title: Interactions between QnrB, QnrB mutants, and DNA gyrase.
abstract: Plasmid-encoded protein QnrB1 protects DNA gyrase from ciprofloxacin inhibition. Using a bacterial two-hybrid system, we evaluated the physical interactions between wild-type and mutant QnrB1, the GyrA and GyrB gyrase subunits, and a GyrBA fusion protein. The interaction of QnrB1 with GyrB and GyrBA was approximately 10-fold higher than that with GyrA, suggesting that domains of GyrB are important for stabilizing QnrB1 interaction with the holoenzyme. Sub-MICs of ciprofloxacin or nalidixic acid reduced the interactions between QnrB1 and GyrA or GyrBA but produced no reduction in the interaction with GyrB or a quinolone-resistant GyrA:S83L (GyrA with S83L substitution) mutant, suggesting that quinolones and QnrB1 compete for binding to gyrase. Of QnrB1 mutants that reduced quinolone resistance, deletions in the C or N terminus of QnrB1 resulted in a marked decrease in interactions with GyrA but limited or no effect on interactions with GyrB and an intermediate effect on interactions with GyrBA. While deletion of loop B and both loops moderately reduced the interaction signal with GyrA, deletion of loop A resulted in only a small reduction in the interaction with GyrB. The loop A deletion also caused a substantial reduction in interaction with GyrBA, with little effect of loop B and dual-loop deletions. Single-amino-acid loop mutations had little effect on physical interactions except for a Δ105I mutant. Therefore, loops A and B may play key roles in the proper positioning of QnrB1 rather than as determinants of the physical interaction of QnrB1 with gyrase.
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea.
authors.forename: Eu Suk
authors.initials: ES
authors.lastname: Kim
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA Institute of Antibiotics, Huashan Hospital, Fudan University, and Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, China.
authors.forename: Chunhui
authors.initials: C
authors.lastname: Chen
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Braun
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea.
authors.forename: Hyo Youl
authors.initials: HY
authors.lastname: Kim
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Okumura
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Lahey Hospital and Medical Center, Burlington, Massachusetts, USA.
authors.forename: George A
authors.initials: GA
authors.lastname: Jacoby
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA dhooper@mgh.harvard.edu.
authors.forename: David C
authors.initials: DC
authors.lastname: Hooper
mesh_terms: D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
chemical_list: D029968:Escherichia coli Proteins; C485384:Qnr protein, E coli; D027081:DNA Gyrase