Cloning a Gene Coding for Norflurazon Resistance in Cyanobacteria
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Cloning a Gene Coding for Norflurazon Resistance in Cyanobacteria Daniel Chamovitz, Iris Pecker, Gerhard Sandmann*, Peter Böger*, and Joseph Hirschberg Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel and * Lehrstuhl für Physiologie und Biochemie der Pflanzen. Universität Konstanz, D-7750 Konstanz, Bundesrepublik Deutschland Z. Naturforsch. 45c, 4 8 2 -4 8 6 (1990); received November 15, 1989 Norflurazon, Phytoene Dehydrogenase (= Phytoene Desaturase), Herbicide Resistance, Cyanobacteria, Gene Cloning The herbicide norflurazon inhibits carotene biosynthesis in photosynthetic organisms by blocking the enzyme phytoene dehydrogenase (= phytoene desaturase). We have isolated nor- flurazon-resistant mutants of the cyanobacterium Synechococcus PCC 7942. The herbicide- resistance gene from the mutant NFZ4 has been cloned by genetic complementation of the resistance trait in wild type cells. The experiment described here illustrates the usefulness of employing cyanobacteria to clone herbicide-resistance genes in a quick and simple way. Introduction Molecular description of carotenogenesis in Studies of herbicide-resistant plants have pro general, and of phytoene dehydrogenase in partic vided an insight on the mode of action of herbi ular has been lacking. Several of the genes in cides and on the biochemical and physiological volved in carotenogenesis have been cloned from properties of their target proteins. The develop Rhodobacter [6-8], among them the gene crtl ment of new techniques for DNA-mediated trans which encodes phytoene dehydrogenase. How formation of plants have paved the way to the pos ever, due to a large phylogenetic gap, this gene did sibility for genetic engineering of herbicide resist not hybridize with DNA from higher plants (J. ance in crop plants. However, a prerequisite of such Hearst, personal communication). studies is the availability of a herbicide-resistant Cyanobacteria (blue-green algae) provide an ex biotype from which it is possible to clone the re cellent system for cloning herbicide-resistance con sistance conferring gene. ferring genes. Their photosynthetic apparatus and A number of bleaching herbicides such as nor metabolism are very similar to those of plants, yet flurazon, fluridone and fluorochloridone block their prokaryotic nature allows sophisticated ge carotenogenesis by inhibiting phytoene dehydro netic manipulations. In addition, herbicide-resis- genase (= phytoene desaturase) which converts tant mutants can be easily isolated [9]. Cloning a phytoene into phytofluene and ^-carotene. Treat resistance-conferring gene from cyanobacteria and ment of plants with these herbicides result in a de analysis of the molecular basis of the resistance are crease of carotenoids and chlorophylls, and a con important first steps towards the study of the gene current accumulation of phytoene [1, 2], Studying also in higher plants. carotenogenesis in a cell-free system using daffodil We describe here the cloning of the gene that chromoplasts or photosynthetic membranes from confers norflurazon resistance in the cyanobacteri the cyanobacterium Synechocystis, showed that um Synechococcus PCC 7942. Our working hy phytoene accumulation originates from a direct in pothesis was that, similar to the case of atrazine terference of norflurazon with the catalytic activity [10] and sulfonylurea [11] resistance, a point muta of phytoene dehydrogenase and not by inhibition tion in the target protein, in our case phytoene of the biosynthesis of the enzyme [3, 4], It has been dehydrogenase, can confer herbicide resistance, demonstrated recently that norflurazon inhibits while still allowing the enzyme to function. phytoene dehydrogenase in a non-competitive manner that involves a reversible binding to the Materials and Methods enzyme [5]. Strains and growth conditions Reprint requests to Dr. J. Hirschberg. Cell cultures of Synechococcus PCC 7942 were Verlag der Zeitschrift für Naturforschung. D-7400 Tübingen grown in BG 11 medium at 35 °C as described [12]. 0341-0382/90/0500-0482 $01.30/0 Norflurazon-resistant mutant strains were grown Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. D. Chamovitz et al. • Norflurazon and Inhibition of Carotenogenesis 483 as above with 4 fiM norflurazon added to the Results growth medium. For selection of kanamycin-re- Cloning the norflurazon resistance gene sistant transformants, cells were plated on solid BG 11 medium containing 1.5% agar and 10 |ig/ml Several norflurazon-resistant mutant strains kanamycin. have been isolated following mutagenesis [15]. Biochemical characterization showed the resist Molecular cloning ance to be manifested at the level of phytoene de hydrogenation [15, 16]. DNA was extracted from Methods for extracting DNA from Synechococ cells of mutant NFZ4 and digested