From the
Department of Biology, Texas A & M University, College Station, Texas 77843-3258,
|| Section of Molecular and Cellular Biology, University of California, Davis, California 95616
Received for publication, December 30, 2002
, and in revised form, March 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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We recently described the cikA gene of the cyanobacterium Synechococcus elongatus PCC 7942, which encodes a phytochrome-related protein with similarity to HPKs of bacterial two component signal transduction systems (24, 25, 26) and is involved in signal perception for resetting the circadian clock in response to environmental cues (27). CikA was first identified from a Tn5 mutant that has subtle alterations in light-responsive regulation of the photosystem II gene psbAII (28). The mutant has an expression level of a psbAII::luxAB reporter fusion strain that is 5080% of that in wild type (WT) under low light conditions and exhibits an exaggerated increase in expression upon exposure to higher light intensities. However, a more striking circadian phenotype was observed, because both the period length and amplitude of circadian rhythms of different luxAB reporter strains (kaiB::luxAB, purF::luxAB, and kaiA::luxAB) were reduced (27, 29, 30). In addition, the relative phasing of expression (timing of circadian peaks) was altered for some genes. The additive effect on period length reduction of cikA inactivation in both short and long period kai mutants of S. elongatus suggested that CikA and the Kai proteins have independent, nonoverlapping functions. A lack of phase resetting in the CikA-free mutant in response to a 5-h dark pulse that resets WT revealed that CikA functions as a sensor component on the input pathway of the circadian clock.
The deduced amino acid sequence of cikA reveals it to be a member of the extended phytochrome family (27). CikA exhibits a typical bacteriophytochrome domain structure with an N-terminal GAF domain (13) and a C-terminal histidine kinase motif possessing H-, N-, D/F-, and G-boxes, as defined previously by Stock et al. (31). By contrast, CikA lacks the PAS domains present in plant phytochromes (27, 32). Relative to the plant phytochromes PhyAE and the cyanobacterial bacteriophytochromes Cph1 of Synechocystis sp. PCC 6803 (18, 33) and RcaE of Fremyella diplosiphon (17), the GAF domain of CikA has a deletion near the usually conserved cysteine ligand for bilin binding; this makes it difficult to evaluate which residues, if any, play a role in bilin association with the CikA apoprotein. Indeed, CikA appears to lack both the conserved Cys residue that serves as the site for bilin attachment in plant phytochrome photoreceptors and the adjacent His residue to which phycocyanobilin (PCB) or biliverdin (BV) binds for some BphPs from nonphotosynthetic bacteria (22, 34).
The C-terminal region of CikA has similarities to receiver domains of two component response regulators (RR) such as PhoB. However, the mode of action of this RR domain may be different, because the conserved Asp residue necessary for phosphoryl transfer is missing (24, 25, 26, 27). In this regard, the CikA structure is similar to the family of so-called pseudo-response regulators (APRR) that has been identified in A. thaliana (35, 36, 37, 38). APRRs also possess N-terminal CONSTANS "Myb-related" motifs, which regulate transcriptional activity in eukaryotes, in addition to their pseudo-receiver domains. In contrast to the authentic response regulator proteins of A. thaliana, APRRs lack two of three invariant residues of receiver domains, which apparently prevents them from accepting phosphoryl groups from A. thaliana histidine kinases in vitro (35). APRR mutants also exhibit significant circadian phenotypes, such as delayed flowering and shortened periods of several rhythmic markers, including expression of the CAB (chlorophyll a/b-binding protein) gene (37, 38).
To determine whether CikA is an input photoreceptor to the cyanobacterial circadian clock and to clarify its mode of action, we made a variety of plasmid constructs for expression of tagged full-length and truncated CikA variants in Escherichia coli and in S. elongatus. After showing functionality of the His-tagged CikA variant in complementation experiments, we investigated the ability of affinity-purified CikA to covalently attach a bilin chromophore in vitro. We also tested the influence of other predicted domains on the autophosphorylation activity of the HPK. The results suggest regulatory roles for the N-terminal, GAF, and pseudo-receiver domains.
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EXPERIMENTAL PROCEDURES |
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E. coliE. coli strains DH10B, SG13009 (Qiagen, Valencia, CA) and BL21DE3 (Novagen, Madison, WI) were hosts for plasmids. The cells were cultivated in liquid or on 1.5% agar in LB medium. Where indicated, the antibiotics were added to the following concentrations: Ap, 150 mg liter1; Cm, 50 mg liter1; Gm, 10 mg liter1; Km, 50 mg liter1; and Sp, 50 mg liter1.
