previously described by Yamagishi et al. (17). Thus, this promoter appears to be weak or inefficient, suggesting that expression of grlA is dependent on the promoter of grlB, which is upstream of grlA. The initiation codon of grlA overlaps the stop codon of grlB (6, 17).

Expression of grlA genes in multicopy alleles. The plasmids with the mutated grlA genes (pBFMTA, pBFC2A, and pBFC4A) were introduced into strain RN4220. Strains derived from RN4220 carried the three different alleles and were obtained by transformation of high-molecular-weight chromosomal DNA as described previously (13): EN20 (grlA [Phe-80]), BF5 (grlA [Pro-116]), and BF6 (grlA [Glu-116]). The plasmid with the wild-type gene (pBFISA) was introduced into each of these strains. The MICs were determined and are presented in Table 3. First, for BF6 and BF5, which carry the mutation at position 116 on the chromosome, the MICs of fluoroquinolone (ciprofloxacin and norfloxacin) were increased (two- to eightfold) and the MICs of coumarins (novobiocin and coumermycin) were decreased (fourfold) in comparison to the MICs for the parent strain RN4220. Second, when the plasmids carrying the mutated grlA genes were introduced into wild-type strain RN4220, an increase in the fluoroquinolone MICs was observed. In addition, RN4220 carrying pBFMTA (grlA [Glu-116]) and pBFC2A (grlA [Pro-116]) exhibited decreased levels of resistance to coumarins (two- to fourfold), in contrast to RN4220 carrying pBFC4A (grlA [Phe-80]), for which no change in the coumarin MICs was found. For the merodiploids of mutant strains EN20, BF5, and BF6 containing plasmid pBFISA (grlA⁺), the opposite effects were observed: decreases in the fluoroquinolone MICs for all strains and increases in the coumarin MICs only for BF5 and BF6 (Table 3). These results confirmed that the mutation at position 116 is responsible for the quinolone resistance and the coumarin hypersusceptibility phenotype of these mutants.

Determination of dominance. Our results also indicate that both mutations in grlA are codominant for quinolone resistance in multicopy alleles (Table 3 and 4). The codominance of fluoroquinolone resistance was also observed for mutations in grlB (7), parC (in E. coli, equivalent to grlA) (8), and parE (in E. coli, equivalent to grlB) (3). For coumarin hypersusceptibility, mutation at position 116 is codominant in multicopy alleles in both gyrB⁺ and gyrB142 backgrounds, as described previously for the mutation grlB (Asp470) (7). Thus, the grlB mutation and grlA mutations at position 116 are also epistatic for the coumarin resistance of GyrB.

Implications for mechanisms of quinolone resistance. Mutations at positions 80 and 116 of GrlA probably have different mechanisms of quinolone resistance. In the crystal structure of a fragment of the GyrA subunit from E. coli (10), Ser83, which is homologous to Ser80 of GrlA, and other previously described quinolone resistance mutations in GyrA, which have not been associated with alterations in coumarin susceptibility, are clustered on a negatively charged surface that bridges the upper (or head) interface of the two subunit monomers. Modeling suggests that this surface is a principal site of binding of the "gate" strand of DNA, which is broken and resealed by enzyme action. The model further predicts that DNA distortion is required for interaction of the active-site tyrosine residues (Tyr122 in E. coli GyrA and Tyr119 in S. aureus GrlA) with the target phosphoryl residues of DNA during initial DNA cleavage. The residues of the quinolone resistance-determining region (QRDR) are localized in the area where this distortion is predicted to occur, suggesting that the binding of quinolones to the gyrase-DNA complex may occur in this region. The finding that complexes of DNA and gyrase reconstituted with a resistant GyrA (Ser83Trp) subunit exhibit substantially reduced levels of quinolone binding (16) is consistent with this model. The catalytic activity of topoisomerase IV reconstituted with GrlA (Ser80Tyr) has also been shown to be similar to that of the wild-type enzyme (2). Thus, the Ser80Phe mutation in GrlA, which is homologous to Ser83 of GyrA, also likely confers resistance by causing a reduction in the level of binding of quinolones to the enzyme-DNA complex and is predicted to have normal or near normal catalytic efficiency.