Cloning a Coding for Norflurazon Resistance in Cyanobacteria Daniel Chamovitz, Iris Pecker, Gerhard Sandmann*, Peter Böger*, and Joseph Hirschberg Department of , The Hebrew University of Jerusalem, Jerusalem 91904, 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 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. Mapping the mutation of norflura- This plasmid, pPD2, contained a 10 kb EcoRl zon resistance (nfzr) on the B a m H l-S a il DNA fragment insert that was able to transfer norflurazon resist­ was done by deletions that were constructed and trans­ ance when used to transfect wild type Synechococ­ fected to wild type cells of Synechococcus PCC 7942. Transformation to nfzr ( + ) was tested. (E-EcoRI; cus PCC 7942 cells. The transformed strain was H -HindllY, P-Psfl; S -S a ll ; different deletions are marked designated PD2. A. B. D .E . 35 and 83). D. Chamovitz et al. • Norflurazon and Inhibition of Carotenogenesis 485

vitro, at the level of phytoene dehydrogenase in [fig /m l p c v ] CAROTENOID CONTENT N FZ4 (Table I).

Table I. Factors of resistance and in vitro activity in Svnechococcus PCC 7942 wild type and resistant strain PD 2.

Strain in vivo in vitro Conversion / jo r f k-, RF rate [%]

WT 0.11 0.09 - 76 PD 2 1.22 12.2 1.37 15.2 37

RF: Resistance factor is the ratio of mutant /50 or ki ver­ CAROTENOID COMPOSITION sus wild type values. The conversion rate was determined as radioactivity [%] from l4C-geranylgeranyl pyrophosphate incorporated into ^- + ß-carotene relative to the radioactivity in phy­ toene + £- + ß-carotene.

Measurement of the efficiency of the phytoene dehydrogenation reaction in the cell-free system revealed a drop of 41% in PD2 relative to wild type (Table I). Phenotypically, this decrease in phytoene dehydrogenase activity is manifested also in carotenoid composition (Fig. 4). In wild type cells, ß-carotene makes up slightly over one echinen- HD ß-carotene one quarter of all carotenoids, with zeaxanthin being Fig. 4. Carotenoid and xanthophyll content and com­ the most abundant (62%). In PD2, there is an position in Svnechococcus PCC 7942 wild type and PD2 18% drop in ß-carotene content, but a large drop strains. In contrast to WT, PD2 accumulated 1 5-cis of 67% in the amount of xanthophylls. Conse­ phytoene up to 20% of total colored carotenoids. quently, ß-carotene in PD2 makes up close to half (48%) of all carotenoids. cient than that of the w'ild type in converting phy­ toene to £- + ß-carotene. This can be explained if Discussion the mutation that confers resistance to nurflurazon We describe here the cloning of a DNA frag­ occurred in the gene for phytoene dehydrogenase, ment from a norflurazon-resistant mutant of Syne- and this alteration also interferes with the en­ chocossus PCC 7942 that confers norflurazon re­ zymatic activity. Since PD 2 is completely isogenic sistance upon transfection to wild type cyanobac­ with the wild type strain, except for the mutation teria. Several lines of evidence indicate that this for herbicide resistance, it is probable that the mu­ fragment may encode the gene for the enzyme phy­ tation has occurred in the gene encoding phytoene toene dehydrogenase. As has previously been dehydrogenase. shown, the resistance to norflurazon in the original The reduced efficiency of phytoene dehydrogen­ mutant strain NFZ4 is manifested both in vivo and ase results also in an altered pattern of carotenoid in vitro at the level of phytoene dehydrogenase composition. While the amount of ß-carotene is [15]. A similar pattern of resistance is found also only slightly lowered in PD 2, there is a significant for PD2, the strain genetically engineered to herbi­ reduction in the amount of xanthophylls. The xan­ cide resistance with the gene cloned from NFZ4. thophylls are biosynthetic products of ß-carotene. Furthermore, norflurazon resistance is accom­ Apparently, a basic concentration of ß-carotene is panied by a drop in the activity of phytoene dehy­ necessary for cell growth, while xanthophyll con­ drogenase. The enzyme in PD 2 is 41% less effi- centration is less critical. It seems that a mecha­ 486 D. Chamovitz et al. ■ Norflurazon and Inhibition of Carotenogenesis nism exists in Synechococcus PCC 7942 that limits did not confere resistance when transfected to wt xanthophyll synthesis under conditions of limited cells. This result is anticipated assuming that the ß-carotene supply. double cross-over mechanism necessitates a several Cyanobacterial cells can be transformed to nor­ hundred base pairs region surrounding the muta­ flurazon resistance by two mechanisms: 1. Integra­ tion on each side in the transforming fragment [17], tion of an additional fragment, containing the her- When the mutation is located too close to the end of bicide-resistance mutation, as a result of a single the transforming DNA fragment, it can no longer crossing-over event between a plasmid and the be transferred to the chromosome by crossing-over chromosome; 2. By a gene replacement due to a of DNA molecules. The deletion analysis, together double crossing-over event between a DNA frag­ with the inability of the internal Pstl and H indlll ment and its homologous sequence in the chromo­ fragments to transform wt cells to norflurazon-re- some. Only in the first case is it possible to reiso- sistance places the mutation in the region of the late the plasmid by the plasmid rescue technique. first Hindlll- Pstl junction. When linear DNA is transfected to cyanobacteria, While the biochemical data indicates that the transformation must occur via the double cross­ norflurazon-resistance gene encodes phytoene de­ over mechanism. This was the case when deletions hydrogenase, the possibility cannot be ruled out of the Sail- BamHl fragment were used (Fig. 3). that norflurazon acts through an as yet unidenti­ In contrast to DNA fragment D, fragment E failed fied protein associated with the carotenogenesis to transfer NFZr. The region that was deleted from complex. Molecular biological studies, including fragment D to give fragment E defines the location the sequencing of the gene, will allow an accurate of the mutation. However, the internal H indlll - identification. Hindlll fragment, that contains that entire region,

