Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones

Abstract

Nalidixic acid, enoxacin, and other antibacterial 4-quinolones inhibit DNA gyrase activity by interrupting DNA breakage and reunion by A subunits of the A2B2 gyrase complex. Despite their clinical importance, the mode of quinolone action and mechanisms of resistance are poorly understood at the molecular level. Using a DNA fragment enrichment procedure, we isolated the gyrA gene from a uropathogenic Escherichia coli strain that encodes a gyrase A protein cross-resistant to a variety of quinolones. When complemented with gyrase B subunit, the purified A protein reconstituted DNA supercoiling activity 100-fold more resistant to inhibition by enoxacin than the susceptible enzyme and failed to mediate quinolone-dependent DNA cleavage. Nucleotide sequence analysis revealed that the gene differed at 58 nucleotide positions compared with the K-12 gyrA sequence. The 875-amino-acid residue-resistant gyrase A protein differed at three positions from its wild-type E. coli K-12 counterpart: tryptophan, glutamate, and serine replaced serine, aspartate, and alanine residues at positions 83, 678, and 828, respectively. By genetic analysis of chimeric gyrA genes in a gyrA(Ts) background, we showed that the Ser-83-*Trp mutation in the gyrase A protein was solely responsible for high-level bacterial resistance to nalidixic acid and fluoroquinolones.Bacterial DNA is maintained in a negatively supercoiled state by DNA gyrase, an ATP-dependent type II DNA topoisomerase (10). Gyrase is essential for cell viability, being implicated in a range of DNA transactions, including DNA replication and recombination, and in the control of gene expression (8,35). Gyrase catalyzes DNA supercoiling by an interesting and unusual mechanism. It passes a duplex DNA segment through a transient double-stranded DNA break made within a 120-to 150-base-pair (bp) loop of DNA wrapped on the surface of the tetrameric A2B2 gyrase complex. DNA strand passage is the salient mechanistic feature that allows DNA supercoiling and ATP-independent DNA relaxation by gyrase and also accounts for its catenation or decatenation and DNA unknotting activities (3,16,18).Antibacterial quinolones such as oxolinic acid inhibit DNA supercoiling by gyrase in vitro and rapidly arrest DNA replication in vivo (2, 9, 12, 28). Addition of detergent to gyrase-DNA complexes formed in the presence of oxolinic acid results in site-specific double-stranded DNA breakage and covalent attachment of the gyrase A subunits to each 5'-phosphate end via tyrosine 7,9,15,22,31). Thus, the A subunits appear to promote DNA breakage and reunion during catalysis, a process interrupted by quinolone inhibitors. In contrast, the B subunits bind ATP and are the locus of action of coumarin antibiotics such as novobiocin (11,20,30). The A and B subunits can be individually purified but must be combined to generate the topoisomerase activities of gyrase (14, 19

Transferable high-level trimethoprim resistance among isolates ofEscherichia colifrom urinary tract infections in Ontario, Canada

“…Two of four isolates resistant to both gentamicin and tobramycin and one of the two resistant to gentamicin but susceptible to tobramycin conjugally transferred their resistances along with TMP to E. coli K-12. Fluoroquinolone resistance was not transferred, these resistances have been reported to be located on the chromosome [20]. Nitrofurantoin resistance was not transferred to E. coli K-12.…”

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

“…The majority of the previously report- ed quinolone-resistance mutations of the gyrA gene have resulted in the conversion of Ser83 to either Leu or Trp (1). These mutants were observed in both clinical isolates (3,13) and laboratory mutants (16,18). 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 gyrA gene that encodes GyrA was cloned from both wild and quinolone-resistant E. coli strains. Nucleotide sequence analysis revealed the Ala67Ser, Gly81Cys, Ser83Leu, Ser83Trp, Ala84Pro, Asp87Asn and Gln106-His mutations in quinolone-resistant strains (3,13,16,18). These mutations are clustered within a narrow region between nucleotide number 199 (Ala67) and 318 (G1n106).…”

“…Plasmid pAW012 allowing expression of E. coli gyrA (12) (2,3,18). KNK453 is viable at 37 C but not at 42 C because that strain carries the mutant gyrA (temperature sensitive) allele.…”

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

“…This enzyme contains a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) in E. coli. Gyrase A catalyzes chromosomal DNA supercoiling during replication (11), is the molecular target of quinolone antimicrobials (12), and can mediate quinolone resistance (13)(14)(15). Eukaryotes also express essential RNase P subunits (10) and gyrase (16) enzymes (the later a target for antineoplastic agents), but they are quite distinct from their bacterial homologs in RNA and protein sequence (17) as well as in some aspects of enzymatic reaction details (10,16).…”