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.

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity

“…One possibility could be that TK1b is also required for chloroplastic DNA repair, and that defects in this process could result in delayed greening. To demonstrate the role of TK1b in the maintenance of organellar genome integrity, we treated Arabidopsis seedlings with ciprofloxacin, a DNA gyrase inhibitor, that induces the formation of double-strand breaks (DSBs) in organellar DNA (Cappadocia et al, 2010;Evans-Roberts et al, 2016). Homozygous tk1b seedlings were hypersensitive to ciprofloxacin compared with wild-type Col-0 seedlings (Figure 4a Figure 3.…”

Structure–Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System

“…Bacterial gyrases use ATP to introduce negative supercoils in DNA. Wall and coworkers discovered that flowering plants encode one gene for gyrA (At3g10690) and two functional genes of gyrB (At3g10270 and At5g04130) [154,155]. AtGyrA is targeted to mitochondria and chloroplast, whereas the product of At5g04130 is targeted to mitochondria and was dubbed AtmtGyrB [154].…”

“…Structural studies of bacterial gyrases show the coordination between gyrA and gyrB that drives cleavage of the DNA strands, strand passage between subunits, and ligation [156][157][158]. Heterologously purified AtGyrA/AtmtGyrB present supercoiling activity [155] and the bacterial origin of the plant organellar AtGyrA/AtmtGyrB makes them a target for the development of new herbicides based on quinolones [155]. Ciprofloxacin, a quinolone drug, is commonly used to induce specific DSBs in plant organelles as the gyrase catalytic cycle is not completed [135,159].…”

RNase H1 Cooperates with DNA Gyrases to Restrict R-Loops and Maintain Genome Integrity in Arabidopsis Chloroplasts

“…They are necessary for introducing negative supercoils and relaxing positive supercoils during transcription and DNA replication in prokaryotes (Aldred et al, 2014;Chang et al, 2013;Higgins, 2014). Plant DNA gyrases have been proposed to have similar functions in the chloroplast (Cho et al, 2004;Evans-Roberts et al, 2016;Wall et al, 2004). The exact functions and regulatory mechanisms of DNA gyrases in plant organelles are not fully understood, although there is evidence that plant organelle-localized DNA gyrases are essential for plant development, as knockout mutations of either AtGyrA or AtGyrBs are lethal (Wall et al, 2004).…”