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.

DNA gyrase, a type II DNA topoisomerase, is an enzyme capable of transforming relaxed closed circular DNA into a negatively supercoiled form (5). The enzyme contains two protein subunits, subunits A and B. The A subunit, coded for by the gyrA gene, is responsible for introducing and rejoining double-stranded breaks in DNA, while the B subunit, coded for by gyrB, mediates energy transduction and ATP hydrolysis during the topological transformation of DNA (28, 40).

Fluoroquinolones are potent broad-spectrum antibacterial agents which inhibit DNA gyrase activity (44). Mutations conferring high-level quinolone resistance have been mapped to a small region of the 5′ end of the gyrA gene, which has been designated the quinolone resistance-determining region (QRDR). Mutations in the Ser-83 and Asp-87 codons in particular have been found in the majority of quinolone-resistant clinical isolates of Escherichia coli (26, 38). Similar mutations were also identified in quinolone-resistant strains from a diverse group of bacteria (1, 6, 7, 12, 14, 19, 21, 22, 24, 35, 39, 41, 47).

Serratia marcescens is recognized as a frequent cause of extraintestinal human infections ranging from simple cystitis to life-threatening bloodstream and central nervous system infections (2, 15, 43). Several strains of S. marcescens causing nosocomial infections were found to acquire resistance to fluoroquinolones at higher frequencies than those for the E. coli strains (4, 30, 31, 42), but nothing has been reported about the gyrA gene structure, the mutation in gyrA responsible for quinolone resistance, or the genetic basis of decreased susceptibility to quinolones in this bacterium. We therefore carried out this study to characterize the wild-type gyrA gene of S. marcescens and also to elucidate the mutations in the gyrA genes of several quinolone-resistant clinical isolates.

Cloning and sequencing of gyrA gene of S. marcescens ATCC 14756.

The type strain of S. marcescens, ATCC 14756, was purchased from the American Type Culture Collection, Rockville, Md. The genomic restriction map for the gyrA gene in ATCC 14756 was determined by Southern hybridization with a randomly digoxigenin-labeled 582-bp SacI-SmaI fragment of pDH24 (9) and a 2.8-kb HindIII fragment of pMK90 (18) corresponding to the 5′ and 3′ ends of E. coli gyrA, respectively (Fig. 1A). In addition, a 648-bp DNA probe which is specific for the 5′ end of the S. marcescens gyrA gene comprising the putative QRDR was generated by PCR and was also used in the hybridization studies. To generate the 648-bp probe, two primers, primers TACACCGGTCAACATTGAGG and TTAATGATTGCCGCCGTCGG, the sequences of which are identical to the nucleotide sequence from positions +24 to +43 of the E. coli gyrase gyrA gene and complementary to positions +652 to +671 of the E. coli gyrA gene, respectively, (26), were selected on the basis of the close genetic relatedness of Serratia spp. to E. coli. Preliminary data showed that the nucleotide sequence of the 648-bp fragment was highly homologous to that of the 5′ end region of the E. coli gyrA gene.

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.

To construct a subgenomic library, genomic DNA digested with BamHI was ligated into bacteriophage lambda EMBL3 and was packaged by using Gigapack III gold packaging extracts (Stratagene Cloning System, La Jolla, Calif.). Replicate plaques were screened with the PCR-amplified QRDR-containing 648-bp probe. Positive clones all contained identical 14-kb BamHI inserts (Fig. 1A). When E. coli KNK453 gyrA43(Ts) (13) was transformed with pBluescript II KS(+) containing a 3.8-kb BamHI-EcoRI fragment of the gyrA gene from a positive clone, bacteriophage lambda clone 6, the bacterium exhibited full growth at the nonpermissive temperature of 42°C. E. coli KNK453 transformed with pBluescript II KS(+) containing no insert DNA grew well at 30°C but did not grow at 42°C, indicating that the complete gyrA gene is present in the 3.8-kb insert DNA and that an S. marcescens GyrA-E. coli GyrB holoenzyme complex is functionally active.

