www.sciencemag.org/cgi/content/full/science.1256963/DC1

Supplementary Materials for

Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light

Fei Gan, Shuyi Zhang, Nathan C. Rockwell, Shelley S. Martin, J. Clark Lagarias,

Donald A. Bryant*

*Corresponding author. E-mail: dab14@psu.edu

Published 21 August 2014 on Science Express DOI: 10.1126/science.1256963

This PDF file includes

Materials and Methods Figs. S1 to S18 and S2 Tables S1 and S2 References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1256963/DC1)

Table S1. Genes encoding subunits of Photosystem I (PS I), Photosystem II (PS II), and phycobilisomes (PBS) and related proteins in strain JSC-1. Table S2. Complete transcription profiling data for transcripts for strain JSC-1 cells grown in white light and far-red light.

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Materials and Methods Source Organism and Cultivation Conditions

Leptolyngbya sp. strain JSC-1 (alternative name, Marsacia ferruginose; ATCC accession BAA-2121 (24); hereafter JSC-1) was isolated from a floating mat at ~45°C in the vicinity of La Duke Hot Springs, near Gardiner, Montana, USA. The roadside effluent channel that was sampled is fed by two hot springs that correspond to thermal features LDHSNN001 and LDHSNN002 in the Yellowstone Park geothermal inventory (24). JSC-1 was routinely grown at 200 to 250 µmol photons m-2 s-1 at 38°C in liquid medium BG-11 plus 4.6 mM HEPES buffer at pH 8.0, which was supplemented with 75 µM ferric ammonium citrate (standard conditions). The strain was maintained on the same medium solidified with 1.5% (w/v) BactoAgar (Difco).

Six different light sources were employed for the growth of strain JSC-1 in this study. Maintenance cultures were kept under continuous, cool-white fluorescent light (denoted “White Light” (WL)) at an irradiance of ~200 µmol photons m-2 s-1. “Red Light” was either provided by light-emitting diodes (LEDs) (Marubeni, Santa Clara, CA) with emission centered at 645 nm (645-nm light) or by filtering fluorescent light with a red plastic filter (RL); the transmittance spectrum of the red filter is shown in Fig. S7. The irradiance value for the 645 nm-LED light was ~80 µmol photons m-2 s-1, and the RL value used was ~67 µmol photons m-2 s-1. White fluorescent light was filtered with a green plastic filter to produce “Green Light” (GL; ~45 µmol photons m-2 s-1); the transmittance spectrum of this filter is also shown in Fig. S7. Finally, “Far-Red Light” was either provided by LEDs (Marubeni, Santa Clara, CA) with emission centered at 710 nm (710-nm light; ~18 µmol photons m-2 s-1) or by filtering tungsten light with a combination of the red and green plastic filters described above (FRL; ~10 µmol photons m-2 s-1). This filter combination transmits light with wavelengths greater than ~690 nm (see Fig. S7). Heterologous Expression of Phytochromes in Escherichia coli and Spectroscopic Analysis of Recombinant Phytochromes

The full-length rfpA gene was amplified from JSC-1 genomic DNA with primers rfpAF (5ʹ′- AGCCAGGATCCGATGCTGGGGAACCGCAACTC-3ʹ′) and rfpAR(5ʹ′-AGCTCGAATTCTCA TAGTGTTGAGTCATGATG-3ʹ′), and the resulting amplicon was inserted into the BamHI and EcoRI sites of plasmid pCDFDuet-1 (Novagen, Madison, WI). The DNA sequence encoding the predicted GAF domain (corresponding to the N-terminal 211 amino acids) was amplified using primers rfpAF and GAFR (5ʹ′-AGCTCGAATTCTCACGCATGCAGCAGAGTGGC-3ʹ′), and the product was also inserted into the BamHI and EcoRI sites of pCDFDuet-1. The resulting recombinant plasmids, either pRfpA or pGAF, were transformed into an E. coli BL21(DE3) strain that produces phycocyanobilin (30). Heterologous expression of His-tagged RfpA or GAF was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside into a freshly grown culture (OD600 nm = ~0.6-0.8) followed by further incubation at 18°C overnight. Cells were harvested and resuspended in Buffer A (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) and disrupted by three passages through a chilled French pressure cell at 138 MPa. The whole-cell lysates were centrifuged at 35,000 × g for 0.5 hour to pellet unbroken cells and larger debris. The supernatants containing recombinant proteins were loaded

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onto pre-equilibrated columns containing Ni2+-NTA affinity resin (Goldbio, St. Louis, MO), and sequentially washed with Buffer A containing 20 mM imidazole and then 40 mM imidazole. Recombinant proteins were eluted with Buffer A containing 250 mM imidazole. Fractions were collected and analyzed by SDS-PAGE and Zn-staining (30). Purified proteins were dialyzed against Buffer A to remove imidazole for further analysis. Phylogenetic Analysis

