Cloning and nucleotide sequence of the DNA gyrase gyrA gene from Serratia marcescens and characterization of mutations in gyrA of quinolone-resistant clinical isolates

Abstract

The sequence of the DNA gyrase gyrA gene of Serratia marcescens ATCC 14756 was determined. An open reading frame of 2,640 nucleotides coding for a polypeptide with a calculated molecular mass of 97,460 was found, and its sequence complemented the sequence of an Escherichia coli gyrA temperature-sensitive mutation. Analysis of the PCR products of the quinolone resistance-determining regions of gyrA genes from six quinolone-resistant clinical isolates revealed a single amino acid substitution, Ser-83 to Arg or Asp-87 to Tyr, in all six mutants, suggesting that a mutational alteration in gyrA is a common mechanism of quinolone resistance in S. marcescens.

FIG. 1

(A) Genomic restriction map for the gyrA locus in S. marcescens ATCC 14756. The gyrA gene is indicated by a thick line. (B) Detailed restriction map and sequencing strategy for the gyrA gene. The open reading frame (ORF) of the S. marcescens gyrA gene is shown by a thick line. The 1.0-kb SalI fragment in pSC6-1 and the 3.2-kb SalI fragment in pSC6-2 are indicated by pSC6-1 and pSC6-2, respectively. A strategy for nucleotide sequencing by the dideoxy method (32) is shown under pSC6-1 and pSC6-2. Three to five independent sequencing reactions were done for each arrow.