title: DNA Gyrase Is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana.
abstract: The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides.
authors.affiliation: From the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Evans-Roberts
authors.affiliation: From the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Mitchenall
authors.affiliation: From the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Melisa K
authors.initials: MK
authors.lastname: Wall
authors.affiliation: the School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, Perth 6009, Australia, and the Australian Research Council Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia.
authors.forename: Julie
authors.initials: J
authors.lastname: Leroux
authors.affiliation: the School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, Perth 6009, Australia, and the Australian Research Council Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Mylne
authors.affiliation: From the Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom, tony.maxwell@jic.ac.uk.
authors.forename: Anthony
authors.initials: A
authors.lastname: Maxwell
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D002939:Ciprofloxacin / Q000494:pharmacology*; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004351:Drug Resistance; D055786:Gene Knockout Techniques; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D053140:Organelle Shape / Q000187:drug effects; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure; D017354:Point Mutation; D011487:Protein Conformation; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D029681:Arabidopsis Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D027081:DNA Gyrase
keywords: DNA gyrase; DNA replication; DNA topoisomerase; chloroplast; plant biochemistry; quinolones