On the basis of the detailed restriction map of the bacteriophage lambda clone 6 constructed by Southern hybridization of the cloned DNA fragment with the three gyrA probes (Fig. 1B), two subclones, pSC6-1 containing a 1.0-kb SalI fragment and pSC6-2 containing a 3.2-kb SalI fragment (Fig. 1B), were constructed in pBluescript II KS(+) and were used for sequence analysis. An open reading frame of 2,640 nucleotides coding for a polypeptide of 880 amino acids with a calculated molecular weight of 97,460 was found. The G+C content of the S. marcescens gyrA gene was 60%, which is in agreement with the overall G+C content (57.5 to 60%) of this bacterium (8). The derived amino acid sequence of the GyrA protein showed 85.8, 85.7, 89, and 75.5% overall identities with the E. coli (34), Klebsiella pneumoniae (3), Erwinia carotovora (29), and Aeromonas salmonicida (25) GyrA proteins, respectively. As expected, a high degree of sequence homology was found in the N-terminal region of the protein. The catalytic Tyr-122 involved in DNA breakage and reunion was also present, as in E. coli and other bacteria (9). Amino acid residues from positions 67 to 106 (ARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQ), which are known to cause quinolone resistance in mutated E. coli GyrA (46), were perfectly conserved in S. marcescens GyrA.

A potential Shine-Dalgarno sequence (GAGGG) was present 12 bp upstream of the ATG translation initiation codon. A putative −10 promoter sequence (TATAGT) similar to the proposed −10 E. coli consensus sequence (TATAAT) was found 46 bp upstream of the ribosome-binding site. The −35 region (TTGACA) was not apparent, but a sequence (ATTTTCC) similar to the conserved sequence (GTTTACC) found in the E. coli (34), K. pneumoniae (3), and Bacillus subtilis (20) gyrA promoter regions was present 26 bp upstream of the −10 region. The sequence is also similar to the sequences GTTTGCC, GTTTCCC, and GTTTAAG found in the gyrA promoter regions of Pseudomonas aeruginosa (14), E. carotovora (29), and A. salmonicida (25), respectively. The formation of a cruciform structure is theoretically possible in the 5-bp inverted repeated sequences AAAGC and GCTTT, located 12 and 5 bp upstream of the −10 region, respectively, as has been predicted to occur in the corresponding region of the K. pneumoniae (3), E. carotovora (29), and A. salmonicida (25) gyrA promoters. The presence of an inverted repeated sequence indicates that the supercoiling-dependent regulation of gyrA transcription may work in S. marcescens, as suggested for E. coli (10, 17). Located 46 bp downstream of the ochre stop codon is another 13-bp inverted repeat with 4 bases in between; this inverted repeat may act as a transcription terminator sequence. The modified relaxation-stimulated transcription sequence (TTGTGATATAGTTTTACACC) which includes the nucleotides surrounding the −10 region (underlined) was also found 38 bp upstream of the ribosome-binding site, as in the gyrA genes of E. coli (33), K. pneumoniae (3), and E. carotovora (29).

In this experiment, approximately 600 bp upstream from the coding region of S. marcescens gyrA gene was sequenced. Analysis of this region, however, failed to identify an open reading frame greater than 74 amino acids long and revealed no homology with the E. coli gyrB sequence. The arrangement of DNA gyrase genes on the S. marcescens chromosome thus appears to be noncontiguous, which is common in many gram-negative bacteria (3, 14, 25, 27, 29, 34, 41, 45).

Characterization of gyrA mutations in quinolone-resistant clinical isolates of S. marcescens.

The susceptibilities of seven uropathogenic clinical isolates of S. marcescens isolated from patients in Dong Suwon Hospital, Suwon, Korea (Table 1), to several quinolones were tested by the agar dilution method established by the National Committee for Clinical Laboratory Standards (23).

TABLE 1.

Susceptibility to quinolones and mutations in the QRDR of gyrA genes of clinical S. marcescens isolates

S. marcescens strain	MIC (μg/ml)a				Mutationsb
	NA	CIP	OFL	NOR	Amino acid substitutions	Silents
ATCC 14756	16	0.1	0.2	0.4	None (wild type)	None
DSWH 101	≥1,024	3.2	25.6	6.4	Asp-87 to Tyr (GAC to TAC)	Pro-79 (CCG to CCA), Gly-105 (GGT to GGC), Gly-110 (GGT to GGC), Ala-117 (GCG to GCC)
DSWH 102	16	0.1	0.2	0.4	None	Gly-110 (GGT to GGC), Ala-117 (GCG to GCC)
DSWH 103	≥1,024	1.6	6.4	3.2	Ser-83 to Arg (AGC to AGA)	Ala-117 (GCG to GCC)
DSWH 104	≥1,024	3.2	25.6	6.4	Asp-87 to Tyr (GAC to TAC)	Pro-79 (CCG to CCA), Gly-105 (GGT to GGC), Gly-110 (GGT to GGC), Ala-117 (GCG to GCC)
DSWH 105	≥1,024	12.8	51.2	25.6	Ser-83 to Arg (AGC to AGA)	Gly-110 (GGT to GGC), Ala-117 (GCG to GCC),
						Lys-129 (AAG to AAA)
DSWH 106	≥1,024	1.6	6.4	3.2	Ser-83 to Arg (AGC to AGA)	Ala-117 (GCG to GCC)
DSWH 107	≥1,024	1.6	3.2	3.2	Ser-83 to Arg (AGC to AGA)	Ala-117 (GCG to GCC)

^aMICs were determined by the agar dilution technique (23) as described in the text. All quinolones were purchased from Sigma Chemical Company, St. Louis, Mo. Abbreviations: NA, nalidixic acid; CIP, ciprofloxacin; OFL, ofloxacin; NOR, norfloxacin. 

