Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori.
PCR was used to amplify a 238-bp region from Helicobacter pylori which corresponded to the quinolone resistance-determining region in Escherichia coli. The gyrA gene of H. pylori was cloned and sequenced. An open reading frame of 2,478 nucleotides coded for a polypeptide of 826 amino acids with a calculated molecular mass of 92,508 Da. The amino acid sequence showed an overall 52% identity with other bacterial gyrA genes but was most closely related to the gyrA subunit of Campylobacter jejuni (76.5% identity). Sequencing of the amplification product from ciprofloxacin-resistant mutants of H. pylori revealed four classes of mutations with substitutions at amino acid 87 (Asn-->Lys), amino acid 88 (Ala-->Val), and amino acid 91 (Asp-->Gly, -->Asn, or -->Tyr) and a double substitution at amino acids 91 and 97 (Ala-->Val). Ciprofloxacin-susceptible strains of H. pylori could be transformed to ciprofloxacin resistance by using the amplified fragment from resistant strains as donor DNA. Of the 11 ciprofloxacin-resistant mutants examined, only one did not have an alteration within the quinolone resistance-determining region, suggesting that, in H. pylori, resistance to quinolones is primarily a result of alterations in gyrA.
Moore
R A
RA
Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada.
Beckthold
B
B
Wong
S
S
Kureishi
A
A
Bryan
L E
LE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002939:Ciprofloxacin / Q000494:pharmacology*; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000187:drug effects*; D004352:Drug Resistance, Microbial; D005784:Gene Amplification; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000187:drug effects; D016133:Polymerase Chain Reaction
D002939:Ciprofloxacin; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II