DAO 67:259-266 (2005)  -  doi:10.3354/dao067259

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda

Soon Bum Shin1, Min Ho Yoo1, Joon Bum Jeong1, Young Min Kim2,Joon-Ki Chung1, Min Do Huh1, Jack L. Komisar3, Hyun Do Jeong1,*

1Department of Aquatic Life Medicine, Pukyong National University, 599-1 Dae Yeon Dong, Nam Ku, Busan 608-737,South Korea
2Department of Biology, Yonsei University, Seoul 120-749, South Korea
3Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring,Maryland 20910-7500, USA
*Corresponding author. Email:

ABSTRACT: Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83→Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83→Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42°C.

KEY WORDS: Edwardsiella tarda · gyrA · Quinolone · Resistance · Mutation

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