Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site Tilman Lamparter*, Norbert Michael, Franz Mittmann, and Berta Esteban Freie Universita¨t Berlin, Pflanzenphysiologie, Ko¨nigin Luise Strasse 12–16, D-14195 Berlin, Germany Edited by Winslow R. Briggs, Carnegie Institution of Washington, Stanford, CA, and approved May 30, 2002 (received for review May 2, 2002) Phytochromes are photochromic photoreceptors with a bilin chro- reversion has so far not been found in bacterial phytochromes. mophore that are found in plants and bacteria. The soil bacterium Cph1 of Synechocystis (17) and CphA of Calothrix (18) have a stable Agrobacterium tumefaciens contains two genes that code for Pfr form; reports on other bacterial orthologs are missing so far. phytochrome-homologous proteins, termed Agrobacterium phyto- Most bacterial phytochromes carry a histidine-kinase module, chrome 1 and 2 (Agp1 and Agp2). To analyze its biochemical and the first component of ‘‘two-component’’ systems. His-kinase spectral properties, Agp1 was purified from the clone of an E. coli activity is light-modulated; cyanobacterial phytochromes are overexpressor. The protein was assembled with the chromophores more active in the Pr form (19–21), whereas phytochrome BphP phycocyanobilin and biliverdin, which is the putative natural chro- from the proteobacterium Pseudomonas aeruginosa is more mophore, to photoactive holoprotein species. Like other bacterial active in the Pfr form (11). In general, His kinases transphos- phytochromes, Agp1 acts as light-regulated His kinase. The biliverdin phorylate particular response regulators (22); this mechanism adduct of Agp1 represents a previously uncharacterized type of also has been shown for bacterial phytochromes (11, 19, 21). phytochrome photoreceptor, because photoreversion from the far- The genome of the soil bacterium Agrobacterium tumefaciens has red absorbing form to the red-absorbing form is very inefficient, a recently been published (23, 24). It contains two genes that code for feature that is combined with a rapid dark reversion. Biliverdin bound phytochrome-homologous proteins (11). Agrobacterium is well covalently to the protein; blocking experiments and site-directed known among plant scientists because it can transform tumor- mutagenesis identified a Cys at position 20 as the binding site. This inducing genes into plants and can be used as a shuttle system for particular position is outside the region where plant and some plant transformation (23). The question of what role a photore- cyanobacterial phytochromes attach their chromophore and thus ceptor might play in an organism of such agricultural and genetic represents a previously uncharacterized binding site. Sequence com- importance prompted us to begin analyzing the biochemical prop- parisons imply that the region around Cys-20 is a ring D binding motif Agrobacterium in phytochromes. erties of recombinant phytochrome. Biliverdin and PCB yielded products with the spectral characteristics of Pr that bilin ͉ biliprotein ͉ photochromic ͉ histidine kinase photoconverted to Pfr. Quite interestingly, both adducts showed Pfr-to-Pr dark-reversion, as is the case for some plant phyto- chromes. Studies on chromophore binding revealed a new Cys- any developmental processes in plants such as seed germi- binding site in the N terminus of the protein. This finding might Mnation, de-etiolation, or flowering are controlled by phyto- have implications for the study of chromophore interaction and chrome photoreceptors (1). The discovery of phytochromes in photoconversion of all phytochromes. bacteria (2, 3) showed that these chromoproteins are of prokaryotic origin, which gave great insight into the evolution of phytochromes. Materials and Methods Prokaryotic phytochromes offer advantages for biochemical and Computer Science. Database searches were performed on Na- biophysical studies (4–6) and help to define the role of protein tional Center for Biotechnology Information (NCBI) BLASTP domains and single amino acids (7). Phytochromes carry a bilin (http:͞͞ncbi.nlm.nih.gov); the Agrobacterium phytochrome ho- chromophore, either phytochromobilin (8), phycocyanobilin (PCB; mologues (Agp1 and Agp2) were found with Cph1 as template ref. 9, 10), or biliverdin (BV), as was recently shown for bacterio- (17). Both Agrobacterium sequences are already mentioned in an phytochrome photoreceptor (BphP) of the bacterium Deinococcus earlier publication (11). Searches for heme oxygenase and bilin radiodurans (ref. 11; chemical structures of chromophores are given in Fig. 1). In plant and most cyanobacterial phytochromes, the reductase genes in the Agrobacterium genome were performed chromophore is bound via its ring A