Construction of Mutant Strains and DNA ManipulationsPlasmid clone analysis, cleavage with restriction endonucleases, agarose gel electrophoresis, ligations, and transformation of E. coli strains were performed according to standard procedures (41). Plasmids, strains, and oligonucleotide primers for PCR amplification with Pwo polymerase (Roche Applied Science) are listed in Table I.
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Expression and Purification of His6- and Trx-tagged CikA and Truncated Versions of CikA in E. coliExpression of recombinant CikA6HIS was achieved using the Qiaexpress overexpression system (Qiagen) for synthesis of N-terminal His6-tagged proteins. A PCR-derived fragment using primers AMO558 and F0748, encoding the entire cikA gene except the first methionine, was cloned into SacI/SalI-digested pQE30/80L expression vector and used to transform E. coli SG13009. The cells were grown to OD600 nm = 0.5, induced with 0.9 mM IPTG, and grown for an additional 35 h at 25 °C to minimize the formation of insoluble protein. The cells were harvested and passed twice through a prechilled French press at 137.9 MPa. This extract was clarified by centrifugation for 30 min at 12,000 x g to prepare a lysate for affinity purification on nickel-nitrilotriacetic acid (Ni-NTA) matrix (Qiagen). For expression of truncated versions of CikA, we used the pET overexpression system (Novagen), generating fusion proteins that carry an N-terminal thioredoxin domain (Trx) for enhanced solubility as well as N- and/or C-terminal His6 tags for purification by a Ni-NTA matrix (Table II).
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Expression and Purification of Cik6HIS in S. elongatus5 ml of stationary phase AMC1006 was added to 500 ml of fresh BG-11M medium, incubated in an aquarium at 30 °C and a light intensity of 200 microeinsteins m2 s1, and bubbled with 1% CO2 in air. When the cells were grown to an OD750 nm of 1.5, production of CikA6HIS was induced with 2 mM IPTG. After 48 h, the cells were harvested, washed, and resuspended in 20 ml of Buffer 1 (25 mM Tris-HCl, pH 7.5, 500 mM NaCl). After passage through a prechilled French press at 137.9 MPa, a cleared lysate was prepared by centrifugation at 100,000 x g for 60 min. CikA6HIS in the soluble protein fraction was diluted to 200 ml in Buffer 1. Protein was bound to Ni-NTA-agarose (Qiagen), washed, and eluted with Buffer 1 that contained 50 and 250 mM imidazole, respectively.
Assay of Bioluminescence in 96-well Microtiter PlatesInocula of AMC913, AMC914, AMC1006, and the corresponding control strains were streaked from solid medium onto 300-µl BG-11 M agar pads in 96-well plates. After applying a 12-h dark pulse to the cultures to synchronize the circadian clock, the psbAI::luxAB and kaiB::luxAB reporter strains were incubated in continuous light, and bioluminescence was measured using a Packard TopCount luminometer (Packard Instrument Company, Meriden, CT) (29). Period length and amplitude were calculated using I&A and FFT-NLLS software (www.scripps.edu/cb/kay/shareware/) (30).
[-32P]ATP Autophosphorylation AssaysCikA autophosphorylation assays were done as previously described (42, 43), except that gels were dried for exposure to phosphorimaging plates in most cases. Briefly, the standard phosphorylation buffer stock to which purified protein was added was 50 mM Tris, pH 7.5, 100 mM KCl, 2 mM dithiothreitol, 0.1 mM ATP (unlabeled), and 0.15 µM [
-32P]ATP. Chemical stability measurements were performed as described by McCleary and Zusman (42) with minor modifications. The assays to test for transphosphorylation from 32P-labeled CikA6HIS or Trx-
RR (Table II) to the receiver domain of CikA were done according to previously published protocols (44). The activities were quantitated using a Fujix BAS 2000 phosphorimaging system.
In Vitro Bilin Lyase Assays with Purified CikAThe assays were performed according to a previously published protocol with purified phycocyanobilin, phytochromobilin, and phycoerythrobilin (12). Biliverdin was obtained as described previously (45). After the reaction, the mixtures were concentrated by acetone precipitation and applied to 0.1% SDS-8% PAGE. The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immun-BlotTM; Bio-Rad) for 3 h at 45 V. After incubation of the PVDF membranes with 1.3 M zinc acetate, the bilin adducts were visualized by fluorescence with a Molecular Dynamics Storm 860 instrument with a setting of red fluorescence and Photomultiplier tube voltage = 1000.