Maximum-likelihood phylogenetic analysis of knotless phytochromes used PhyML-Structure (49) with knotted phytochromes from Synechocystis sp. PCC 6803 (50) and from Leptolyngbya sp. JSC-1 as an outgroup (n=3). The PAS-GAF-PHY and GAF-PHY crystal structures for knotted and knotless phytochromes from Synechocystis sp. PCC 6803 (51, 52) were used as structural references (PDB accessions 2VEA and 4BWI, respectively). MUSCLE (53) was used to construct the protein sequence alignment, and secondary structure assignments and solvent accessibility were evaluated using the DSSP (Dictionary of Secondary Structure of Proteins) algorithm. An in-house script was then used to match the alignment and DSSP output, to remove positions with ≥5% gaps, and to generate formatted alignment files for input to PhyML-Structure (Supp. Fig. S4 shows this alignment with working sequence names replaced with accession information). The maximum-likelihood tree (Fig. 1) was calculated using an EX_EHO substitution model with four substitution categories, and with maximum-likelihood estimates used for proportion of invariable sites and for the gamma distribution shape parameter. Domain architecture of full-length proteins was assigned using Genbank accession records and/or BLAST searches with full-length protein sequences. Pigment Extraction, HPLC Analysis, Mass Spectrometry

Pigments were extracted from cells and analyzed by reversed-phase HPLC on an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) with an analytical Discovery C18 column (4.6 mm × 25 cm) (Supelco, Sigma-Aldrich, St. Louis, MO). The gradient elution program [B, minutes] using Solvent A (methanol:acetonitrile:H2O = 42:33:25) and Solvent B (methanol: acetonitrile: ethyl acetate = 50: 20: 30) was set as [30%, 0], [100%, 50], [100%, 58], [30%, 60] at a flow rate 1 mL min-1. Elution of pigments was monitored at 705 nm, the absorbance maximum of Chl f, or at other wavelengths (e.g., 667 nm for Chl a, 695 nm for Chl d) as desired. For HPLC-MS and MS/MS analysis, pigments were extracted with methanol, dried under nitrogen gas, and redissolved in methanol:diethyl ether (1:1, v/v). Double-distilled H2O was added until phase separation occurred. The colored ether phase was collected and dried again under nitrogen gas. Pigments re-dissolved in methanol:acetone (2:7, v/v) were separated with the same HPLC system using a semi-preparative Discovery C18 column (10 mm × 25 cm) (Supelco, Sigma-Aldrich, St. Louis, MO) and a similar elution program at a flow rate 3.5 mL min-1. Fractions containing desired Chls were collected and acidified with 2% HCl (v/v) to convert the Chls into the corresponding pheophytins. The resulting pheophytins were extracted with diethyl ether and washed with 4% (w/v) NaHCO3 and double-distilled H2O. Concentrated pheophytin samples were submitted for mass determination on an LCT Premier mass spectrometer (Waters, Milford, MA). MS/MS analysis for ions of selected masses was performed on a 3200 QTRAP mass spectrometer

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(Applied Biosystems, Foster City, CA). The MS and MS/MS analyses of Chls and pheophytins was performed in the Proteomics and Mass Spectrometry Core Facility, the Huck Institutes of the Life Sciences, Penn State University, University Park. Absorption and Fluorescence Spectroscopy

Absorption spectra were recorded with a GENESYS 10 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) or a Cary 14 UV-Vis-NIR spectrophotometer modified for computer-controlled operation by OLIS, Inc. (Bogart, GA). Solutions of RfpA or its N-terminal GAF domain were treated with continuous illumination at 645 nm and 700 nm for 10 to 30 minutes, and the absorption was immediately measured. Whole cells of JSC-1 were homogenized prior to recording the absorption spectrum, and spectra were recorded through the opal glass side of the cuvettes to allow correction for scattering. Fluorescence emission spectra of whole cells at 77 K were measured with an SLM 8000C spectrofluorometer, modified for computer-controlled operation by OLIS, Inc. (Bogart, GA). Cells in exponential growth phase (OD750 nm = ~0.6 to 0.7 mL-1) were collected and resuspended in 50 mM Tris–HCl, pH 8.0 buffer. Glycerol was added to a final concentration of 60% (v/v). Cells were adjusted to a concentration of ~0.5 OD750 nm ml-1 and quickly frozen in liquid nitrogen. The excitation wavelength was set to 440 nm to excite Chls. For some experiments, the excitation wavelength was set to 550 nm or 590 nm, depending on the phycobiliprotein content of the cells (550 nm was used when cells contained large amounts of phycoerythrin).

Characterization of full-length RfpA was also carried out using a Cary 50 spectrophotometer modified to permit top-down illumination of the sample cuvette using actinic light from a filtered xenon source (54). For characterization, a 580 ± 20 nm bandpass interference filter and a long-pass filter (Schott Glass 695LP) were used. Protein samples were prepared using the heterologous expression system described above, and cell pellets were stored at -80°C until used. For purification, pellets were thawed at room temperature, resuspended in 20 mM HEPES-NaOH pH 7.5, 0.1 M NaCl, 10 % (w/v) glycerol, and lysed with five passages through a microfluidizer (M-110Y, Microfluidics, Westwood, MA) at 15,000 psi. The protein was then purified on Ni2+-NTA resin using an imidazole gradient (30-430 mM) with final dialysis into 20 mM sodium phosphate (pH 7.5), 50 mM NaCl, 1 mM EDTA. For comparison of RfpA to the knotless phytochrome photosensory core module of protein Npun_R4776 from N. punctiforme, a DNA fragment encoding amino acids 1-420 of Npun_R4776 was cloned into a previously described pBAD-intein-CBD system (54). Expression and purification of NpR4776-PCM followed previously described procedures (54), with final dialysis into TKKG buffer (25 mM TES-KOH, pH 7.5; 25 mM KCl; 10% glycerol).