^bMutations detected in QRDR. 

Two-step nested PCR (11) and direct DNA sequencing were carried out to analyze mutations in the gyrA genes of the clinical isolates. For the first step, a 866-bp region of the gyrA gene was amplified by using two 25-mer oligonucleotide primers, GGGCTTGTCTGGCTCCTTGTTCTCG and GGGAAGTCCGGCCCCGGGATGTGTT, the sequences of which are identical to the sequences at nucleotide positions 212 to 187 bp upstream of the ATG translation start codon and complementary to the sequence at positions 626 to 651 bp downstream of the ATG codon, respectively. A 245-bp gyrA fragment was then amplified from the 866-bp PCR product by using two 17-mer primers, TTGGGTAACGACTGGAA and GCCAACAGTTCGTGAGC, the sequences of which are identical to the sequences at nucleotide positions 160 to 176 bp and 388 to 404 bp downstream of the translation initiation codon, respectively. After removal of primers and free nucleotides, PCR products were directly sequenced by the dideoxy method (32).

Table 1 presents the nucleotide and amino acid changes in the QRDR of the PCR-amplified fragments of the gyrA genes from clinical isolates. In six of the seven isolates, a nucleotide change leading to an amino acid substitution was observed. No amino acid substitution was found in strain DSWH 102. Among the six mutants, four (DSWH 103, DSWH 105, DSWH 106, and DSWH 107) were found to have the same amino acid substitution at position 83. These four mutants were characterized by a C-to-A mutation at nucleotide position 249, resulting in a Ser-to-Arg substitution. Two other mutants, DSWH 101 and DSWH 104, were characterized by a G-to-T mutation at codon 87, leading to a Asp-to-Tyr substitution. All other nucleotide changes detected in the seven isolates were silent. A silent mutation at the wobble position of codon 117 (GCG to GCC) was observed in all isolates. The substitution of Ser-83 with Arg has also been found in quinolone-resistant clinical isolates of Enterococcus faecalis (12, 36) and A. salmonicida (24). The Ser-83-to-Arg substitution might lead to high-level quinolone resistance in the S. marcescens GyrA protein by introducing a bulky amino acid residue into the protein and also by decreasing the hydrogen-bonding capacity between amino acid residues. The Asp-87-to-Tyr substitution, which results in the exchange of a negatively charged residue for a larger polar amino acid, has also been observed in quinolone-resistant P. aeruginosa (14), E. coli (38), and Salmonella saint-paul (7).

In most previous reports on the resistance of clinical isolates or spontaneous mutants of S. marcescens to quinolones, the decreased susceptibilities have been attributed to alterations in outer membrane proteins, resulting in the decreased permeation of quinolones (30, 37, 42). Although mutations resulting in quinolone resistance have not yet been described at the nucleotide level for the S. marcescens gyrA gene, it has been demonstrated in reconstitution experiments with mutant GyrA and wild-type GyrB proteins that an alteration in the GyrA protein is sufficient to render the DNA gyrase resistant to quinolones in vitro (4, 16). This, together with the present observation that an amino acid substitution at position 83 or 87 was present in all clinical isolates for which the ciprofloxacin MIC was high (Table 1), strongly suggests that DNA gyrase is the primary target of quinolones in S. marcescens and that these two residues of the GyrA protein are especially important in the formation of the quinolone-gyrase-DNA complex. The possibility that other mutations in the gyrA gene or that other mechanisms of resistance (e.g., changes in outer membrane proteins and mutations in gyrB or parC) may modulate the ultimate MICs cannot be excluded, however, since we have examined only a small portion of the gyrA gene sequence from clinical isolates. The unique MIC pattern for DSWH 105 among the four Ser-83-to-Arg mutants supports this possibility (Table 1).

Nucleotide sequence accession number.

The nucleotide sequence of the gyrA gene of S. marcescens ATCC 14756 has been assigned accession number U56906 in the GenBank/EMBL database.