Nucleotide sequence analysis of the gyrA genes of 10 spontaneous quinolone-resistant gyrA mutants of Escherichia coli KL16, including four mutants examined previously, disclosed that quinolone resistance was caused by a point mutation within the region between amino acids 67 and 106, especially in the vicinity of amino acid 83, of the GyrA protein.Quinolones are considered to exert antibacterial activity by inhibiting DNA gyrase (EC 5.99.1.3), which catalyzes topological changes of DNA (4, 11). DNA gyrase of Escherichia coli consists of subunits A and B, which are the products of the gyrA and gyrB genes, respectively. Mutations in either gene can cause quinolone resistance (4,(15)(16)(17) were determined by dideoxy-chain termination (9) with phage M13mpl8 and M13mpl9 vectors. Table 1 shows the sites and types of mutations and the levels of resistance to quinolones of 10 quinolone-resistant gyrA mutants of E. coli KL16. Four mutants (N-51, P-18, P-10, and N-89) were analyzed previously (17). All 10 point mutations were considered to be solely responsible for quinolone resistance, because replacement of the 0.6-kilobase Sacl-SmaI fragment containing the mutations by the corresponding fragment from wild-type gyrA gene resulted in complete loss of quinolone resistance (data not shown). Sequencing of the 0.6-kilobase SacI-SmaI fragments of the mutant gyrA genes revealed that these mutations were located within a relatively small region (amino acids 67 through 106) of the A subunit, which we call a quinolone resistance-determining region. There were no other mutations in all of the sequenced fragments. Eight of the 10 mutations were in a limited area (amino acids 81 through 87) of the region; surprisingly, five mutations were situated at the same site of amino acid 83. The levels of resistance to quinolones seemed to be related to the mutation sites, because quinolone MICs were high in the decreasing order of MICs for mutants with mutations at amino acids 83, 87, 81, 84, 67, and 106. This result suggests the importance of an area around amino acid 83 of the gyrase A subunit for determining quinolone resistance.Amino acid changes detected at amino acid 83 were Ser to

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The The increase in methicillin-resistant Staphylococcus aureus is a serious problem because only a few effective agents are clinically available. Some quinolones have been used for the treatment of methicillin-resistant S. aureus infections, but the emergence of quinolone resistance has been reported elsewhere (32). Unlike the mechanism underlying the quinolone resistance of gram-negative bacteria such as Escherichia coli (2,7,9,11,12,15,27,31,(36)(37)(38)(39) and Pseudomonas aeruginosa (4,13,16,29,30,36,40)

Thirteen spontaneous quinolone-resistant gyrB mutants of Escherichia coli KL16, including two that were examined previously, were divided into two types according to their quinolone resistance patterns. Type 1 mutants were resistant to all the quinolones tested, while type 2 mutants were resistant to acidic quinolones and were hypersusceptible to amphoteric quinolones. Nucleotide sequence analysis disclosed that all nine type 1 mutants had a point mutation from aspartic acid to asparagine at amino acid 426 and that all four type 2 mutants had a point mutation from lysine to glutamic acid at amino acid 447. Quinolones are a group of antibacterial agents whose target is DNA gyrase (EC 5.99.1.3), an enzyme that catalyzes topological changes of DNA (4). The DNA gyrase of Escherichia coli consists of two A and two B subunits, which are the products of the gyrA (48 min) and gyrB (83 min) genes, respectively (3,7,11,21,29). Mutations in the gyrA gene are as frequent as those in the gyrB gene in spontaneous quinolone-resistant mutants of E. coli KL16, although the majority of quinolone-resistant clinical E. coli isolates have gyrA mutations (23). In the gyrA gene, quinolone resistance is caused by a point mutation within the relatively narrow region of amino acids 67 to 106, which is called the quinolone resistance-determining region (34, 35). In the gyrB gene, two quinolone resistance-determining sites (amino acids 426 and 447) have been found (32,33). To obtain more information on the region responsible for quinolone resistance in the gyrB gene, 11 additional quinolone-resistant gyrB mutants of E. coli KL16 were analyzed.MATERIALS AND METHODS Strains. Quinolone-resistant mutants of E. coli KL16 were isolated by plating the organism on LB agar (18) containing nalidixic acid or enoxacin at four times the MIC, and gyrB mutants were identified by transformation with the wild-type gyrB gene as described previously (23).Reagents, plasmids, and phages. Nalidixic acid (14) Cloning and sequencing of the E. coli gyrB genes. HindIII DNA fragments of about 13 kb in size containing the gyrB gene were cloned from quinolone-resistant gyrB mutants of E. coli KL16 as described previously (33). Nucleotide sequences were determined by the dideoxy-chain termination method (17) by using phage M13mpl8 and M13mpl9 vectors. RESULTS AND DISCUSSIONThe levels of resistance or hypersusceptibility (the increase or decrease in MIC compared with that for E. coli KL16) to various quinolones of 13 quinolone-resistant gyrB mutants of E. coli KL16 are given in Table 1. The MICs of some quinolones for N-24 and N-31 were not identical to those reported previously (23, 32) but were within experimental fluctuations. All the mutants could be divided into two types with respect to their quinolone resistance. Type 1 mutants were resistant to all the quinolones tested, while type 2 mutants were resistant to acidic quinolones, such as nalidixic acid, oxolinic acid, cinoxacin, piromidic acid, and flumequine but were hypersusceptible to amphoteric quinolones, ...

