Cyanobacteriochrome CcaS is the green lightreceptor that induces the expression ofphycobilisome linker proteinYuu Hirose*, Takashi Shimada†, Rei Narikawa*, Mitsunori Katayama‡, and Masahiko Ikeuchi*§

*Department of Life Sciences (Biology), University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan; †Life Science Research Center, ShimadzuCorporation, 3-1 Kanda-Nishikicho, Chiyoda-ku, Tokyo 108-8639, Japan; and ‡College of Industrial Technology, Nihon University, 1-2-1 Izumicho, Narashino,Chiba 275-8575, Japan

Edited by J. Clark Lagarias, University of California, Davis, CA, and approved May 8, 2008 (received for review February 28, 2008)

Cyanobacteriochromes are a newly recognized group of photore-ceptors that are distinct relatives of phytochromes but are foundonly in cyanobacteria. A putative cyanobacteriochrome, CcaS, isknown to chromatically regulate the expression of the phycobili-some linker gene (cpcG2) in Synechocystis sp. PCC 6803. In thisstudy, we isolated the chromophore-binding domain of CcaS fromSynechocystis as well as from phycocyanobilin-producing Esche-richia coli. Both preparations showed the same reversible photo-conversion between a green-absorbing form (Pg, �max � 535 nm)and a red-absorbing form (Pr, �max � 672 nm). Mass spectrometryand denaturation analyses suggested that Pg and Pr bind phyco-cyanobilin in a double-bond configuration of C15-Z and C15-E,respectively. Autophosphorylation activity of the histidine kinasedomain in nearly full-length CcaS was up-regulated by preirradia-tion with green light. Similarly, phosphotransfer to the cognateresponse regulator, CcaR, was higher in Pr than in Pg. From theseresults, we conclude that CcaS phosphorylates CcaR under greenlight and induces expression of cpcG2, leading to accumulationof CpcG2-phycobilisome as a chromatic acclimation system. CcaSis the first recognized green light receptor in the expandedphytochrome superfamily, which includes phytochromes andcyanobacteriochromes.

chromatic adaptation � phycocyanobilin � phytochrome � cyanobacteria �photoreceptor

Phytochromes (Phys) are photoreceptors that typically per-ceive red and far-red light and regulate a wide range of

physiological responses in plants, bacteria, cyanobacteria, andfungi (1). They exhibit reversible photoconversion between twodistinct forms: the red-absorbing form (Pr) and the far-red-absorbing form (Pfr). Their N-terminal photosensory region,which consists of Per-ARNT-Sim (PAS), cGMP phosphodies-terase/adenylyl cyclase/FhlA (GAF), and phytochrome domains,is highly conserved, but there are variations in the chromophoreof the linear tetrapyrrole, such as phytochromobilin, phycocya-nobilin (PCB), and biliverdin. It is reported that phytochromo-bilin or PCB is covalently anchored at a conserved cysteineresidue in the GAF domain (2, 3), whereas biliverdin is anchoredat another conserved cysteine residue in the N terminus of thePAS domain (4). The perception of light by Phys triggers a Z toE isomerization of the C15–C16 double bond between the C andD pyrrole rings as well as subsequent conformational changes ofthe chromophore and the apoprotein [supporting information(SI) Fig. S1] (5) which signal to downstream processes. Recentcrystallographic analyses of bacterial Phys (DrBphP and RpB-phP3) have revealed the three-dimensional structure of PAS andGAF domains in the Pr form (6–8). The biliverdin chromophoreis buried deep within a pocket in the GAF domain with aconfiguration of C5-Z,syn/C10-Z,syn/C15-Z,anti. Because theresidues in the chromophore-binding pocket are highly con-served, it was proposed that Phys share a common photocon-version mechanism, albeit with certain variations.

