Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter

Abstract

Responding to green and red light, certain cyanobacteria change the composition of their light-harvesting pigments, phycoerythrin (PE) and phycocyanin (PC). Although this phenomenon-complementary chromatic adaptation-is well known, the green light-sensing mechanism for PE accumulation is unclear. The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 (N. punctiforme) regulates PE synthesis in response to green and red light (group II chromatic adaptation). We disrupted the green/red-perceiving histidine-kinase gene (ccaS) or the cognate response regulator gene (ccaR), which are clustered with several PE and PC genes (cpeC-cpcG2-cpeR1 operon) in N. punctiforme. Under green light, wild-type cells accumulated a significant amount of PE upon induction of cpeC-cpcG2-cpeR1 expression, whereas they accumulated little PE with suppression of cpeC-cpcG2-cpeR1 expression under red light. Under both green and red light, the ccaS mutant constitutively accumulated some PE with constitutively low cpeC-cpcG2-cpeR1 expression, whereas the ccaR mutant accumulated little PE with suppression of cpeC-cpcG2-cpeR1 expression. The results of an electrophoretic mobility shift assay suggest that CcaR binds to the promoter region of cpeC-cpcG2-cpeR1, which contains a conserved direct-repeat motif. Taken together, the results suggest that CcaS phosphorylates CcaR under green light and that phosphorylated CcaR then induces cpeC-cpcG2-cpeR1 expression, leading to PE accumulation. In contrast, CcaS probably represses cpeC-cpcG2-cpeR1 expression by dephosphorylation of CcaR under red light. We also found that the cpeB-cpeA operon is partially regulated by green and red light, suggesting that the green light-induced regulatory protein CpeR1 activates cpeB-cpeA expression together with constitutively induced CpeR2.

Fig. 1.

Gene arrangement, domain organization, and purification of CcaS and CcaR. (A) Arrangement of cyanobacteriochrome ccaS and cognate response regulator ccaR in the N. punctiforme genome. They are clustered with a rod linker of PE (cpeC), a rod-core linker of PC (cpcG2), and a transcriptional regulator of PE (cpeR1). (B) Domain architecture of CcaS (Upper) and CcaR (Lower). Phycocyanobilin is covalently bound to Cys147 of CcaS. His537 of CcaS and Asp51 of CcaR are conserved phosphorylation residues of the histidine–asparagine (His–Asp) phosphorelay system. DB, DNA-binding domain; His kinase, histidine-kinase domain; REC, receiver domain; TM, transmembrane. (C) Absorption spectra of the chromophore-binding GAF domain of N. punctiforme CcaS, which was purified from phycocyanobilin-producing E. coli. The spectrum of the green-absorbing form (Pg; red line) has a maximum at 536 nm, whereas that of the red-absorbing form (Pr; green line) has a maximum at 672 nm. (D) Photographs of Pg (Left) and Pr (Right). Pg is converted to Pr by irradiation with GL. Pr is converted to Pg by irradiation with RL. These photoconversions were fully reversible. (E) An SDS polyacrylamide gel showing the purified GAF domain of CcaS (lane 1) and full-length CcaR (lane 2). Fluorescence of covalently bound phycocyanobilin was detected for CcaS (lane 3) but not for CcaR (lane 4).

Fig. 2.

Pigmentation of PE and PC. Absorption spectra of wild type (A), ccaS (B), and ccaR (C) mutants that had been incubated under GL (green line) or RL (red line). The spectra were normalized to the PC peak maxima. The four major peaks are those for the absorbances of PE, PC, and Chl. Their photographs in liquid medium also are included in each graph.

Fig. 3.

Transcription of PE and PC genes. Northern blots of transcribed PE and PC genes in the wild type and in the ccaS (ΔccaS) and ccaR (ΔccaR) mutants that had been incubated under GL (green bars) or RL (red bars). Extracted RNA was separated, blotted, stained with methylene blue for control of rRNA, and then hybridized with ³²P-labeled probes of cpeC (A), cpcG2 (B), cpeB-cpeA (C), cpeE (D), cpcG1 (E), or cpcB-cpcA (F). Quantitation of each band is shown by the height of the corresponding bar below the gel lane. Wild type under GL was set to 100% to compare relative expression levels. Error bars indicate SD of three independent experiments. (G) RT-PCR analysis of cpeR1, cpcG2-cpeR1, cpeC-cpcG2-cpeR1, and cpeR2 using spanning primer sets shown in Fig S1. RT-PCR analysis of 16S rRNA also was included as positive (+RT) and negative (−RT) controls.

Fig. 4.

Specific binding of CcaR to cpeC-cpcG2-cpeR1 promoter. (A) Electrophoretic mobility shift assay of CcaR. Purified CcaR [0 ng (lane 1), 100 ng (lane 2), 200 ng (lane 3), or 300 ng (lane 4)] was mixed with the radiolabeled DNA fragment that encodes the promoter region of cpeC-cpcG2-cpeR1. For the competition assay, 300 ng of CcaR was mixed with the radiolabeled probe and 1 μg of unlabeled probe (lane 5) or 1 μg of a nonspecific DNA fragment (lane 6). (B) Alignment of the promoter regions of N. punctiforme (N. pun) cpeC and S. 6803 (S.6803) cpcG2. A conserved direct-repeat motif of [CTTTNCNATTT] × 2 (gray box) is designated the “G-box.“ The numbers to the right of the sequences indicate the nucleotide positions with respect to the start codon.

Fig. 5.

Model of complementary chromatic adaptation in N. punctiforme. (Left) Under GL, CcaS phosphorylates CcaR, and phosphorylated CcaR binds to the G-box of the cpeC-cpcG2-cpeR1 promoter, thus activating its expression. Both GL-induced CpeR1 and constitutively induced CpeR2 activate cpeB-cpeA expression. (Right) Under RL, CcaS dephosphorylates CcaR; dephosphorylated CcaR does not bind to the cpeC-cpcG2-cpeR1 promoter, and its expression remains suppressed. In this case, only CpeR2 activates cpeB-cpeA expression. GL- and RL-activated pathways are shown as green and red lines, respectively; the constitutively activated pathway is shown as a black line.