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RESULTS |
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Assaying GAF as a Chromophore Attachment Domain CikA6HIS, Thioredoxin-tagged CikA (Trx-CikA) and its variants were assayed for chromophore ligation with purified PCB in vitro. In this experiment, we decreased the PCB concentration to 2.0 µM to reduce nonspecific binding. As shown in Fig. 4 (A and B), recombinant proteins that included the GAF domain (CikA6HIS, Trx-CikA, RR,
ATP,
HPK, N+GAF, and
N) emitted obvious zinc-induced fluorescence, indicating the presence of protein-chromophore interaction. However, the
GAF variant had a reduced signal, only slightly higher than background negative controls (Fig. 4B). We concluded that GAF, but no other single motif, is important for bilin attachment. However, a Trx-GAF variant, composed of only GAF and the Trx tag, produced no signal above that obtained with a Trx-RR, in which the only the receiver-like domain of CikA is fused to Trx (Fig. 4C). These results suggest that the GAF domain we defined is not sufficient for bilin lyase activity or that the domain does not fold stably when removed from its natural protein context.
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Although lyase assays showed that CikA can bind a bilin in vitro, CikA6HIS isolated from S. elongatus gave no fluorescent signal on a zinc blot (Fig. 4D) and showed no spectral evidence of a chromophore (data not shown). This negative result was obtained whether the protein was isolated from cells under normal illumination or in darkness. We reasoned that CikA might associate noncovalently with a bilin in vivo and that the co-factor might be lost during purification. However, co-expression of the protein with either PCB or BV in E. coli, under conditions in which other bacteriophytochromes form photoactive adducts (46, 47), showed no evidence of association between CikA and the chromophores (Fig. 5). We concluded from these various lines of data that CikA is unlikely to form a biliprotein complex naturally in S. elongatus.
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Autophosphorylation Assays with Overexpressed and Partially Purified CikA6HISWe performed in vitro autophosphorylation assays with affinity-purified CikA6HIS apoprotein under various conditions to test whether the protein has authentic HPK activity as predicted from its sequence. CikA6HIS showed strong autophosphorylation, with a temperature optimum around 25 °C and detectable activity at 0 °C (Fig. 6A). Maximum phosphorylation was detected after 10 min of incubation at 25 °C under these conditions (Fig. 6B). Autophosphorylation activity showed a strong dependence on substrate concentration (Fig. 6C), and cold ATP competed with [-32P]ATP as expected for an enzymatic activity (Fig. 6D). The phosphoryl linkage remained stable under basic conditions (100% signal remaining; Fig. 6E, lane 1) and decreased after treatment with hydroxylamine (14% signal remaining; Fig. 6E, lane 2) and under acidic conditions (11% signal remaining; Fig. 6E, lane 3). This result is consistent with a phosphoryl linkage to the His393 determined as an H-box by sequence alignment (27). The holoprotein generated in vitro by addition of 2 or 4 µM PCB showed the same autophosphorylation activity as the apoprotein purified from E. coli, and CikA6HIS purified directly from the cyanobacterium showed autokinase activity comparable with that observed for E. coli-derived protein (data not shown).
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Influence of other CikA domains on HPK ActivityThe assay conditions described for partially purified CikA did not produce robust activity when the protein was more highly purified, either as a His6 or Trx fusion. The addition of 2 µg/µl (w/v) BSA to the reaction mixture allowed a linear increase of autophosphorylation of CikA variants up to 120 min at 30 °C, whereas the mixture without BSA reached a plateau at 15 min (data not shown). Therefore, the following reactions were carried out in the presence of 2 µg/µl BSA at 30 °C for 1 h.
To identify which regions of the protein influence autophosphorylation activity, we produced variants that are missing specific residues or motifs and performed in vitro autophosphorylation assays. Because truncated CikA variants were soluble only if fused to Trx, we first compared autophosphorylation of full-length CikA6HIS and Trx-CikA (Fig. 7). The addition of the Trx tag dropped autophosphorylation to 2025%; this variant served as the control for the following mutant derivatives.
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All of the recombinant proteins that have a critical deficiency of the HPK domain (HPK and Trx-RR) or a point mutation at the expected phosphoryl acceptor His residue in the kinase domain (Trx-H393A) were completely defective in autophosphorylation activity (Fig. 7, B and C).