The extent of formation of the far-red-absorbing (Pfr) form of RfpA at photoequilibrium was examined using a series of interference bandpass filters (40 nm FWHM) at varying center wavelengths: 500 nm, 580 nm, 600 nm, 650 nm, 670 nm, 700 nm, and 750 nm. RfpA was driven to photoequilibrium from either the red-absorbing (Pr) form (center wavelength <650 nm) or the Pfr form (center wavelength ≥650 nm). No photoconversion occurred with 750 nm light (Fig. S6B).

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Transcription Profiling by RNAseq Analysis Cells grown continuously in white light (WL) were inoculated into fresh B-

HEPES medium at OD750 nm = 0.05 and sparged with 1% (v/v) CO2. When cells grew to OD750 nm = 0.3~0.4, a 50-mL aliquot of culture was collected, rapidly centrifuged at 4°C, and the resulting cell pellet was quickly frozen with liquid nitrogen and stored at -80°C (WL sample). The remainder of the culture was switched to irradiation with far-red light (FRL). An aliquot (50 mL) of cells was collected after 24 hours, and the cells were harvested as described above (24-h far-red light). For RNA isolation, cell pellets were suspended in 50 mM Tris-HCl, pH 8.0. An equal volume of glass beads was mixed with the cell suspension, which was then subjected to a brief bead beating (4200 rpm for 10 seconds) with a mini-beadbeater (Biospec Products, Bartlesville, OK) to fragment the long filaments. Total RNA was extracted with phenol and further purified as described (55) with High Pure RNA Isolation Kit (Roche, Indianapolis, IN).

Following the manufacturer’s instructions, ribosomal RNA was removed using Ribo-ZeroTM rRNA Removal Kit for bacteria (Epicentre, Madison WI) to obtain enriched mRNA. Construction of cDNA library and Illumina sequencing was performed in the Genomics Core Facility, Penn State University, University Park. Libraries were prepared from enriched mRNA using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer's instructions with the exception that the poly-A selection steps at the beginning of the protocol were omitted. Samples were sequenced on an Illumina HiSeq 2500 instrument in Rapid Run mode by performing 50-nt single read sequencing according to the instructions of the manufacturer. Mapping against the Leptolyngbya sp. strain JSC-1 genome was performed using the BWA software package using scripts modified to accommodate the Illumina sequences (56). The resulting alignment files were further analyzed with self-developed scripts to extract expression levels for each gene as described previously (55). In the rRNA depleted WL sample, 26,308,915 reads were obtained, of which 24,775,560 reads (94%) were uniquely mapped to mRNA. For the rRNA-depleted FRL sample, 17,865,505 reads were obtained and 16,624,014 reads (93%) were uniquely mapped to mRNA. The draft genome sequence of strain JSC-1 was determined at the DOE Joint Genome Institutes, and the sequence has been deposited in GenBank under the accession number JMKF00000000. The RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRA) under accession number SRP041154. Isolation of Photosystem I, Photosystem II, and PBS on Sucrose Gradients

To isolate Photosystem I and Photosystem II, cells grown under different light conditions were harvested and resuspended in 50 mM Tris-HCl, pH 8.0 buffer. After homogenization, cells were broken by three passages through a chilled French pressure cell at 138 MPa. After removing unbroken cells and larger debris by centrifugation, thylakoid membranes were pelleted by ultracentrifugation at 125,800 × g for 1 hour. Membranes were resuspended, adjusted with 50 mM Tris-HCl, pH 8.0 buffer to ~0.4 to 0.5 mg chlorophyll mL-1, and solubilized by addition of 1% (w/v) n-dodecyl-β-D-maltoside (DDM) at 4°C for 1 hour. The resulting solution was centrifuged at 22,000 × g for 10 minutes to remove insoluble material. The supernatant was then loaded onto 5% to 20% (w/v) sucrose gradients containing 0.05% DDM which were centrifuged at 140,000

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× g for 12-16 hours at 4°C. Chlorophyll-containing fractions were collected from the resulting gradients (see Fig. 4A).

For phycobilisome isolation, cells were resuspended in 0.75 M K2HPO4-KH2PO4 buffer, pH 7.0, and disrupted by three passages through a French pressure cell at 138 MPa at room temperature. Triton X-100 (2% w/v, final concentration) was added to the lysed cell suspension, which was stirred until it became homogenous (~30 min). The resulting solution was centrifuged at 17,210 × g for 20 minutes to remove unbroken cells and large cell debris. The phycobilisome-containing supernatant fraction was loaded onto sucrose gradients made with sucrose solutions prepared with 0.75 M K2HPO4-KH2PO4 buffer, pH 7.0. The sucrose solutions and amounts were 3.0 mL of 2.0 M sucrose; 5.0 mL of 1.0 M sucrose; 7.0 mL of 0.75 M sucrose; and 7.0 mL of 0.5 M sucrose. The resulting gradients were centrifuged at 125,800 × g for ~16 to 20 hours at 20°C. Blue-colored fractions (see Fig. 5A) were collected from the sucrose gradients, dialyzed, and concentrated by ultrafiltration for further analyses.