‘‘Cyanobacteriochromes’’ are a newly recognized group ofphotoreceptors that possess putative chromophore-binding GAFdomains that are related to but distinct from those of Phys (9).They were initially identified by mutational studies of somecyanobacteria, including a chromatic acclimation of phycobilip-roteins in Fremyella diplosiphon (10), a light-induced reset of thecircadian rhythm in Synechococcus elongatus PCC 7942 (11), andlight-induced mixotrophic growth (12) and phototaxis (13) inSynechocystis sp. PCC 6803. The pigment-binding and spectralproperties were first revealed in the protein SyPixJ1, which isessential for positive phototaxis in a motile substrain of Syn-echocystis. SyPixJ1, isolated from Synechocystis, bound a lineartetrapyrrole covalently and showed reversible photoconversionbetween the blue-absorbing form and the green-absorbing form(Pg), in contrast with the red-absorbing Pr and far-red-absorbingPfr of Phys (9). At first, the chromophore of SyPixJ1 wasassumed to be PCB because its GAF domain showed similarphotoconversion to the native protein when expressed in PCB-producing E. coli (14). However, subsequent denaturation anal-ysis of PixJ homolog in Thermosynechococcus elongatus, TePixJ,revealed that the chromophore is not PCB but its isomerphycoviolobilin, suggesting autoisomerization of PCB to phyco-violobilin (15, 16). The disconnection of the conjugated �-elec-trons between pyrrole rings A and B in phycoviolobilin issuggested to be one of the reasons why TePixJ absorbs consid-erably shorter wavelength compared with Phys.

Many cyanobacteria modulate the biosynthesis of a photosyn-thetic light-harvesting antenna, phycobilisome, in response toambient light conditions (17). It is reported that in some speciesgreen-light irradiation induces accumulation of a green-absorbing pigment, phycoerythrin, whereas red-light irradiationinduces accumulation of a red-absorbing pigment, phycocyanin,in the phycobilisome (18). Because this process, termed com-plementary chromatic adaptation, is photoreversible, phyto-chrome-class photoreceptor(s) has been postulated to regulatethis acclimation and has been explored by many laboratories. Inthe 1970s, photochromic pigments that showed slight green/redreversible conversion were found in extracts of some cyanobac-teria (phycochromes), but they proved to be partially disinte-grated phycobiliproteins (19, 20). Genetic studies of F. diplosi-phon suggested that a putative cyanobacteriochrome, FdRcaE, is

Author contributions: Y.H. and M.I. designed research; Y.H., T.S., R.N., and M.K. performedresearch; Y.H. and T.S. analyzed data; and Y.H. and M.I. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

The sequences reported in this paper have been deposited in Cyanobase or GenBank[accession nos. sll1473–5, ABI83649 (SyCcaS), slr0473, Q55168 (Cph1), ZP�00106627(NpCcaS), AAB08575 (FdRcaE), NP�285374 (DrBphP), and NP�948356 (RpBphP3)].

§To whom correspondence should be addressed. E-mail: mikeuchi@bio.c.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0801826105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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responsible for the red-light-induced expression of phycocyaningenes (21); however, its spectral properties and chemical speciesof the chromophore remain unknown (22). Further analysissuggested that another green-light-sensing system mainly regu-lates the expression of phycoerythrin genes (23).

Recently, the expression of phycobilisome linker gene cpcG2has been reported to be chromatically regulated by a cyanobac-teriochrome gene, ccaS, and a response regulator gene, ccaR, inSynechocystis sp. PCC 6803 (24). It has been shown that (i)expression of cpcG2 is up-regulated by green-orange light at550–600 nm, (ii) disruption of either ccaS or ccaR results in adramatic decrease in cpcG2 expression, and (iii) CcaR directlybinds to the promoter region of cpcG2 (M.K., X. X. Geng, M.Kobayashi, F. Yano, M. Kanehisa, and M.I, unpublished data).As CpcG2 forms atypical phycobilisome and is involved inenergy transfer to photosystem I, this chromatic regulation ofcpcG2 expression is considered as a new type of chromaticacclimation of Synechocystis (25, 26).

In this study, we demonstrate that CcaS undergoes photocon-version between the Pg and the Pr. The chromophore of CcaSappears to be PCB in a configuration of C15-Z and C15-E for Pgand Pr, respectively. The autophosphorylation of CcaS and thephosphotransfer to CcaR are up-regulated by green light. These

data provide insight into how cyanobacteria control the expres-sion of genes for phycobilisomes in response to changes in lightconditions.