GAF, which conserves an intact HPK domain (Fig. 2), also had no autophosphorylation signal (Fig. 7, B and C). Additionally, only an extremely weak signal was detected after 60 min from
N (Fig. 7, B and C), which retains all recognizable motifs of the protein (Fig. 2), and was lower than the detectable sensitivity at 30 min (data not shown). In sharp contrast, removal of the receiver domain enhanced autophosphorylation activity by about 2-fold over CikA6HIS, and more than 10-fold over the more comparable Trx-CikA full-length control. We confirmed the linearity of these single time point experiments by performing time courses for the active CikA variants (CikA6HIS, Trx-CikA, and
RR; Fig. 8A), except for
N, which needs longer than 60 min to visualize activity. The assays showed a linear increase of autophosphorylation as expected for kinase activity dependent on the HPK domain, as depicted for CikA6HIS in Fig. 8B.
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Absence of Internal Phosphoryl Transfer in CikAWe tested whether CikA transfers a phosphoryl group from the HPK domain to the atypical receiver domain in transphosphorylation assays. In the first strategy, we used autophosphorylated full-length CikA6HIS or RR as a phosphodonor and Trx-RR as a potential acceptor. No phosphorylation of Trx-RR was observed (data not shown). Because HPKs tend to dimerize, we reasoned that if phosphoryl transfer occurs in CikA, it might involve the H-box of one monomer and the receiver domain of the other. To test this possibility, we used
RR as a donor that is lacking its own receiver and Trx-H393A as a potential receiver that is incapable of autophosphorylation (Fig. 9A). No phosphorylation of Trx-H393A was observed (Fig. 9B). The same negative result was obtained when the Trx-H393A sequence was modified to substitute Asp for Ala at the site that most closely aligns with Asp of bona fide receivers of two-component phosphorelay systems (data not shown).
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DISCUSSION |
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No spectral activity was detected from CikA in the presence of PCB or PB, and recombinant CikA isolated from S. elongatus did not co-purify with a bound bilin. Recent studies with phytochromes and bacteriophytochromes that lack the conserved cysteine site of attachment have established that noncovalent adducts are photochromic and could serve as photoreceptors (48, 49, 50). However, co-expression of CikA with BV or PCB in E. coli did not produce a pigmented holoprotein (Fig. 5). It is possible that CikA does not serve as a photoreceptor itself but oligomerizes with a partner that binds a photoactive co-factor. One of the potential CikA partners identified in a yeast two-hybrid screen is a protein that carries a canonical GAF.2 Whether or not CikA naturally binds a co-factor that would serve as a chromophore, the GAF domain likely serves a regulatory role, as evidenced by the negative effect of GAF removal on HPK activity. We favor interaction with a partner as the regulatory role of the CikA GAF domain.
As is implicit in the name of the gene cikA (circadian input kinase A), the deduced amino acid sequence predicts a well conserved transmitter module typical of the sensor kinases of bacterial two-component signal transduction systems and is expected to supply a phosphoryl group to a specific, yet unidentified, response regulator(s) (27, 31). A histidyl residue in the region designated as the H-box is the site of autophosphorylation in HPKs. In CikA, this residue is His393, and a single point mutation at that site completely abolished the autophosphorylation with [-32P]ATP (H393A in Fig. 7). Additionally, the 32P-labeled protein obtained by in vitro autophosphorylation was alkali-tolerant in 3 M NaOH on PVDF membrane but was not stable to hydroxylamine or acidic conditions. These results also support the expectation that the histidyl residue in the H-box works as a typical phosphoacceptor residue (42).
In vitro autophosphorylation assays with a series of truncated CikA variants identified regions that affect autokinase activity. Purified full-length CikA tagged by His6 or Trx and some of the Trx-tagged truncated variants that carry the HPK domain showed clear autophosphorylation activity. However, deletion of the GAF domain or the N-terminal region adjacent to the GAF domain dramatically reduced the autophosphorylation despite the existence of an intact HPK domain (N and
GAF; Fig. 7, B and C). These variants were soluble and expected to be folded correctly. From this, we conclude that both the GAF domain and the undefined N-terminal region are important for activating the kinase (Fig. 7D). Interaction of each domain with a protein partner or small organic molecule (bilin or other) could provide a mechanism to modulate the activating functions. Fusion of Trx to the CikA N terminus, which was used to achieve soluble protein expressed in E. coli, reduced the activity as compared with CikA6HIS. Because Trx is a larger tag than His6, the physical structure of the N-terminal region of CikA may have been modified. This is consistent with the finding that a functional domain that influences kinase activity resides in this portion of the protein.