Trypsin Digestion and Proteomics Analysis

To identify the protein composition of chlorophyll-containing fractions from sucrose gradient centrifugation by mass spectrometry, aliquots of gradient fractions containing an estimated total protein content of 20-30 µg were mixed with 90% (v/v) methanol to extract pigments and precipitate proteins. Protein pellets were collected by centrifugation and were resuspended with 100 µl of 50 mM NH4HCO3, pH 7.8, containing 6 M urea and 0.1% PPS Silent Surfactant (Expedeon, San Diego, CA), followed by sequential reduction by addition of 5 mM dithiothreitol and alkylation with 15 mM iodoacetamide. Trypsin/Lys-C Mix (Mass-spec grade, Promega, Madison, WI) was added into the protein solution in a ratio of ~1:25 (w/w) and digestion was allowed to proceed at 37°C for 4 hours. The resulting digests were diluted six-fold with 50 mM NH4HCO3, pH 7.8, and the digestion was continued overnight. For identification of proteins in isolated phycobilisomes, an aliquot of phycobilisomes representing about ~20-30 µg total protein was precipitated with 10% (w/v) trichloroacetic acid. The precipitate was collected by centrifugation and washed twice with acetone. The resulting pellet was resuspended with 100 µl of 50 mM NH4HCO3, pH 7.8, containing 6 M urea, and digested with trypsin after reduction and alkylation as described above. MS analysis of all peptides was performed at the Proteomics and Mass Spectrometry Core Facility, College of Medicine, Penn State University, Hershey, PA. Samples were analyzed on the 5600 Triple TOF mass spectrometer (AB SCIEX Framingham, MA) and peptides and proteins were identified using the Paragon algorithm (57), in ProteinPilot 4.5 beta software (AB SCIEX Framingham, MA), using settings of Thorough Search, carbamidomethylation, and biological modifications. The peptide spectra were searched against a protein database that contained 6942 predicted protein sequences created from the draft genome sequence for JSC-1, and which was concatenated to a list of 536 common laboratory contaminants that also included a reversed ‘decoy” version of the same database. In Table S1, “Unused ProtScore” values for identified proteins are presented. The “Unused ProtScore” is an easier-to-read form of the calculated confidence of the protein identification, with ProtScore essentially equal to -log(1-[(Percent Confidence)/100]). Each individual peptide identified can contribute at most 2.0 to the Unused ProtScore (2.0 would come from a 99% confident peptide identification,

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calculated from the number of matches of MS/MS peaks observed to theoretical MS/MS peaks for each peptide). Thus, an “Unused ProtScore” >1.3 indicates that a protein was positively identified with 95% confidence, and an “Unused ProtScore” >2.0 indicates that a protein was positively identified with 99% confidence by using evidence from at least two peptides. An “Unused ProtScore” >4.0 indicates that a protein was positively identified with 99.99% confidence by using evidence that included at least two highly confident peptide identifications. Oxygen Evolution Assay

Oxygen evolution rates for JSC-1 cells that had been continuously grown in 645-nm light and 710-nm light were measured using a Clark-type electrode as described (58). The temperature of the sample cell was maintained at 30°C with a circulating water bath. White actinic light was provided with a halogen lamp. A combination of red and green filters and the halogen light source were used to produce far-red actinic light as described in Fig. S7. The combination of a green filter (similar to GamColor655, Gam Products, Inc., Los Angeles, CA) and a red filter (GamColor250, Gam Products, Inc.) transmits light at wavelengths longer than 695 nm. Cells were washed with fresh growth medium and adjusted to ~28–30 µg ml-1 or ~8–10 µg ml-1 total chlorophyll for assays with white or far-red actinic light, respectively. The total chlorophyll content was calculated as described (59). Sodium bicarbonate (10 mM) was added to the cell suspensions as final electron acceptor.

References 49. S. Q. Le, O. Gascuel, Systematic Biol. 59, 277-287 (2010). 50. K. C. Yeh, S. H. Wu, J. T. Murphy, J. C. Lagarias, Science 277, 1505-1508. 51. L. O. Essen, J. Mailliet, J. Hughes, Proc. Natl. Acad. Sci. USA 105, 14709-14714

(2008). 52. K. Anders, G. Daminelli-Widany, M. A. Mroginski, D. von Stetten, L. O. Essen,

J. Biol. Chem. 288, 35714-35725 (2013). 53. R. C. Edgar, Nucl. Acids Res. 32, 1792-1797 (2004). 54. N. C. Rockwell, L. Shang, S. S. Martin, J. C. Lagarias, Proc. Natl. Acad. Sci. USA

106, 6123-6127 (2009). 55. M. Ludwig, D. A. Bryant, Front. Microbio. 2, 41 (2011). 56. H. Li, R. Durbin, Bioinformatics 25, 1754-1760 (2009). 57. I. V. Shilov et al., Mol. Cell Proteomics 6, 1638-1655 (2007). 58. C. T. Nomura, S. Persson, G. Shen, K. Inoue-Sakamoto, D. A. Bryant,

Photosynth. Res. 87, 215-228 (2006). 59. Y. Li, Y. Lin, P. C. Loughlin, M. Chen, Front. Plant Sci. 5, 1 (2014). 60. R. M. Alvey, A. Biswas, W. M. Schluchter, D. A. Bryant, J. Bacteriol. 193, 1663-

1671 (2011).