ResultsGene Arrangement and Domain Architecture. The Cca gene clusteris composed of the four genes cpcG2 (Cyanobase ID sll1471), ahypothetical gene (sll1472), ccaS (sll1473–5), and an upstreamregulator gene ccaR (slr1584) with opposite orientation (Fig.1A). Note that ccaS is interrupted by insertion sequenceISY203g, generating two inactive ORFs (sll1473 and sll1475) inthe sequenced substrain, Kazusa, but is functional in the originalstrain, PCC (27, 28). The predicted CcaS protein consists of anN-terminal transmembrane helix, the cyanobacteriochrome-typeGAF domain, two PAS domains, and a C-terminal histidinekinase domain (Fig. 1B). It should be mentioned that a chro-mophore ligation motif in the GAF domain is Cys-Leu for CcaSinstead of Cys-His, as seen in plant Phys and other cyanobac-teriochromes. The second PAS domain is similar to the light/oxygen/voltage domain of plant phototropins, but it lacks thecysteine residue that forms a flavin-cysteine adduct upon blue-light excitation (29). CcaR is a typical response regulator of theOmpR class, which consists of an N-terminal receiver domainand a C-terminal DNA-binding domain. His-534 of CcaS andAsp-51 of CcaR are predicted to be involved in autophosphor-ylation and phosphotransfer of the two-component phosphore-lay system, respectively.

Purification and Spectral Analysis of the GAF Domain. We firstexpressed the GAF domain of CcaS (CcaS-GAF) with a His-tagin Synechocystis by using the trc promoter system (15). The bandof purified CcaS-GAF in SDS/PAGE strongly fluoresced afterincubation with Zn2� (Fig. 2A), indicating covalent binding of alinear tetrapyrrole chromophore (30). The purified proteinshowed no obvious peaks for CcaS in the absorption spectrumdue to the relatively abundant chlorophylls and some carote-noids. However, irradiation of this preparation with green lightcaused a significant decrease in the green region and a concom-itant increase in the red region of the absorption spectrum (Fig.S2). Irradiation with red light had the opposite effect on theabsorption spectrum. The difference spectrum of green-minus-red showed a photoconversion between the two distinct spectralforms: the Pg peaked at 535 nm and the Pr peaked at 672 nm(Fig. 2B, upper line). Such photoconversion was repeated severaltimes without any changes, indicating full photoreversibility.

We also purified CcaS-GAF from E. coli by using the chro-

Fig. 1. Molecular characterization of CcaS and CcaR. (A) Arrangement ofcpcG2, ccaS, and ccaR on the chromosome of Synechocystis. (B) Predicteddomain architecture of CcaS and CcaR. CcaS-GAF comprises 185 residues fromposition 53 to 237. �N-CcaS comprises 730 residues from position 24 to 753.TM, transmembrane; GAF, GAF domain; PAS, PAS domain; His-kinase, histi-dine kinase domain; REC, receiver domain; DNA-binding, DNA-bindingdomain.

Fig. 2. Purification and spectral analysis of the GAF domain. (A) CcaS-GAF was purified from both Synechocystis and PCB-producing E. coli. Cys141Ala mutant(CA) was prepared from PCB-producing E. coli. The SDS/PAGE gel was stained with Coomassie brilliant blue (CBB) after monitoring for Zn2�-induced fluorescence(Zn). (B) Difference absorption spectra of green-light irradiation minus red-light irradiation of CcaS-GAF isolated from Synechocystis (upper line) andPCB-producing E. coli (lower line). (C) Absorption spectra of the Pg (dashed line) and Pr (solid line) of CcaS-GAF from E. coli. (D) Photographs of solutions of Pgand Pr of CcaS-GAF from E. coli.