The C terminus of CikA contains a similar sequence to the receiver domains of response regulators involved in two-component signal transduction systems. Although this cryptic receiver domain conserves other key residues of the motif, the invariable aspartyl residue that is phosphorylated by a cognate HPK in bona fide receivers is absent from the sequence (27). From the negative results of various in vitro trans-phosphorelay experiments using the HPK domain and the C-terminal domain of CikA (such as in Fig. 9), we conclude that the latter is a pseudo-receiver and does not accept a phosphoryl group from the kinase domain of CikA. The N-terminal domain of the circadian clock protein KaiA has been identified structurally as a pseudo-receiver that is unlikely to be involved in phosphorelay activity (51). Sequence also predicts a pseudo-receiver, unrelated to phosphoryl transfer, in the A. thaliana putative clock component TOC1 and its APRR family members (35, 36, 37, 38). We predict that each of these acts as a protein-protein interaction domain that induces conformational changes in another domain to modulate its activity. Unlike the N-terminal region and GAF domain, elimination of the pseudo-receiver domain increased autokinase activity 10-fold over full-length Trx-CikA. These results suggest that the receiver domain functions as an autophosphorylation suppressor of the CikA HPK transmitter but not as a phosphoryl receiver. This is consistent with a role for the pseudo-receiver similar to that in AmiR, an antiterminator whose RNA-binding domain is regulated by interaction of the pseudo-receiver with its ligand-binding partner AmiC (52).
Bacteriophytochrome two-component systems are present in other cyanobacterial species and have been characterized best from Synechocystis sp. PCC 6803. Cph1 carries a typical GAF domain that includes the critical cysteine residue for bilin attachment and has an HPK domain; Rcp1 is its response regulator (19). Like plant phytochromes, Cph1 exists in photo-convertible Pr and Pfr forms that are generated by absorption of red or far-red light, respectively. Cph1 autophosphorylation is attenuated by red light and increased by far-red light; the phosphorylated Pr form transfers a phosphoryl group to Rcp1 (19). In the case of plant phytochromes, light affects a Ser/Thr protein kinase activity (15, 16). CikA reconstituted in vitro with purified PCB had the same level of autophosphorylation activity as CikA apoprotein (data not shown) and showed no spectral activity. These differences between CikA and phytochromes/bacteriophytochromes suggest a very distinct mechanism for modulating signal transduction, despite an expected shared reliance on phosphoryl transfer to a partner response regulator protein.
CikA plays a central role in transducing environmental information to the cyanobacterial circadian clock. In addition to acting as a mediator for light/dark signals (27), it is required for response to temperature and clock-generated signals as well.3 Whether multiple signals converge on CikA as an integrator before transduction to the clock or CikA is part of a complex that can sense various inputs directly remains to determined. The basic function of the protein as a kinase has been established; further understanding its role depends on discovering the other molecules with which it directly interacts.
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FOOTNOTES |
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These authors contributed equally to this work.
¶ Present address: Fakultaet Fuer Biologie, Universitaet Bielefeld, D-33615 Bielefeld, Germany.
** Supported by National Science Foundation Grant MCB 96-04511 and United States Department of Agriculture Grant AMD-9801768 to J. Clark Lagarias (University of California, Davis). Present address: Dept. of Biology, Indiana University, Bloomington, IN 47405.
To whom correspondence should be addressed: Dept. of Biology, 3258 TAMU, College Station, TX 77843-3258. Tel.: 979-845-9824; Fax: 979-862-7659; E-mail: sgolden{at}tamu.edu.
1 The abbreviations used are: HPK, histidine-protein kinase(s); WT, wild type; RR, response regulator(s); APRR, Arabidopsis pseudo-response regulator(s); Ap, ampicillin; Cm, chloramphenicol; Gm, gentamycin; Km, kanamycin; Sp, spectinomycin; IPTG, isopropyl--D-thiogalactopyranoside; Ni-NTA, nickel-nitrilotriacetic acid; PVDF, polyvinylidene difluoride; PCB, phycocyanobilin; BSA, bovine serum albumin.
2 J.-S. Choi, S. Canales, and S. S. Golden, unpublished data.
3 M. Katayama, S. S. Golden, H. Iwasaki, and T. Kondo, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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