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Figure S1. Leptolyngbya sp. strain JSC-1 and Synechococcus sp. PCC 7335 exhibiting Type-III complementary chromatic acclimation (CCA). Cells were grown in red light (RL) and or in green light (GL) using the filters described in Figure S7.

RL

Synechococcus sp. PCC 7335

GL

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Figure S2. Physical map of a 21-gene region of the Leptolyngbya sp. strain JSC-1 genome that is expressed in far-red light. and absorption spectra of JSC-1 cells after acclimation to different light conditions. Color-coding for genes: core subunits of Photosystem I (psa, red); core subunits of Photosystem II (psb, green); core subunits of the PBS (apc, blue); knotless phytochrome photoreceptor (rfpA) and response regulators rfpB and rfpC (brown); conserved hypothetical protein (ORF, black).

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Figure S3. Gene clusters similar to the 21-gene cluster in Leptolyngbya sp. strain JSC-1 occur in 12 other cyanobacteria. Color-coding for genes: psa genes for core subunits of Photosystem I (red); psb genes for core subunits of Photosystem II (green); apc genes for core subunits of the PBS (blue); rfp genes for the knotless phytochrome (RfpA) and response regulators RfpB and RfpC (brown); conserved hypothetical protein (Hyp, black); all other genes are shown in gray. The locus designations for the genes for JSC-1 are the same as those in Fig. S2 and are included here to assist the reader in interpreting the gene patterns. The rfpA genes in Fischerella thermalis and Fischerella muscicola are divided into two open reading frames because of sequencing or assembly errors. In

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Chroococcidiopsis thermalis PCC 7203, the psaF2-psaJ2 genes are located more than 1 Mb from this gene cluster and are apparently cotranscribed with psaB3 (see Table S1). Two strains, Synechococcus sp. PCC 7335 and Leptolyngbya sp. JSC-1, exhibit Type-III complementary chromatic acclimation (CCA; see Fig. S1). Pleurocapsa sp. PCC 7327 synthesizes phycoerythrin constitutively. Mastigocoleus testarum is predicted to synthesize phycoerythrin, has a homolog of rcaE and multiple genes for phycocyanin, and thus is also predicted to perform Type-III CCA. The other strains shown are predicted to synthesize phycoerythrocyanin and thus cannot perform CCA. Brief source information concerning the origins of these organisms follows: Synechococcus sp. PCC 7335, snail shell, intertidal zone, Puerto Penasco, Mexico; Chroococcidiopsis thermalis PCC 7203, soil sample, near Greifswald, Germany; Pleurocapsa sp. PCC 7327, Hunters Hot Spring, Oregon, USA; Leptolyngbya sp. JSC-1, microbial mat, La Duke Hot Spring, Gardiner, MT USA; Oscillatoriales strain JSC-12, microbial mat, La Duke Hot Spring, Gardiner, MT USA; Calothrix sp. PCC 7507, sphagnum bog, near Kastanienbaum, Vierwaldstättersee, Switzerland; Chlorogloeopsis sp. PCC 9212, thermal spring water, Orense, Spain; Chlorogloeopsis sp. PCC 6912, soil sample, Allahabad, India; Fischerella sp. PCC 9605, no information available; Fischerella sp. strain JSC-11, microbial mat, La Duke Hot Spring, Gardiner, MT USA; Fischerella musicola PCC 7414, hot spring, New Zealand; Fischerella thermalis PCC 7521, hot spring Sinkhole II, Mammoth Hot Springs area, Yellowstone National Park, WY, USA; Mastigocoleus testarum strain BC008, marine snail shell, Cabo Rojo, Puerto Rico.