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mophore coexpression system (31). When PCB was coexpressed,the chromophore-binding holoprotein was purified as a singleband by SDS/PAGE that strongly fluoresced after incubationwith Zn2� (Fig. 2 A). On the other hand, only the chromophore-free apoprotein was isolated when biliverdin, a precursor ofPCB, was coexpressed (Fig. S3A). Irradiation with green lightyielded a peak at 672 nm with a slight shoulder at 614 nm (Pr),whereas irradiation with the red light yielded a peak at 535 nmwith a shoulder at 570 nm (Pg) (Fig. 2C). Fig. 2D shows a solutionof CcaS-GAF in Pg and Pr forms. When CcaS-GAF wasexpressed in the dark, E. coli cells turned brownish-red, indi-cating that Pg was initially formed as a ground state (Fig. S3B).The difference spectrum of Pr-minus-Pg showed three majorpeaks: positive peaks at 672 nm and 370 nm and a negative peakat 535 nm (Fig. 2B, lower line). These are identical to peaks seen

in the samples prepared from Synechocystis, suggesting thatCcaS-GAF binds PCB as a natural chromophore.

Identification of Chromophore, Its Binding Site, and Configuration. Toidentify the chromophore and its binding site, we isolated atryptic chromopeptide by HPLC and analyzed it by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-f light mass spectrometry (MALDI-QIT-TOF MS) (Fig. 3A).We detected signals of a desorbed chromophore at a m/z of587.30, a chromophore-desorbed peptide at m/z 2151.23, and achromophore-bound peptide at m/z 2736.51 (Fig. 3B). Thechromophore signal corresponds to the calculated mass of aprotonated PCB or its isomer (587.28). Further MS/MS analysisof these peptide fragments indicated that the peptide sequenceis AINDIDQDDIEICLADFVK, which contains a conservedcysteine residue (Cys-141) (Fig. S4). These signals also indicatethat the thiol group of Cys-141 was not carbamidomethylated,suggesting that Cys-141 forms a thioether bond with the chro-mophore. Site-directed mutagenesis of this cysteine to alanine(Cys141Ala) resulted in a loss of covalent binding of the chro-mophore even by detection of sensitive Zn2�-induced fluores-cence (Fig. 2 A). These results strongly suggest that PCB or itsisomer is covalently anchored through Cys-141 via a thioetherbond.

It is accepted that the tetrapyrrole configurations of Pr and Pfrof Phys are C5-Z/C10-Z/C15-Z (ZZZ) and C5-Z/C10-Z/C15-E(ZZE), respectively (32). These configurations can be distin-guished by absorption spectra after denaturation of holoproteins(16). To identify the chemical species and configuration of theCcaS chromophore, we denatured the holoprotein isolated fromE. coli with acidic urea and compared it with the cyanobacterialphytochrome Cph1, for which PCB is a natural chromophore (33,34). The spectrum of the denatured Pg of CcaS-GAF (�max � 661nm) matched well with that of denatured Pr of Cph1 (�max � 661nm, Fig. 4 A and B). The spectrum of the denatured Pr form ofCcaS-GAF (�max � 584 nm) matched closely with that of thedenatured Pfr of Cph1 (�max � 593 nm) if incomplete photo-conversion from Pr to Pfr is taken into consideration for Cph1.The difference spectra demonstrated that the chromophorephotoconversion of CcaS-GAF between Pg and Pr was identicalto that of Cph1 between Pr and Pfr (Fig. 4C). Denaturationanalysis of CcaS-GAF and Cph1 at neutral pH is consistent withthe result at acidic pH (Fig. S5). These results suggest that Pg andPr of CcaS-GAF bind PCB in the configuration ZZZ and ZZE,respectively.

Protein Kinase Assay. Because CcaS has a histidine kinase domainin the C-terminal region, we studied the effects of light irradi-ation on CcaS autophosphorylation activity. A truncated form of

Fig. 3. MALDI-QIT-TOF MS spectrum of the chromopeptide isolated by HPLC.(A) Whole MS spectrum of the purified chromopeptide. (B) Three major signalsindicated with black triangles in A are enlarged. The ion signal at m/z 587.30corresponds to the calculated mass of protonated PCB or its isomer (587.29).The m/z 2150.23 and 2736.51 correspond to the calculated mass of the chro-mophore-desorbed (2150.03) and chromophore-bound (2736.31) peptideAINDIDQDDIEICLADFVK, respectively.

