Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli

Abstract

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, ...

Analysis of the NH2‐Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone‐Resistance

“…Resistance, due to substitutions of Ser83, is attributed to the loss of the hydroxyl group of Ser and the bulkiness and hydrophobicity of the Leu and Trp residues (6). The bulkier hydrophobic group results in a physical obstruction for quinolones to enter into 'the quinolone pocket' proposed by Yoshida et al (17).…”

Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast

“…This motif corresponds to a region of topoisomerase II that shares homology with the DNA gyrase GyrB protein [48], and which in the crystal structure of an S. cere isiae topoisomerase II fragment lies above the DNA breakage-reunion domain [10]. Mutation of the GyrB EGDSA motif to EGNSA has been reported to confer resistance to quinolone anti-bacterial agents in E. coli [49]. Thus it appears that the conserved aspartate in the EGDSA structure can be replaced with other residues without complete impairment of enzyme function.…”

Restoration of antibiotic susceptibility in fluoroquinolone-resistant Escherichia coli by targeting acrB with antisense phosphorothioate oligonucleotide encapsulated in novel anion liposome

“…Furthermore, in contrast to the steric-blocker oligonucleotides, RNase H-dependent oligonucleotides, such as phosphorothioate oligonucleotides, can inhibit protein expression when targeted to widely separated areas in the coding region. [18][19][20] Mechanisms in fluoroquinolone resistance of E. coli fall into two principal categories: alterations in drug targets (for example DNA gyrase or topoisomerase IV) [21][22][23][24][25][26] and decreased cellular accumulation of quinolones. The latter involves the major and constitutively expressed multi-drug efflux pump, AcrAB-TolC.…”