<em>Arabidopsis thaliana</em> DNA gyrase is targeted to chloroplasts and mitochondria

Melisa Wall

Lesley Mitchenall

Anthony Maxwell

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

^{To whom correspondence should be addressed. E-mail:}ku.ca.crsbb@llewxam.ynot.

Edited by Martin Gellert, National Institutes of Health, Bethesda, MD

Received 2004 Feb 5; Accepted 2004 Apr 5.

Abstract

DNA gyrase is the bacterial DNA topoisomerase (topo) that supercoils DNA by using the free energy of ATP hydrolysis. The enzyme, an A₂B₂ tetramer encoded by the gyrA and gyrB genes, catalyses topological changes in DNA during replication and transcription, and is the only topo that is able to introduce negative supercoils. Gyrase is essential in bacteria and apparently absent from eukaryotes and is, consequently, an important target for antibacterial agents (e.g., quinolones and coumarins). We have identified four putative gyrase genes in the model plant Arabidopsis thaliana; one gyrA and three gyrB homologues. DNA gyrase protein A (GyrA) has a dual translational initiation site targeting the mature protein to both chloroplasts and mitochondria, and there are individual targeting sequences for two of the DNA gyrase protein B (GyrB) homologues. N-terminal fusions of the organellar targeting sequences to GFPs support the hypothesis that one enzyme is targeted to the chloroplast and another to the mitochondrion, which correlates with supercoiling activity in isolated organelles. Treatment of seedlings and cultured cells with gyrase-specific drugs leads to growth inhibition. Knockout of A. thaliana gyrA is embryo-lethal whereas knockouts in the gyrB genes lead to seedling-lethal phenotypes or severely stunted growth and development. The A. thaliana genes have been cloned in Escherichia coli and found to complement gyrase temperature-sensitive strains. This report confirms the existence of DNA gyrase in eukaryotes and has important implications for drug targeting, organelle replication, and the evolution of topos in plants.

Abstract

DNA topoisomerases (topos) are key enzymes present in all cells that control the topological state of DNA (1). There are two types, topos I and II, distinguished by whether they transiently break one or both strands of the DNA. DNA gyrase is a type II enzyme that is essential for the processes of replication and transcription in prokaryotes. It is the only enzyme of this type that is able to catalyze ATP-dependent DNA supercoiling (2). The best-studied gyrase is that from Escherichia coli, which consists of two subunits, DNA gyrase protein A (GyrA; 97 kDa) and DNA gyrase protein B (GyrB; 90 kDa), which form an A₂B₂ complex. Due to its essential role in prokaryotes and its apparent absence from eukaryotes, gyrase is the target of a number of antibacterial agents, including quinolones, e.g., nalidixic acid (NAL) and ciprofloxacin (CFX), and coumarins, e.g., novobiocin (NOV) and coumermycin A₁ (3).

Although thought to be a uniquely bacterial enzyme, there have been previous indications that there may be a gyrase in plants. Thompson and Mosig (4) found that Chlamydomonas reinhardtii contains an ATP-dependent topo activity that can supercoil DNA in vitro. The supercoiling activity was weak and, although unable to purify the enzyme, they found that gyrase-specific drugs inhibited chloroplast transcription. Further work showed that NOV could inhibit chloroplast DNA replication in vivo (5). In Nicotiana tabacum, NAL was shown to have a greater inhibitory effect on plastid than nuclear DNA synthesis at low drug concentrations (6); high concentrations affected both plastid and nuclear DNA synthesis. NAL was also found to inhibit DNA synthesis in both the chloroplast and mitochondrion of the unicellular alga Cyanidioschyzon merolae (7). Similarly, Mills et al. (8) found that NAL and NOV inhibited thymidine incorporation in pea chloroplasts; Lam and Chua (9) found that NOV affected transcription in pea chloroplasts. Ebringer et al. (10) reported that, in Euglena gracilis, the second-generation quinolone ofloxacin caused mass aberrations and subsequent loss of chloroplasts and ultrastructural changes in mitochondria. Pyke et al. (11) reported the presence of two proteins in wheat chloroplasts with molecular masses similar to gyrase, which crossreacted to yeast topo II antibodies.

These results suggest that there might be DNA gyrase activity in the chloroplasts and mitochondria of plant cells. In principle, eukaryotes do not require gyrase because negative supercoiling can be established by the wrapping of DNA around histones and the relaxation of the internucleosomal DNA by topos I and II; topo II is evolutionarily related to gyrase but lacks the ability to supercoil DNA (1). However, chloroplasts and mitochondria lack histones and their genomes resemble those of their bacterial ancestors in a number of respects, raising the possibility that these organelles may organize their DNA differently from nuclear DNA and could both require DNA gyrase activity.

With these considerations in mind, we have examined the genome sequence of Arabidopsis thaliana (12) and found four putative gyrase genes. By using a combination of in vivo and in vitro experiments we show that there is gyrase activity in A. thaliana cells that is targeted to chloroplasts and mitochondria. We find that the A. thaliana gyrase subunits are able to complement their E. coli counterparts and we speculate on the role of gyrase in organelle replication.

cp, chloroplast; mt, mitochondrial; nt, not targeted; NA, not applicable; +++, strong complementation; +, weak complementation; -, no complementation.

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Acknowledgments

We thank K. Roberts and K. Sugimoto for helpful suggestions and J. Mylne and F. Corke for reagents. This work was supported by grants from the Wellcome Trust (U.K.) and the Biotechnology and Biological Sciences Research Council (U.K.). M.K.W. is a Biotechnology and Biological Sciences Research Council-funded Postdoctoral Researcher. Funding for the Salk Institute Genomic Analysis Laboratory (La Jolla, CA) indexed insertion mutant collection was provided by the National Science Foundation.

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: CFX, ciprofloxacin; GyrA, DNA gyrase protein A; GyrB, DNA gyrase protein B; NAL, nalidixic acid; NOV, novobiocin; topo, topoisomerase.

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: CFX, ciprofloxacin; GyrA, DNA gyrase protein A; GyrB, DNA gyrase protein B; NAL, nalidixic acid; NOV, novobiocin; topo, topoisomerase.

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