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CYJSC1_DRAFT_41490 (Leptolyngbya sp. JSC-1) KFYHQVKGTITYIQQAATLVEMCQLVAKEIRRITGFNRVMVYRFDQEESGSVIAEDT-- DQETPYLGLHYPASDIPPAARHLYTRNWLRLVPDASYQPVPLIPAPVTHQPLSVLRSVSPCHLEYLHNMGVTASMSISLI QDQKLWGLIACHHTSAKHVPYRIRTACEFIGQVMSVELANKAVTEDLDYKHRLKSLQTQFVGALSQAEHLLMVQLQAQLL ELVNATGAVIC-GKRYVRGQTPSEDEIHALLDWIKPQQQNLFETQSLSKH--- YPAAQSFQAVASGVLALEISQVHHNYILWFRPEVIQTVNWGGNPNEGSRHLSPRQSFDLWQETVRGCSLSWEAAEIEAVA ELRSLIVGIVLRQADE CYJSC1_DRAFT_40400 (Leptolyngbya sp. JSC-1) NFYHLARASINQLEATSNLQDFCQIIVREVRKVTGFDRVMLYRFDQDGHGEVVAEEKLAEMEPYLGLHYPESDIPQPARK MFLSNWIRVIPDAKASPVELYPHPITNQSLSILRSAYPCHLEYLHNMGVGASLTISLIKDQTLWGLIACHHRTPKQVPYE LRKACEFLGRVIFAEISTREETADHNYRIKLAQVQSALVEWMSQEDNFILTQHEPNLLELANASGAAICFSGHWTTGRTP PEDELNYLVQWLAKTDGEVFYTDSL---PLVYSDAERFKDVASGLLAIPISKR-- SYVLWFRPEVIQTVNWGGDPNANDLKLCPRKSFELWKETVRLKSLPWKPVEIKATLELRKAIVNIVLRQAEE Cph1 (Synechocystis sp. PCC 6803) NFYHMANAALNRL---- NLRDFYDVIVEEVRRMTGFDRVMLYRFDENNHGDVIAEDKRDDMEPYLGLHYPESDIPQPARRLFIHNPIRVIPDVYGVA VPLTPAPSTNRAESILRSAYHCHLTYLKNMGVGASLTISLIKDGHLWGLIACHHQTPKVIPFELRKACEFFGRVVFSNIS AQEDTETFDYRVQLAEHEAVLLDKMTTAADFVLTNHPDRLLGLTGSQGAAICFEKLILVGETPDEKAVQYLLQWL----- ENREVQDVFFTSSLSQIAVNFKSVASGLLAIPIARH-- NFLLWFRPEVLQTVNWGGDPNAQKIELHPRQSFDLWKEIVRLQSLPWQSVEIQSALALKKAIVNLILR---- Cph2 (Synechocystis sp. PCC 6803) L-EDFLRNVINKFHRALTLRETLQVIVEEARIFLGVDRVKIYKFASDGSGEVLAEAVNRAALPLLGLHFPVEDIPPQARE ELGNQRKMIAVDVAHRRKKSHELSPTEHSNGHYTTVDSCHIQYLLAMGVLSSLTVPVMQDQQLWGIMAVHHSKPRRFTEQ EWETMALLSKEVSLAITQSQLSRQVHQQQVQEALVQRLETTVAQYGDRPWQYALETVGQAVEADGAVLYIAQHYQWNLRF NWLETSLWQELMRGQVPHGYTLGELEQRSDWIAPPESLSAENQSFLIVPLAADQQGSLILLRKEKSLVKHWAGKRGIDRR NILPRLSFEAWEETQK-LVPTWNRSERKLAQVASTQLYMAITQQFVT PCC7424_3673 (Cyanothece sp. PCC 7424) NQEQLLLKITNLIHHNLDFTDTIQKITQAAQDFLKIDRVTIYQFAQDGSGEVIAESTTPTRLPLLGLHFPASDIPSKSLE QFAKLRSSSIIDVSAKRKTINSFYSDGKSIPTYSTVDPCHLQYLLAMGVLSSLSMPIFYWDQLWGLLIAHHSEPRRFSRE ELWTIELWCRQISLALAQNTLVSQFQKQKQQEHFIETINRLIDNQSNLSWQKILLETLKTLQADGARLYIDQLYTYGSQP QLEEQPQWQQLMKGKRPFTCTLAQLSADPQYHLLSKAFGNQSQSFLFIPLRSRSQGCLTLFRQEQEWETLWAGQQKLDQH NSTPRQSFSPWCEIQRG-VREWSTDELKLAEVLGLHLYMIVIQQRLT Cyan7822_1063 (Cyanothece sp. PCC 7822) NQEQLLLKITNFIHHSRDFKDSLQKIAQAARDFLKVDRVKIYQFAEDGSGEVIAESTNLERLPLLGLHFPGTDIPLKIRE QFAKARQGVIIDVSAKRKTINILYSDLKQQPSYSTVHPCHLRYLLAMGVLSSLAIPIFYWDKLWGLLVAHHSEPRRFSQE