Mutation in the DNA Gyrase A Gene of Escherichia coli That Expands the Quinolone Resistance-Determining Region

Abstract

In three Escherichia coli mutants, a change (Ala-51 to Val) in the gyrase A protein outside the standard quinolone resistance-determining region (QRDR) lowered the level of quinolone susceptibility more than changes at amino acids 67, 82, 84, and 106 did. Revision of the QRDR to include amino acid 51 is indicated.

The quinolones are antibacterial agents that act by forming ternary complexes with DNA gyrase and DNA topoisomerase IV on chromosomal DNA. Resistance to the compounds is generally associated with amino acid substitutions in portions of the GyrA (gyrase) and ParC (topoisomerase IV) proteins called the quinolone resistance-determining regions (QRDRs) (20). In Escherichia coli, the GyrA QRDR spans amino acids 67 to 106, with alteration at positions 83 and 87 often associated with clinical resistance (19). A similar association with resistance has been observed for a variety of pathogens (1, 6, 12, 16, 17), suggesting that resistance is due to altered drug targets. The use of fluoroquinolones that mitigate the protective effects of alterations at positions 83 and 87 (7, 21) may cause other alleles to assume a more important role in reducing the level of susceptibility (4). To help define additional sites that may contribute to resistance, we examined three nalidixic acid-resistant mutants of E. coli that are also thermotolerant (2, 3).

Independent, nalidixic acid-resistant (Nal^r) strains of E. coli CGSC 6353 (3) were obtained by selecting for growth on Luria-Bertani (LB) agar (9) containing 20 μg of nalidixic acid per ml, and members of the thermotolerant (T/r⁺) subset were identified by growth on LB agar plates at 48°C. These strains were designated MF1, MF3, and MF4-1; a Nal^r strain that lacked thermotolerance was designated MF13. Nal^r was mapped by P1-mediated transduction (18), DNA was isolated by phenol extraction, PCR was used to amplify regions of the gyrA gene, and nucleotide sequences in amplified regions were determined by automated sequencing. The protective effect of the gyrA mutations was compared by determining the fluoroquinolone concentration required to inhibit colony formation by 99% (MIC₉₉) rather than by standard MIC determinations to focus more sharply on bacteriostatic activity. For this measurement cells grown to the stationary phase in LB medium were diluted and applied to quinolone-containing agar plates; the colonies were counted after incubation at 37°C for 1 day. Preliminary determinations with twofold dilutions of the fluoroquinolone provided an approximate value for the MIC₉₉; a second measurement, plus a replicate, then used linear drug concentration increments that were 10 to 20% of the MIC₉₉. The numbers of colonies recovered were plotted against the drug concentration to determine the MIC₉₉ by interpolation.

P1-mediated transduction showed that a gyrA mutation was associated with nalidixic acid resistance. In this experiment a zfa-3145::Tn10 Kan^r marker, which is 70% cotransducible with gyrA, was first transferred by transduction from strain CAG 12183 (Nal^s Kan^r) (14) into strain MF4-1 (Nal^r T/r⁺). About 30% of the Kan^r transductants retained the Nal^r phenotype, indicating that a mutation at or near gyrA can confer nalidixic acid resistance. Then, the Nal^r marker was transferred into wild-type cells. Two Kan^r Nal^r transductants (KD1719 and KD1720) from the initial transduction were used to prepare phage lysates that were used to infect the parental Nal^s strain (CGSC 6353) and two other wild-type strains, DM4100 (15) and C600 (5). Cotransduction frequencies between Kan^r and Nal^r were 70, 80, and 70% for the three recipient strains, respectively. Thus, a mutation at or near gyrA was sufficient to confer nalidixic acid resistance.

The change associated with Nal^r in the gyrA QRDR was determined by nucleotide sequence analysis following PCR with primers 1037 and 1038 (Table 1). As shown in Table 2, the parental strain (CGSC 6353) had a predicted amino acid sequence identical to a sequence found in GenBank (accession no. X06744). In contrast, Nal^r T/r⁺ mutants (strains MF4-1, MF1, and MF3) contained a change that altered amino acid 51 from alanine to valine. The Nal^r mutant that was not thermotolerant, strain MF-13, had a 5′ base change (G to T) in codon 87 expected to reduce the level of quinolone susceptibility by substituting tyrosine for aspartic acid (11).

TABLE 1.