[1] P. G. Bartels and C. McCullough, Biochem. [9] J. Hirschberg, N. Ohad, I. Pecker, and A. Rahat, Z. Biophys. Res. Commun. 48, 16 (1972). NaturforschT 42 c, 102-112(1987). [2] G. Sandmann and P. Böger, in: Target Sites of Her­ [10] J. Hirschberg, A. Ben Yehuda, I. Pecker, and N. bicides of Herbicide Action (G. Sandmann and P. Ohad, in: Plant (D. von Wett- Böger, eds.), pp. 25-44, CRC Press, Boca Raton, stein and N. H. Chua, eds.), pp. 357-366, Plenum FI. 1989. Publ., 1987. [3] P. Beyer, K. Kreuz, and H. Kleinig, Planta 150, 435 [11] K. Y. Lee, J. Townsed, J. Tepperman, M. M. Black (1980). e ta /., EM BO J. 7, 1241-1248(1988). [4] I. E. C larke, G. Sandm ann, P. M. Bramley, and P. [12] J. G. K. Williams and A. A. Szalay, Gene 24, 37 Böger, FEBS Lett. 140, 203 (1982). (1983). [5] G. Sandmann, H. Linden, and P. Böger, Z. Natur- [13] T. Maniatis, E. F. Fritsch, and J. Sambrook, Molec­ forsch. 44c, 787(1989). ular Cloning a Laboratory Manual, Cold Spring [6] J. M. Pemberton and C. M. Harding, Curr. Micro­ H arbor L aboratory 1982. biol. 14,25-29(1986). [14] A. O ka et al., J. Mol. Biol. 147,217(1981). [7] G. Giuliano, D. Pollock, and P. A. Scolnik, J. Biol. [15] H. Linden, G. Sandmann, D. Chamovitz, J. Hirsch- Chem. 261 (28), 12925- 12929 (1986). berg, and P. Böger, Pestic. Biochem. Physiol. 36, [8] G. A. Armstrong, M. Alberti, F. Leach, and J. E. 46-51 (1990). Hearst, Mol. Gen. Genet. 216, 254-268 (1989). [16] G. Sandmann, Methods Enzymol. 167, 329 (1988). [17] S. S. Golden, M ethods Enzymol. 167,714(1988).