QLWTIELLCRQISLALAQNTLIAQFQKQKQQEEFIQKINTIIDNSPNLSWQQVLSEILKTLEADGARLYIDQLYTYGIQP ELEAQPQWQALMKGKRPLIYTLAELCNDPQYHLLSKAFSNQSQSFLFIPLRSRSQGCLTLFRQEREWETLWAGRENPDQR NQMPRQSFVAWCEIQRG-VPDWTGEQLKLAEILGLHLYMILTQQRLT Npun_R1759 (Nostoc punctiforme) LKEALLARMANRIRQSLNLQEILDATVVELRAFLQTDRTKVYRFDRDGHGHVVAEASAPNRLPLLGLHYPADDIPPQARA LFVKARTRSIVNVSEQRIILNSLTTAIGDDILSRPVDPCHVDYLTQMGVQSSLVVPIIYQQELWGLLISHHAEPREITSE DLLVVQLLADQVSLAITQASLLSQVQEQQQRDAIVNQIATMLHAPLTPELHNVLEQAVKASGSSGGRLYLAHLYTYGNQP LLENHPLWQEVRKIPKPALRVVINLQQEPRLHQLIESFQTTPRGLIVQPLYYSKEGYLTFFRDQISTKNLWPGYEEADER QQRPRQSMAQWQALKEDKAHPWSIEEMELIQSLNIHLSLAVLQNRLY WP_006453393 (Synechococcus sp. PCC 7335) ------------------ MPEILEATVDEMRQFLKTDRVKIYQFHPDGSGEVVAEAIYQQRLPLLGQRFPAEDIPEQARELFLKLKQRSIVNVSTQEI GISPLSEAKVATIVFRSVDPCHVEYLTAMGVQSSLVVPILYRHRLWGLMVAHHSVPKRFGDRELEIVQLITDQVTVAISH ASLLQLTRLQGQHEAIINQTVSYLHSTVEDPLQRALQHVVNELRCAGGRLYIKDRWVYGNQPELEQSIEWQSWLQDDVAN LWEISDIETAQIPQAIANALAHARRSLLIVKLTSQNQGYLSLFRQPVDIETVWAGRLDADPRQQRPRQSFEAWQEIRKNQ AHAWEPRDIGLVQDLADRFASVIYQTQLY Oscil6304_2970 (Oscillatoria acuminata PCC 6304) SRENLLNRIASRIRQSLELNEILETTAREIRAFLNTHRVKIYRFEEDGSGEVIAESIQDDRLPMLGLRFPATDIPVKSRELFLTARQRVIVDVVSGRKTTEELSDNGESGIRYWPVDPCHSEYLTNMGVSSSLAIPILHQHHLWGLLVSHHGKPRRYSER EIEVVQLLVDQMSIAIAQSDLLNRTRHQAHDEAVLNKISGLLHSPLDLKRQTVLKETVLALRADAGRLYIAQIYTYGIQP WIEETDCWQQLINGGNGHSVSIGNIYNFPAFNPLVYAFQATRRSVLIVPLKYNKQGCLTLFRNEIETATLWAGRNDWDER NRRPRASFEAWREIKKGETKIWKIEEIKLAESVGTHLYMAVMQRRVE Glo7428_1357 (Gloeocapsa sp. PCC 7428) KHEGVLNRITSRIRKSLELQEILTTTVEEVRAFLNTERVKIYRFHPDGSGEVIAESIHGKRLPLLGLHFPADDIPPQARE MFVKTRQRVIVDVIGQRKALNQLSETGESDIRYSPIDPCHAEYLSNMGVYSSLTLPILYQNQLWGLLVSHHSQRRQVSQK ELEVVQLLVDQLSIAISQSNLLQQTRQQVQHEATLNQISCLLHSPVSVRRQTVLEEIVKALQGAGGRLYIAQLYTYGEQP ELEESAYWQQIMGFPMPFLRTISDLYQEPQFDLASAFAATARSILIMPLQYRQNGCFTIFRNEIETETLWAGRVNHDERNLRPRESFAAWREIKKGQAQEWSPNEVKLAQA LGNHLYMAVMQRRVE FisPCC7414_2922 (Fischerella muscicola PCC 7414) PRQDLLHRITLRIRQSLELQEILETTAAEVRSLLETDRVKIYRFHADGSGEVIAESIHGDRLPLLGLNFPADDIPPYARE LFIKSRQRVVVDIVTQTTGMGLINQSTTPQLRYRPVDPCHVEYLTAMGVRSSVVVPIVHDHRLWGLLVSHHSQQRHVSEV ELQFIQAVTDQVEVAIAQSTLLSQMREQAEREAKINQLTTVLHRLPTVQLQAALETVVEIFQASGGRLYLDQFYVYGEQP QIEQHLLWQQYLRSPNPNCWAINDIYQESIFRTIVTSFASTQRGLLIVPLRYGKQGCLTIFRNEIATERLWAGRCDNDTR