Fig. 4. Denaturation analysis of CcaS and Cph1. Both absorbing forms of CcaS and Cph1 were denatured with 8 M urea at pH 2.0 in the dark. (A) Absorptionspectra of denatured Pg (dashed line) and Pr (solid line) of CcaS-GAF. (B) Absorption spectra of denatured Pr (dashed line) and Pfr (solid line) of Cph1. (C)Difference absorption spectra of CcaS-GAF (upper line) and Cph1 (lower line).

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CcaS (�N-CcaS) lacking the N-terminal transmembrane helix(Fig. 1B) was expressed in Synechocystis. We obtained near-pure�N-CcaS and confirmed the covalently bound tetrapyrrole byZn2�-induced fluorescence (Fig. S6A). The light-induced dif-ference spectrum was identical to that of the GAF domain alone(Fig. S6B). In the dark, no conversion of either Pg to Pr or Pr toPg was observed in incubation for 3 h at room temperature (datanot shown). When �N-CcaS was incubated with [�-32P]ATP, thelabeled phosphate was covalently incorporated into the proteinin a time-dependent manner. When phosphorylation was per-formed in the dark after full photoconversion, we observed thatthe Pr form was more efficiently phosphorylated (by �2.5-fold)than the Pg form (Fig. 5 A and B). When autophosphorylatedCcaS was incubated with the putative cognate response regulatorCcaR, the phosphate was rapidly transferred to CcaR (Fig. 5C).Again, CcaR was more efficiently phosphorylated (by �1.5-fold)than the Pg form. These results clearly demonstrate that greenlight enhances the CcaS autophosphorylation and phosphotrans-fer to CcaR.

DiscussionIn this study, we demonstrated that CcaS undergoes reversiblephotoconversion between Pg and Pr forms. The chromophore of

CcaS is suggested to be PCB in configurations ZZZ for Pg andZZE for Pr, which are covalently anchored at Cys-141. Theautophosphorylation of �N-CcaS and phosphotransfer to CcaRwere enhanced by green light.

CcaS shows unique green/red photoreversibility that contrastswith conventional red/far-red photoreversibility of Phys. Ourdenaturation analyses revealed that the chromophore configu-ration is ZZZ for Pg and ZZE for Pr in CcaS, whereas it is ZZZfor Pr and ZZE for Pfr in Phys. This finding indicates thatabsorption peaks of both forms of CcaS are ‘‘blue-shifted’’ fromthose of Phys, suggesting that the conjugated �-electrons of thechromophore are shortened or distorted in the GAF domain ofCcaS. GAF domains that are homologous to CcaS are found inthe ortholog NpCcaS of the filamentous N2-fixing cyanobacte-rium Nostoc punctiforme PCC 73102 and in a putative cyanobac-teriochrome FdRcaE of another filamentous cyanobacterium, F.diplosiphon. We compared these sequences with the GAF do-mains of DrBphP and RpBphP3, whose three-dimensional struc-ture has been solved (Fig. 6).

CcaSs lack the ‘‘lasso’’ sequence in the figure-of-eight knotstructure, which stabilizes the interaction of the preceding PASwith the GAF domains in Phys (7) (Fig. 6, dotted box). Theabsence of the lasso sequence and the PAS domain in CcaS isconsistent with that the GAF domain alone is sufficient for thecomplete photocycle. Notably, all of the cyanobacteriochromes,including the phototaxis regulator, PixJ, lack the lasso sequenceas well as the preceding PAS domain.

In regard to the chromophore-binding motifs, CcaSs are highlydivergent from Phys but retain several key features. CcaSs havethe Cys residue (Cys-141 in CcaS) that covalently ligates thechromophore as in plant Phys but not in bacterial Phys (Fig. 6,asterisk). His-184 of CcaS is assumed to form a hydrogen bondwith the C19-carbonyl oxygen of ring D in Pg as found in Pr ofDrBphP and RpBphP3 (Fig. 6, filled triangle). Three aromaticresidues (Tyr-82, Phe-109, and Phe-145 in CcaS) are also con-served as the hydrophobic pocket that encompasses ring D (Fig.6, open triangles). These facts suggest that Pg of CcaS arepractically identical to Pr of Phys with regard to the configura-tion of ring D and its interaction with the apoprotein.