Oligonucleotides used for PCR and sequence determination

Oligonucleotide no.	Nucleotide sequence	Position in gyrA
1037	5′-AGTGGATCCGTAATTGGCAAG ACAAACGAG-3′	−110 to −134
1038	5′-CGGCCATCAGTTCATGGGCA-3′	390 to 409
1042	5′-TTGAATTCAAACAAGGGAGAT AGCTCC-3′	2651 to 2674
1043	5′-TACGGAAATCCGTCTGGCGA-3′	366 to 385
1044	5′-TCATTAACGGTCGTCGCGGT-3′	665 to 684
1045	5′-TCCCAGACCCAGTTGCAGGT-3′	964 to 984
1046	5′-TGCTCGAACGTGCTGGCGAC-3′	1268 to 1288
1047	5′-CGCCAACAGCGCAGACATCA-3′	1565 to 1585
1048	5′-GTCAACCTGCTGCCGCTGGA-3′	1858 to 1878
1049	5′-TCGCGGTATTCGCTTAGGTG-3′	2163 to 2183
1050	5′-CAGGGCGTGATCCTCATCCG-3′	2458 to 2478

TABLE 2.

Properties of nalidixic acid-resistant mutants

Strain	Relevant phenotype	Nucleotide sequence (amino acid) in GyrA
		Codon 51	Codon 87
CGSC 6353	Nals T/r−	GCC (Ala)	GAC (Asp)
MF4-1	Nalr T/r+	GTC (Val)	GAC (Asp)
MF1	Nalr T/r+	GTC (Val)	GAC (Asp)
MF3	Nalr T/r+	GTC (Val)	GAC (Asp)
MF13	Nalr T/r−	GCC (Ala)	TAC (Tyr)

To determine whether a Nal^r T/r⁺ isolate contains additional changes in gyrA, we determined the sequence of the entire gene for parental (CGSC 6353) and mutant (MF4-1) strains. The gyrA gene was amplified with primers 1037 and 1042 (Table 1) and sequenced with primers 1043 through 1050 (Table 1). No further differences were observed between these two strains. Thus, an Ala-51-to-Val change in GyrA is sufficient to confer nalidixic acid resistance.

The Ala-51-to-Val substitution was neither necessary nor sufficient to confer thermotolerance: a Nal^s transductant of MF4-1 (strain KD1567) retained thermotolerance. Moreover, the Nal^r transductants of CGSC 6353, DM4100, and C600 were not thermotolerant: Nal^r transductants and their parental strains were indistinguishable with respect to growth on salt-free LB agar plates at both 43.5 and 46°C. In previous work (3), we reported that a plasmid-borne, wild-type gyrA gene suppressed both Nal^r and T/r⁺, suggesting that a GyrA alteration was necessary for thermotolerance. Suppression of thermotolerance by expression of GyrA from a plasmid probably arises from the presence of multiple copies of the gyrA gene. The genetic basis for thermotolerance remains unclear.

We compared the loss of quinolone susceptibility associated with the Ala-51-to-Val change to that seen with other GyrA variants. As shown in Fig. 1, Val-51 was associated with intermediate susceptibility to ciprofloxacin and gatifloxacin that was greater than that seen for four other amino acid changes generally considered to be within the QRDR (Ala-67 to Ser, Asp-82 to Ala, and Gln-106 to His). In the case of ciprofloxacin, this was also true for the Ala-84-to-Pro change. We conclude that the QRDR should be expanded to include position 51.

FIG. 1.

Relative susceptibilites of gyrase mutants to fluoroquinolones. The MIC₉₉s (see text) of ciprofloxacin (A) and gatifloxacin (B) were determined for a series of GyrA variants and are indicated above each bar (each determination was made twice with similar results). For illustrative purposes, the results for the mutant with the Ala-51-to-Val substitution are shown in white and are indicated by the arrows. All strains are gyrA Nal^r transductants of wild-type strain DM4100, as described in the text or elsewhere (8). Amino acid changes in the GyrA QRDR and strain numbers (in parentheses) are as follows: S83L (KD66), A51V (KD1721), A67S (KD1911), G81C (KD1915), S83W (KD1909), D87N (KD1913), Q106H (KD1917), D82A (KD1973), A84P (KD1975), and D87Y (KD1977).

Examination of the crystal structure of the GyrA59 dimer (10) reveals that Ala-51 lies in helix 2, which is below the DNA recognition helix (helix 4; Fig. 2). Changes at positions 83 and 87 that cause the greatest loss in quinolone susceptibility are located on the surface of the recognition helix (Fig. 2), where quinolone binding may occur (8, 10, 13, 22). We speculate that the Ala-51-to-Val substitution distorts the region, altering its ability to interact with fluoroquinolones.

FIG. 2.

Structure of the GyrA59 dimer. The figure shows a ribbon representation (generated in RasMol) of the GyrA59 fragment (10), courtesy of J. G. Heddle (John Innes Centre, Norwich, United Kingdom). (A) The entire GyrA59 dimer. (B) An enlargement of the boxed region in panel A. Amino acids that change to confer quinolone resistance are indicated in black and by the amino acid numbers. Amino acid 51 is in helix 2, amino acid 67 is in helix 3, and amino acids 83 and 87 are in helix 4.