Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones.
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 approximately 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.
Cullen
M E
ME
Department of Biochemistry, St. George's Hospital Medical School, University of London, United Kingdom.
Wyke
A W
AW
Kuroda
R
R
Fisher
L M
LM
D042462:4-Quinolones; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004269:DNA, Bacterial / Q000096:biosynthesis; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data
D042462:4-Quinolones; D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D004250:DNA Topoisomerases, Type II