On the other hand, CcaSs lack the conserved Asp residue inthe DIP motif that forms hydrogen bonds with nitrogen atomsof the pyrrole rings A, B, and C via the pyrrole water moleculein Phys (6–8) and is involved in protonation of the chromophore(3, 35) (Fig. 6, boxed type). CcaSs also lack the highly conservedHis residue that is positioned next to the chromophore-ligatingCys residue and directly interacts with the pyrrole water from theopposite side of the Asp residue in Phys (Fig. 6, highlightedtype). These observations suggest that the environment aroundrings A, B, and C of CcaS may differ substantially from that of

Fig. 5. Protein kinase assays. (A) Autophosphorylation of Pg and Pr of�N-CcaS with [�-32P]ATP in the dark. The reaction products were subjected toSDS/PAGE followed by autoradiography. (B) Quantitation of the radiolabelincorporated into Pg (open circles) and Pr (solid circles) in A. (C) Transfer of thephosphate from �N-CcaS to CcaR. Autophosphorylated Pg and Pr of �N-CcaSwere incubated with the same amount of CcaR for 10 min in the dark.

Fig. 6. Protein alignment (CLUSTALX) of the chromophore-binding GAF domain of CcaS, its related cyanobacteriochromes, and Phys. CcaSs lack the sequencesthat form a part of the figure-of-eight knot in Phys (dashed-line boxed type). Cys-141 of CcaS (asterisk) is the chromophore-binding site. His-184 of CcaS (solidtriangle) may form a hydrogen bond with the nitrogen of the D ring of PCB. Three aromatic residues (Tyr-82, Phe-109, and Phe-145 in CcaS, open triangles) forma hydrophobic cavity to hold ring D. A highly conserved Asp (solid-line boxed type) and His (highlighted type) in Phys are replaced with Val and Leu in CcaSs,respectively. SyCcaS is CcaS of Synechocystis in this study. NpCcaS is the ortholog of SyCcaS in N. punctiforme. FdRcaE is a putative cyanobacteriochrome of F.diplosiphon. Cph1, DrBphP, and RpBphP3 are phytochromes of Synechocystis, Deinococcus radiodurans R1, and Pseudomonas palustris CGA009, respectively.

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the Phys. We speculate that these differences may cause distor-tion in the coplanar geometry of rings A, B, and C of PCB, whichdecouples the �-conjugation system, leading to the blue-shift inthe absorption of both forms of CcaS. Alternatively, it might bepossible that an unstable second bond shortens the �-conjuga-tion system of the chromophore in the native protein. To gaindirect information of the chromophore structure, it is necessaryto crystallize CcaS-GAF in both Pg and Pr forms.

We have demonstrated that the Pr form of �N-CcaS hashigher autophosphorylation activity than Pg. This contrasts withmany bacterial and cyanobacterial Phys such as Cph1, where thePr form shows higher autophosphorylation than Pfr (34).Namely, the Pfr is the active state for CcaS. We also demon-strated that CcaS directly transfers the incorporated phosphateto CcaR. These results suggest that CcaS phosphorylates CcaRunder green light, which may change the DNA-binding affinityof CcaR. Phosphorylated CcaR would bind to the promoterregion of cpcG2 and activate its transcription (24).

CpcG2 is a unique variant of the rod-core linker polypeptideof phycobilisome, CpcG1. CpcG1 assembles a typical phycobili-some supercomplex (CpcG1-PBS) consisting of central corecylinders and several peripheral rods, whereas CpcG2 containsa hydrophobic segment at the C terminus and forms the atypicalphycobilisome CpcG2-PBS consisting of peripheral rods but nocentral core (25). Fluorescence energy transfer analysis sug-gested that CpcG2-PBS preferentially transfers light energy tophotosystem I, in contrast to the predominant transfer from thetypical CpcG1-PBS to photosystem II (26). Considering theseprevious studies, the green-light-induced expression of cpcG2can be interpreted as the accumulation of CpcG2-PBS to com-pensate for the reduced light harvesting by photosystem Ichlorophylls because the green light excites PBS more efficientlythan chlorophylls. Thus, the chromatic regulation of CpcG2-PBSby CcaS and CcaR enables Synechocystis to coordinate theexcitation of the two photosystems to efficiently drive linearelectron transport.