ethylidene side chain to a by using known protein sequences as templates (25, 26). Phyto- particular Cys residue. However, proteobacteria, Deinococcus, and chrome protein domains were identified by the SMART computer tool at the European Molecular Biology Laboratory (EMBL; some cyanobacteria have leucin, valin, isoleucin, or methionine at ͞͞ that position. For Deinococcus BphP, it was postulated that the http: smart.embl-heidelberg.de); the ‘‘PHY’’ domain was iden- tified by the PFAM tool of the Sanger Centre (www.sanger.ac.uk͞ chromophore is covalently attached to the neighboring, highly ͞ ͞ conserved, His (3). Phytochromes are synthesized in a red- Software Pfam ). Protein alignments were performed with absorbing form (Pr); a second thermostable far-red absorbing form CLUSTALX V.1.8 (27) with the default parameters, except in the (Pfr) that absorbs in the longer wavelength region is part of the case of ‘‘gap opening’’ and ‘‘gap extension’’, which were set to 50 photocycle. As a result, phytochromes appear as photoreversibly and 0.5, respectively. For secondary structure prediction, the photochromic pigments. For this reason phytochromes were the protein sequences were analyzed with the program PHD (28, 29) first plant photoreceptors to be detected and characterized (12). However, Pfr is not always infinitely stable. Some plant phyto- chromes revert from Pfr to Pr in darkness on a time scale of hours This paper was submitted directly (Track II) to the PNAS office. (13, 14). Dark-reversion has significant effects on the activity of Abbreviations: PCB, phycocyanobilin; BV, biliverdin; BphP, bacteriophytochrome photore- ceptor; Pr, red-absorbing form of phytochrome; Pfr, far-red absorbing form of phyto- plant phytochromes. The physiological activity of Arabidopsis phy- chrome; Agp1, -2, Agrobacterium phytochrome 1, -2; EMBL, European Molecular Biology tochrome B is reduced if dark-reversion is accelerated by a partic- Laboratory; Apo-Agp1, Agp1 apoprotein; SEC, size-exclusion chromatography; DTNB, ular mutation (15), and it is increased if dark-reversion is hindered 5,5Ј-dithiobis(2-nitrobenzoic acid); PEB, phycoerythrobilin; BV-Agp1, BV adduct of Agp1. by overexpressed interacting proteins (16). A comparable dark- *To whom reprint requests should be addressed. E-mail: [email protected]. 11628–11633 ͉ PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.152263999 Downloaded by guest on September 29, 2021 methanol stock solution were mixed and incubated for 5 min in darkness at room temperature. An aliquot of the protein then was separated from free bilin by using NAP-5 desalting-columns (Am- ersham Pharmacia). The columns were equilibrated in TE buffer; thereafter, 0.5 ml of the protein–chromophore mix was applied and allowed to enter the gel. The solution that eluted after application of 1 ml buffer was collected. This fraction contained protein and was depleted of low-molecular compounds such as free bilin. Spectra were recorded from 250 to 900 nm with a Uvikon 931 photometer (Kontron͞Biotek, Milano) and compared with those of the control (the sample before the separation). The protein peak at 280 nm was used to normalize both samples; the chromophore peak in the range above 550 nm was used for estimating the ratio of chromophore that eluted together with the protein. To test for covalent chromophore–protein interaction, the chromophore– protein mix was incubated with 1% SDS (final concentration) before the separation to dissociate noncovalently bound chro- mophore. The desalting columns were run in the presence of 1% SDS; the procedure was otherwise as described above. In control runs without the protein, 7 Ϯ 2% of the bilin appeared in the front Fig. 1. Chemical structure of biliverdin, phycocyanobilin, and fraction. This background value and slight variations between phycoerythrobilin. different runs made exact quantifications difficult if the fraction of bound chromophore was very low. However, comparisons between the different bilins were possible, and the results were confirmed by at the EMBL Predict-Protein server (www.embl-heidelberg.de͞ ͞ repeated experiments. For data presentation, the background value predictprotein ). was subtracted in those cases where the apparent value was low. The measurements performed without SDS were not critical in this Cloning of Agp1 and Mutants. Agp1 was amplified by PCR from respect, because free chromophore bound tightly to the matrix, and BIOCHEMISTRY A. tumefaciens, strain C58, by using TaKaRa Ex Taq polymerase it was thus quantitatively separated from the protein. (Takara Shozu, Otsu, Japan). The primers were GGAATTCA- TTAAAGAGGAGAAATTAACTATGCAAAGAGAGCG- Photoconversion and Extinction Coefficients. Spectra were recorded