title: QnrS1 structure-activity relationships.
abstract: OBJECTIVE
  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.
authors.affiliation: Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Microbiología, Departamento de Ciencias Clínicas, Universidad de Las Palmas de Gran Canaria, Las Palmas de G.C., España mtavio@dcc.ulpgc.es.
authors.forename: María M
authors.initials: MM
authors.lastname: Tavío
authors.affiliation: Lahey Hospital and Medical Center, Burlington, MA, USA.
authors.forename: George A
authors.initials: GA
authors.lastname: Jacoby
authors.affiliation: Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Hooper
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology*; D027081:DNA Gyrase / Q000187:drug effects / Q000235:genetics; D004278:DNA, Superhelical / Q000187:drug effects; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009154:Mutation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; C485384:Qnr protein, E coli; D007544:Isopropyl Thiogalactoside; D002939:Ciprofloxacin; D027081:DNA Gyrase
keywords: QnrS; pentapeptide repeat proteins; quinolone resistance
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