The green-light-induced accumulation of phycoerythrin andthe red-light-induced accumulation of phycocyanin are typicalacclimations found in some cyanobacteria (18). This phenome-non (complementary chromatic adaptation) has been exten-sively studied in F. diplosiphon. FdRcaE of F. diplosiphon is aputative cyanobacteriochrome that is responsible for expressionof the cpc2 operon, which encodes inducible phycocyanin and itsassociated linkers (21). High sequence similarity in the GAFdomain between FdRcaE and SyCcaS suggests that FdRcaE alsoperceives green or red light reversibly, as does SyCcaS. However,genetic studies have suggested that FdRcaE phosphorylatesFdRcaF and FdRcaC under red light and induces expression ofcpc2, leading to accumulation of inducible phycocyanin (21).These findings suggest that FdRcaE is a red light receptor thatis distinct from the green-light-perceiving SyCcaS and NpCcaS.On the other hand, green-light-induced expression of phyco-erythrin genes cpeBA and cpeCDESTR is mainly controlled byyet an unidentified green-light-sensing system (23).

N. punctiforme is an N2-fixing cyanobacterium that accumu-lates phycoerythrin under green light but not phycocyanin underred light (a phenomenon characteristic of group II chromaticadaptation) (18). Interestingly, rod linker and regulator genes ofphycoerythrin (cpeC and cpeR) are clustered with NpccaS andNpccaR in the genome of N. punctiforme, which would supportthe idea that NpCcaS regulates green-light-induced accumula-tion of phycoerythrin. It is tempting to speculate that the CcaSortholog (or another green/red-reversible cyanobacterio-chrome) regulates phycoerythrin accumulation in other cya-nobacteria including F. diplosiphon.

Materials and MethodsBacterial Strains and Growth Media. The original motile strain of the unicellularcyanobacterium Synechocystis sp. PCC 6803 was obtained from the PasteurCulture Collection. A clone showing positive phototaxis (substrain PCC-P) wasused for genetic engineering (36). Cells were grown in liquid BG11 medium(37) bubbled with air containing 1% (vol/vol) CO2 at 31°C. E. coli strain JM109was used for DNA cloning, and E. coli BL21 (DE3) was used for proteinexpression.

Plasmid Construction. A compatible plasmid, pKT271, which harbors a hemeoxygenase gene, ho1, and a ferredoxin phycocyanobilin oxidoreductase gene,pcyA, of Synechocystis, was used for PCB biosynthesis in E. coli (31). pKT270,which harbors only ho1, was used for biliverdin biosynthesis in E. coli (31).Coding DNAs were amplified by PCR with the following primers: 5�-GGCATATGCGCCAATCTTTAAACTTGG-3� and 5�-GCTCGAGATCTCATTGCT-GGGTGCGTTTTTC-3� for CcaS-GAF, 5�-GGCATATGCGCCAATCTTTAAACT-TGG-3� and 5�-GCGTCGACATGTTTCTACGCCTA-3� for �N-CcaS, and 5�-TTCATCTCCAGAGACTTC-3� and 5�-GAGTGAAGCAGAAGTCAC-3� for CcaR.The cloned PCR products were confirmed by nucleotide sequencing and thenexcised with NdeI and BamHI. They were subcloned into a pET28a vector(Novagen) for expression in E. coli and into pTCH vector for expression inSynechocystis (15). A plasmid, pKT214, was used for the expression of His-tagged Cph1 (31). In all cases, a polyhistidine tag was fused to the N terminus.Cys141Ala mutant was created by a QuickChange site-directed mutagenesiskit (Stratagene) according to the manufacturer’s instruction.