The proportion of DNA gyrase mutants among quinolone-resistant strains of Pseudomonas aeruginosa was examined by introducing the cloned wild-type Escherichia coli gyrA and gyrB genes. Of 101 spontaneous mutants of P. aeruginosa PA0505, 33 (33%) were found to have gyrA mutations. Among 17 clinical isolates, 12 (71%) had gyrA mutations and 1 (6%) had a gyrB mutation.Quinolone-resistant organisms are increasing as quinolone derivatives are used clinically for various kinds of infections. Quinolone resistance is caused by chromosomal mutations affecting permeability (1, 2, 6-9, 15, 16, 18) or drug susceptibility of DNA gyrase (EC 5.99.1.3) (3-7, 10, 14-20). In Escherichia coli, it has been revealed by transformation with cloned wild-type gyrA and gyrB genes that mutations in the gyrA and gyrB genes are equally frequent in spontaneous quinolone-resistant mutants, whereas mutations in the former gene predominate in quinolone-resistant clinical isolates (14). In Pseudomonas aeruginosa, what kind(s) of mutation predominates is not yet known. Since we found that the cloned wild-type E. coli gyrA gene could make a quinolone-resistant nalA mutant of P. aeruginosa quinolone susceptible just as with a quinolone-resistant gyrA mutant of E. coli, the proportion of gyr mutations in quinolone-resistant P. aeruginosa was examined by transformation with cloned E. coli gyrA and gyrB genes.P. aeruginosa PA0505 (15), its naLA mutant PAO51S (15), and its transport mutant, PA06002 (nalB) (15) and plasmid pME294, which multiplies in P. aeruginosa and was constructed by Y. Itoh (11), were used. They were kindly supplied by S. Iyobe. Spontaneous quinolone-resistant mutants of P. aeruginosa PAO505 were isolated at mutation frequencies of about 10-9 on LB agar (13) containing nalidixic acid at 8 or 16 times the MIC (400 or 800 ,ug/ml, respectively) or enoxacin at 8 or 12 times the MIC (6.25 or 9.4 ,ug/ml, respectively). Quinolone-resistant clinical isolates were obtained from patients with urinary or respiratory tract infections. Plasmid pPAW207 carrying the wild-type E. coli gyrA gene was constructed by inserting a 4.5-kilobase filledin StuI-Spll fragment containing the gyrA gene from pAW011 (20) into the SmaI site of pME294 (11). Plasmid pPBW801 carrying the wild-type E. coli gyrB gene was made by inserting a 3.6-kilobase SspI-BalI fraginent containing the gyrB gene from pJBll (19) into the same site of pME294. These plasmids have a carbenicillin resistance gene as a selective marker. The quinolones used were synthesized in our laboratories. Carbenicillin, novobiocin, and chloramphenicol were purchased from Sigma Chemical Co., St.Louis, Mo., and gentamicin was purchased from Shionogi & Co., Osaka, Japan. Transformation was done by the CaCl2 method (12), and transformants were selected on LB agar containing carbenicillin at 200 jig/ml for carbenicillin-suscep-* Corresponding author. tible strains and at 3,200 ,ug/ml for carbenicillin-resistant strains.The nalA mutant PAO51S was 16-fold or more resistant than its parent, PAOS05, to nalidix...

The mechanism of action of quinolones was investigated by use of various DNA gyrases reconstituted from wild-type and mutant GyrA and GyrB proteins of Escherichia coli. The quinolone sensitivities of the DNA supercoiling activity of the gyrases were generally parallel to the quinolone susceptibilities of strains having the corresponding enzymes and depended on gyrase subunits but not on substrate DNA.[3lH]Enoxacin did not bind to gyrase alone or DNA alone but bound to gyrase-DNA complexes when measured by a gel filtration method.There appeared to be two enoxacin binding phases, at low and high enoxacin concentrations, for the wild-type gyrase-DNA and type 2 GyrB (Lys-447 to Glu) mutant gyrase-DNA complexes but only one enoxacin binding phase at the concentrations used for the GyrA (Ser-83 to Leu) mutant gyrase-DNA and type 1 GyrB (Asp-426 to Asn) mutant gyrase-DNA complexes. New enoxacin binding sites appeared in the presence of enoxacin, and the enoxacin binding affinities for the sites, especially at low enoxacin concentrations, near the MICs for the strains having the corresponding gyrases, correlated well with the enoxacin sensitivities of the gyrases and the MICs. From the results obtained, we propose a quinolone pocket model as the mechanism of action of quinolones, in which quinolones exert their action through binding to a gyrase-DNA complex and the quinolone binding affinities for the complex are determined by both GyrA and GyrB subunits in concert.

It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1, a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) was still functional in the cytochrome c1-deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171-->Asn170Asn171 or Asp170Asp171-->Asn170Lys171) made the cytochrome c1-deficient cells respiration-competent. On the other hand, mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) or altered charge distribution in the second region (Asp170Asp171-->Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1 but also the QCR6 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The His-44 and Met-164 residues of yeast cytochrome c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome c reductase activity in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.