QnrS1 structure-activity relationships

Abstract

Objectives: Loop B is important for low-level quinolone resistance conferred by Qnr proteins. The role of individual amino acids within QnrS1 loop B in quinolone resistance and gyrase protection was assessed.

Methods: qnrS1 and 11 qnrS1 alleles with site-directed Ala mutations in loop B were expressed in Escherichia coli BL21(DE3) and proteins were purified by affinity chromatography. Ciprofloxacin MICs were determined with and without IPTG. Gyrase DNA supercoiling was measured with and without ciprofloxacin IC50 and with various concentrations of QnrS1 proteins.

Results: Wild-type QnrS1 and QnrS1 with Asn-110→Ala and Arg-111→Ala substitutions increased the ciprofloxacin MIC 12-fold in BL21(DE3), although QnrS1 with Gln-107→Ala replacement increased it 2-fold more than wild-type did. However, QnrS1 with Ala substitutions at His-106, Val-108, Ser-109, Met-112, Tyr-113, Phe-114, Cys-115 and Ser-116 increased ciprofloxacin MIC 1.4- to 8-fold less than wild-type QnrS1. Induction by 10-1000 μM IPTG increased ciprofloxacin MICs for all mutants, reaching values similar to those for wild-type. Purified wild-type and mutated proteins differed in protection of gyrase from ciprofloxacin action. Wild-type QnrS1 produced complete protection of gyrase supercoiling from ciprofloxacin (1.8 μM) action at 0.05 nM and half protection at 0.5 pM, whereas QnrS1 with Ala replacements that conferred the least increase in ciprofloxacin MICs also required the highest QnrS1 concentrations for protection.

Conclusions: Key individual residues in QnrS1 loop B affect ciprofloxacin resistance and gyrase protection from ciprofloxacin action, supporting the concept that loop B is key for interaction with gyrase necessary for quinolone resistance.

Keywords: QnrS; pentapeptide repeat proteins; quinolone resistance.

Figure 1.

Comparison of plasmid-encoded homologues of QnrS1 loop B amino acid sequence. Amino acid residues that are coincident with those of QnrS1 loop B are underlined. GenBank accession number for proteins are available in the qnr numbering and sequence web page.

Figure 2.

DNA gyrase protection assays with QnrS1 purified proteins. Reactions of 25 μL were analysed by agarose gel electrophoresis. Reactions mixtures (40 μL) contained 0.5 μg of relaxed pHOT-1 DNA (lanes 1–12), 2 U of purified E. coli DNA gyrase (lanes 2–12) and 1.8 μM (0.6 mg/L) ciprofloxacin (lanes 3–12). The concentrations of the 12 different QnrS1 purified proteins are expressed in nM above each lane; the numbers in parentheses refer to the power of 10 that multiplies the value. (a) QnrS1 wild-type (lanes 4–7) and QnrS1 Q107A (lanes 8–12). (b) QnrS1 H106A (lanes 4–7) and QnrS1 V108A (lanes 8–12). (c) QnrS1 S109A (lanes 4–7) and QnrS1 N110A (lanes 8–11). (d) QnrS1 R111A (lanes 4–7) and QnrS1 Y113A (lanes 8–12). (e) QnrS1 M112A (lanes 4–8). (f) QnrS1 F114A (lanes 4–7) and QnrS1 C115A (lanes 8–11). (g) QnrS1 wild-type (lanes 4–7) and QnrS1 S116A (lanes 8–12). nc, l and sc indicate positions of the nicked circular, linear and supercoiled forms of pHOT-1, respectively.