Protein Expression and Purification. Synechocystis cells expressing the His-tagged CcaS-GAF and �N-CcaS were grown in 8 liters of BG11 medium with 20�g�ml�1 chloramphenicol for 10 days and were harvested by centrifugation.Cells were resuspended in disruption buffer (0.1 M NaCl, 10% glycerol, 20 mMHepes-NaOH, pH 7.5) and stored at �80°C. Cells were thawed on ice and thenbroken by French press (no. 5501-M, Ohtake) three times at 1,500 kg�cm�2. Thehomogenate was centrifuged at 12,000 � g for 10 min, and its supernatantwas centrifuged again at 146,000 � g for 60 min at 4°C. The supernatant wasloaded onto a nickel-affinity His Trap chelating column (GE healthcare).Proteins were eluted by a linear gradient of imidazole.

His-tagged CcaS-GAF was expressed in E. coli BL21 (DE3) carrying pKT271 orpKT270, which was grown in LB containing 0.05 mM 5-aminolevulinic acid,0.05 mM FeCl3, 20 �g�ml�1 kanamycin, and 20 �g�ml�1 chloramphenicol. Whencells were grown at 37°C to OD600 � 0.4–0.8, 10 �M isopropyl thio-�-D-galactoside was added, and cells were incubated at 25°C overnight. His-tagged CcaR was expressed in E. coli BL21 (DE3) as described above withoutaddition of 5-aminolevulinic acid, FeCl3, or isopropyl thio-�-D-galactoside.His-tagged Cph1 was expressed as described (31). Cells were harvested bycentrifugation and stored at �80°C. Cells were thawed in disruption buffer onice and then broken by French press twice at 1,500 kg�cm�2. The homogenatewas centrifuged at 146,000 � g for 30 min at 4°C, and the supernatant wassubjected to nickel-affinity chromatograph as described above.

SDS/PAGE and Zinc-Induced Fluorescence Assay. Purified proteins were solubi-lized with 2% lithium dodecyl sulfate, 60 mM DTT, and 60 mM Tris�HCl (pH 8.0)and were subjected to SDS/PAGE on a 15% (wt/vol) polyacrylamide gel. For thezinc-induced fluorescence assay, the gel was soaked with 20 mM zinc acetateat room temperature for 30 min in the dark, and fluorescence was detectedthrough a 605-nm filter (FMBIO II, Takara Bio) upon excitation at 532 nm. Thegel was then stained with Coomassie brilliant blue R-250 (Bio-Rad).

Spectral Analysis. Absorption spectra were measured at room temperature byusing a UV-2400PC spectrophotometer (Shimadzu). For excitation, light-emitting diodes emitting at 515 nm (E1L53-AG0A, Toyoda Gousei) were usedas green light, and light-emitting diodes emitting at 700 nm (L700–03AU,Epitex) were used as red light. For denaturation analysis, each spectral form ofCcaS and Cph1 was denatured in 8 M urea (pH 2.0 or 7.5) for a few minutes atroom temperature in the dark.

Mass Spectrometry. In-gel digestion of CcaS-GAF prepared from PCB-producing E. coli with trypsin, isolation of the chromopeptide by HPLC, andmass spectrometric analysis were performed as reported in refs. 14 and 15).

Protein Kinase Assays. For autophosphorylation, the preirradiated Pg or Prform of �N-CcaS (2 �g) was incubated in a reaction buffer (50 mM Tris�HCl(pH 8.0), 50 mM NaCl, 10 mM MgCl2, and 2.5 �M ATP containing 185 kBq of

9532 � www.pnas.org�cgi�doi�10.1073�pnas.0801826105 Hirose et al.

[�-32P]ATP) at 25°C in the dark. The kinase reaction was stopped by additionof 2% lithium dodecyl sulfate, 60 mM DTT, and 60 mM Tris�HCl (pH 8.0). Forphosphotransfer, 2 �g of �N-CcaS was autophosphorylated for 25 min andthen incubated with 2 �g of CcaR and unlabeled ATP (final 0.5 mM) for 10min at 25°C in the dark. Samples were then subjected to SDS/PAGE, and thegel was washed with 45% methanol and 10% acetic acid, dried, and

exposed onto an image plate. The intensity of each band was measuredwith BAS-2500 (Fuji).

ACKNOWLEDGMENTS. We thank Prof. Takayuki Kohchi (Kyoto University) forthe gift of pKT271, pKT270, and pKT214. This work was supported by Grants-in-Aid for Scientific Research (to M.I.).

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