with the re cus PCC 7942 and for transformation of cyanobac- striction enzyme £coRI. The cleaved DNA was terial cells were according to Williams and Szalay transfected into cells of the wild type (wt) strain. [12]. Restriction enzyme digestion, Southern hy Homologous recombination, involving a double bridization and cloning followed conventional crossing-over event between the transfected DNA protocols [13] except where noted below. The vec and the cyanobacterial chromosome, could result tor pBR329k was constructed as follows: a 1.2 kb in a particular exchange that replaces the sequence Pstl fragment containing the aminoglycoside in the chromosome with the herbicide resistant 3'-phosphotransferase gene from Tn903 [14] was mutation [17]. Norflurazon-resistant colonies have inserted into the Pstl site of pBR329 (Fig. 1). This appeared following selection on plates containing was necessary since ampicillin resistance is a poor 1.0 |im norflurazon at the estimated frequency of selectable marker in cyanobacteria. Deletions were 10-5. The recovery of the norflurazon-resistant constructed using the Promega Erase-a-base kit. transformants indicates that the resistance in The E. coli strain MV 1190 was used as a host for NFZ4 is a genetic trait that is most probably en the plasmid vectors pBR329K and pUC118. Re coded by a single gene. striction and modifying enzymes were purchased In order to clone this gene, a genomic DNA from United States Biochemical Co. (Cleveland, OH). library of mutant NFZ4 was constructed in the £coRI site of the E. coli plasmid pBR329k. The li Biochemical analysis brary was amplified in E. coli and was transfected in the form of closed circular plasmids into wild Procedures for carotenoid determination in type cells of Synechococcus PCC 7942. Transfor Synechococcus PCC 7942, as well as in vivo and in mants were selected for growth on medium con vitro carotenogenesis assays have been previously taining kanamycin and norflurazon. The double described [15, 16]. resistance phenotype acquired by wt cells is a result of a stable transformation of the cells with both the kanamycin and norflurazon-resistance genes. Since pBR329k is unable to replicate autonomously in cyanobacteria, the only way that these two genes could be maintained in the transformant was by integration into the cyanobacterial genome by a single crossing-over event between the cyanobac terial DNA insert in the recombinant pBR329k and its homologous sequence in the cyanobacterial chromosome. As a result, the entire plasmid was integrated into the genome, creating a merodiploid for the segment it carried (Fig. 2). DNA was extracted from one of the partial di ploid strains and cut with the restriction enzyme SalI. The Sail resulting fragments were then self ligated and used to transfect E. coli cells. The Fig. 1. Structure of plasmid pBR329k. See text for transformants, which were selected for growth on details. kanamycin-containing LB medium, contained a 484 D. Chamovitz et al. • Norflurazon and Inhibition of Carotenogenesis Localization of the norflurazon-resistance mutation A restriction map of the 10 kb insert of pPD2 was constructed (Fig. 3). In order to localize the mutation that confers norflurazon resistance, each of the restriction fragments shown in Fig. 3 was checked for its ability to transfer norflurazon resistance via DNA transformation. The S a l\- BamW\ 2.8 kb fragment was found positive and B was further analyzed to localize the mutation E S N FZr E S NFZ (Fig. 3). tetr ori kanr Analysis ofphvtoene dehydrogenase from PD2 C S S Similar to NFZ 4, norflurazon resistance in PD2 was found to be manifested, both in vivo and in s mTTTTT*— E i—i S V s nfz-resistant E S transformants \ ori kanr Fig. 2. Strategy used to clone the norflurazon-resistance (NFZr) gene. A genomic library from herbicide-resistant mutant NFZ4 in the £coRI site of pBR329k was trans HP H P —t—i— -l- H fected to wild type cells. A single crossover event be tween the transfected plasmid and the cyanobacterial chromosome (A) lead to its integration into the cyano bacterial genome next to the homologous sequence of the insert (B). A kanamycin-resistant (kanr) and NFZr transformant was selected. “Self ligation” of the Sail fragments of the transformant DNA, followed by trans formation to E. coli, “rescued” the plasmid along with a H P HP portion of the original insert. (E-£coRI; S-Sall; ori- 35 -H- origin of DNA replication in E. coli). H P 83 HP h+- HP portion of the pBR329k vector along with part of —H the original £coRI fragment (Fig. 2). This plasmid H P was designated pPD 6. —t—i The S a ll-E co R l 800bp insert in pPD6 was 1 Kb used as a probe to screen the original NFZ 4 gen omic library for the plasmid clone containing the Fig. 3. A restriction map of the 10 kb ZTcoRI fragment cloned from the genome of Synechococcus PCC 7942 in complete norflurazon-resistance conferring insert. the plasmid pPD2.