scientific correspondence A prokaryotic phytochrome hytochrome photoreceptors are almost tive phy gene product by expression-cloning tively, and an isosbestic point at 677 nm. Pcertainly ubiquitous in green plants, in E.coli using the vector pQE1 2. This is the first report of a spectrally regulating numerous aspects of develop Expression of the Synechocystis PHY apo functional prokaryotic phytochrome. In ment throughout their life cycle. Phyto protein was very efficient. Products of rela most lower organisms light is detected by chromes were thought to exist only in tive molecular mass 85,000 accumulated to retinal or coumaric acid-based photorecep plants, but the recently described sequence of about 50% total soluble protein (Fig. la, lane tors. However, a Fremyella gene, rcaE, the chromosome from the cyanobacterium 1). This contrasts with results from similar involved in chromatic adaptation and Synechocystis revealed a gene that seemed to approaches with plant phytochrome genes encoding a putative histidine-kinase sensor 3 encode a phytochrome-like protein 1• By in E. coli which are usually very weakly protein was recently described' • Although expressing this gene in Escherichia coli and expressed or give rise to largely insoluble the conceptual gene product shows local feeding appropriate chromophores we show products&--10• The clone was engineered to similarities to PHYE in Arabidopsis, no sig that it encodes a phytochrome, which may express a C-terminal polyhistidine tag for nificant homology to the phytochrome offer an excellent starting material for crys nickel-affinity purification. The product chromophore-binding domain is apparent13 tallization and X-ray diffraction analysis. bound quantitatively to Ni-NTA (lane 2) and and photoreceptor activity has yet to be The carboxy-terminal amphiphilic struc was eluted as a homogeneous apoprotein demonstrated. The Synechocystis phy gene ture of phytochrome resembles that of (lane 3), which could be concentrated to a product is the most phytochrome-like of bacterial sensory histidine-kinases, a group 5- 10 mg rn1 - 1 solution. the Synechocystis genome and among all of enzymes used by prokaryotes to monitor Plant phytochrome apoproteins auto known prokaryotic sequences. and react to various aspects of their envir catalytically attach linear tetrapyrrole chro A simple prokaryote has advantages for onment2-4. Conceptual translation of the mophores such as phycocyanobilin (PCB)11 , basic studies of phytochrome biology. For Synechocystis sp. PCC 6803 open reading abundant in the cytoplasm of cyanobacte example, co-expression in the heterologous frame slr0473 (the putative Synechocystis phy ria. Indeed, the Synechocystis PHY apopro E. coli host might be useful in studying gene) yields a product that shows similarity tein attached purified PCB, producing subsequent components of the signal trans to plant phytochromes throughout its length visibly photochromic holoprotein (phy*, duction pathway. Furthermore, milligram and to bacterial sensory kinases towards the Fig. lb). In contrast, plant phytochromes amounts of homogeneous, spectrally active C terminus1•5• In particular, the chromo expressed in E. coli show poor autoassembly, Synechocystis phytochrome holoprotein can phore-binding domain, highly conserved in folding incorrectly8'9' 12• Synechocystis phy* be produced easily in our system. As concen all phytochromes, is clearly represented in was analysed spectrophotometrically after trations of 10 mg ml-' can be achieved read the product (residues Val 246-Asp 280). exposure to saturating monochromatic 657 ily - at least ten times higher than in other This, however, does not prove that the gene nm (red) and 731 nm (far-red) irradiation phytochrome overexpression systems known product is a genuine phytochrome. Phyco (Fig. le). The spectra are reminiscent of to us - there remains no barrier in principle cyanin levels prevent spectral photoreversi plant phytochrome-PCB adducts'' with to obtaining crystals for X-ray diffraction bility measurements6'7 of phytochrome in absorb-ance maxima at 658 and 702 nm analysis of phytochrome molecular structure. cyanobacteria, so we investigated the puta- after red and far-red irradiation, respec- Jon Hughes, Tihnan Lamparter a Franz Mittmann, Elmar Hartmann Institut fiir Pjlanzenphysiologie und Mikrobiologie, M,(K) 2 3 Freie Un iversitiit Berlin, 205- Konigin-Luise-Strasse 12- I 6, D-141 95 Berlin, Germany e-mail: [email protected] Wolfgang Gartner - Max-Planck-Institut fur Strahlenchemie, Postfach 101365, D-45413 Mulheim/Ruhr, Germany Annegret Wilde, Thomas Borner lnstitut fur Biologie, Humboldt Universitiit Berlin, Chausseestrasse 11 7, D-10115 Berlin, Germany
I. Kaneko, T. et al. DNA Res. 3, l 09- 136 ( 1996). Figure I a, Expression and affinity purification 2. Schneider-Poetsch, H. A. W., Braun, B., Marx, S. & of recombinant PHY apoprotein in E. coli. SD~ Schaumburg, A. f'EBS Left. 281, 245- 249 ( 1991 ). 3. Parkinson, J. S. & Kofoid, E. C. Annu. Rev. Genet. 26, 7 1- 11 2 PAGE 10% gel stained with Coomassie. Lane l, (1992). total soluble protein in lysate; 2, after adsorption 4. Quail, P.H. t't al. Science 268, 675-680 ( 1996). to Ni-NTA matrix; 3,250 mM imidazole eluate. 5. Hughes, J., Lamparter, T. & Mittmann, F. Plant Physiol. 11 2,446 C b, Photochromicity of phy* holoprotein. ( 1996). 0.20 6. Butler, W. L., Norris, H. W., Sicgelman, H. W. & Hendricks, S. 8. of PCB were added to Stoichiometric amounts Proc. Natl Arnd. Sci. USA 45, 1703- 1708 ( 1959). Q) 0.15 PHY (3 mg m1 - '). After autoassembly (20 min in 7. Lam parter, T., Hughes, J. & Hartmann, E. Phorochem. Photobiol. () C darkness) the sample was divided and each 60, 179- 183 ( 1994). ro 0.10 .0 8. Wahleith ner, J. A. . Li, L & Lagarias, J.C. Proc. Na tl Acad. Sci. 0 portion irradiated with 731 nm (far-red, left) or (J) USA 88, 10387- 1039 1 (199 1). .0 0.05 657 nm (red, right) light. Note the blue or green <( 9. lOmizawa, K.-1., Stockhaus, J., Chua, N.-H. & Furuya, M. Pfnnt 0.00 transition associated with phytochrome Cell Pliysiol. 36, 511 - 51 6 ( 199 1) . photoconversion. c, Absorbance characteristics 10. Lamparter, T ct al.}. JJ/a 11t Physiol. 147, 426-4341 ( 995). E, Palma, L.A. & Lagaria.s, J.C. -0.05 of phy* after irradiation with saturating red (R) 11. Elich, T. D., Md)onagh, A. 300 400 500 600 700 800 /. lliol. Chem. 264, 183-189 (1989). Wavelength (nm) or far-red (FR) light, and the calculated difference 12. Hill, C. et ,1/. E11 r. ]. Biod 1em. 223, 69- 77 ( 1994 ). spectrum. 13. Kehoe, D. M. & Grossman, R. Science 273, 1409-14 12 ( 1996).
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