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QIIAQISFETWRELRRGQAKPWTENEKKLAEVIAGHLVMAVQQYRLY PCC7521_4687 (Fischerella thermalis PCC 7521) PRQDLLHRITLRIRQSLELQEILETTTAEVRSLLGTDRVKIYRFHADGSGEVIAESIHGDRLPLLGLNFPADDIPPYARE LFIKSRQRVVVDIVTQTTGASLRNESTTQQLRYRPVDPCHVEYLTAMGVRSSVVVPILHDDRLWGLLVSHHSQQRHVSEV ELQFIQAVTDQVEVAIAQSTLLSQMREQAEREAKINQLTTVLHRLPTVQLQAALETVVEIFQASGGRLYLDQFYVYGEQP QIEQHLLWQQYLRSPNPNCWAINDIYQEPIFRTIVTSFQSTQRGLLIVPLRYGKQGCLTIFRNEIETERLWAGRCDNDTR QIITQISFETWRELRRGQAKPWTENEKKLAEVIGGHLVMAVQQYRLY CYJSC1_DRAFT_30990 (Leptolyngbya sp. JSC-1) MQDRLLHRITSQIRQSLNLEEILNTAATEVRLFLDTDRVKIYQFYSDGHGQVIAESIHQNRLPLLGLHFPADDIPPQARE LFLTARQRSIVNLETQQIGLSCAQHAS--EIHYRPVDPCHIEYLRAMGVQSSVVVPILQAEQLWGLLVSHHSGPRLVTETELQFLQSVVNQLEIAITQATLVKTIQQQV EHEAKVNRISKLLHADPRIHFQRALEETTIALQGTGSRLYLPELYTSGEQPLLEQHYLWQSYLRSDAQLPWAIRDLYQEP TCRTLSSVFQATSRGFLVIPLHYGQQGCLTVFRPTVETETLWAGQFDSDRRQTMPRESFLAWRELKGGQTKPWAEADLKL AQALGEHFSMAVQQYYLY Npun_AF142 (Nostoc punctiforme) NQEILLHRIASRIRQSLELQEILSATVAEVRSFLGTDRIKIYQFQADGHGLVIAESIQEDRLPLLGLNFPADDIPPYARE LFVRARQRCIVDLTTQEIGISPLPETGKPDIRYRPVDPCHLEYLTAMGVKSSVVVPIVLSSQLWGLLVSHHSQARVVTQQ ELLLIQSVVDQVAIAISQSILLTQVREQARQEAIINKVTEQLHSTPVAQLQTALEETVAAFNGSGGRLYLAKLYTFGLQP PIEEHRLWQKYLFASDSNLWAIADLYKEPLLRSVAPCFQTTQRGLLIVPLQHGSTGCLTIFRDEVDIETIWAGCVDTDSR QLMPRQSFAAWRELRTGQAQQWSESEIKLAQALGERFATAVKQHRLY PCC7110_joined_7652 (Scytonema hofmanni PCC 7110) NRESLLRRITYRIRRSLNLQEILSVTVAELSSFLKTDRIKIYKFHPDGSGQVIAEYIREQRLPLYGLNFPVDDIPLSARE LFIKAQVRVVVDVDSCQLGQSILPETGEFDLRFRPVDPCHNEYLTAMGVKTSLVLPILHHDQLWGLLVSHHAESRMFSEY ELMVVQLVTDQLCVAIAQSILLAEAQEKAQREAVINRVAMLLHSQSAIELQAALEETVAAFGGSGGRLCFIRLYTCGSQP MLEEYHIWYEHFKSNNYQAWAISDLHLISSLRNLQPLFESTKRSLLIVPLSYRQQGYLSIFRNEFETDILWAGQFDPDER QLYPRQSFAVWRQSDRLQIQQWKTVEIELAQTLATHFATAIQQYEMC FisPCC7414_3003 (Fischerella muscicola PCC 7414) MQKSLLRQLTSYIRQKLELQVILTATVAELRSFLEIDRVKIYKFNPDGSGQVIAESVDHNRLPVLGLHFPGDEIPPQYRD LYLKVRVRSIVNVDTKQIGQSPLSETGEMEFRYRPVDPCHLEYLTAMGVKSSLVLPILHDDQLWGLLASHHAEPRSIAES EVEVVQMVVDQLSVAIAQSALLSQVPQTASQEATIKHIVTLLHSLSTVEFQSALAQTVTAFGGSGGRLCMLKVYAYGKQP LMEQYSIWQEHYKFGNYDVWAISDIYQIHEFRFLQVAFTSSKRSILMIPLVYRQQGYLSIFRDETQTEPLWAGECDSDER LLYPRQSFNAWRESTKAQTREWTTEEIELATEVGKQFAFAIHEHELN FisPCC73103_3839 (Fischerella muscicola PCC 73103) MPETLRLRIANRIRLSSELQDVLTTTVAELRSFLASDRVKIYKFHPDQSGQVIAESVDSNKLPLLGLNFPADDIPPEARE LFINSRVRSIVNVDTQQIGRIPLLEDAENQDFYRPVDPCHVEYLTAMGVKSSVVVPIMHHNQLWGLLVSHDSESRLIPES ELEVLQMVVDQLSLAIAQSTLTIQAKEKATRETTSKRIATLLHSLPTIEYQSALAETIAAFGGSGGRLCIIKVYAYGKQP HMEQYNVWQERYKSGKYHIWAISDVYKIPGLRTLQVAFRATKRSILMIPLNYRQEGYLSIFRDEVETKILWAGEFDPDGR QLYPRQSFEVWQELKTAQPRDWSVEEVELARELGQQFAFAIHENEQS FisPCC7414_5516 (Fischerella muscicola PCC 7414) MAETLRLRIANRIRLSSELQDVFTTTVAELRSFLGSDRVKIYKFHPDQSGQVVAESVDSNKLPLLGLNFPADDIPPEARE LFIKLRVRSIVNVDTQQIGRIPLLEDEETENYYRPVDPCHVEYLTAMGVKSSLVVPIMHHDQLWGLLVSHHTESRSISES ELEVVQMVSDQLSLAIAQSTLLTQASEKAKREATIKRIAILLHSLPTIEYQSALAETIAALGGSGGRLCIIKVYAYGKQP LIEQYSVWQERYKSGKYNIWAISDVYKIPGLRNLQVAFRPTKRSMLMIPLHYRQEGYLSIFRDEIETKILWAGEFDSDER QLYPRQSFEVWQESKTAQAWEWNIDEVELARELGQQFAFAIHENEQF PCC7110_joined_3585 (Scytonema hofmanni PCC 7110) LQENLLRRIVNNIRRTLSLEEILTTTVAEVRSLLLIDRVLIYKFHPDSSGQVIAEAIHNYRLPLLGLNFPADDIPPHARE LFIKARVRSVAHVESRQIGHSPLLETGETNIRYRPIDPCHLEYLSAMGVTSSLVTPIFHQDQLWGLLVSHDSGFHVFGED DVEAVQRVVDQLSVAIAQSNILTQAHEKSQREAIVNRVATLLHSMPTVELQPALEAAVTAFGGSGGRLCIVKIYIYGHQP LMEQYSVWQEHYNESDCDIWVIPDIYKTPQLRNLQVAFRPTKRSILMLPLQYRQQGYLSIFRDEVDTETLWAGRFDRDER QLYPRLSFEVWREYKKAQPREWTAEELELAQALGKQFSLAIQQREIY UYC_00004 (Chlorogloeopsis fritschii PCC6912) LQENLMRRIINRIRLSLDLEEILTTTVAELRSFLGTDRVMIYKFNVDDSGQVIAESINDNQLPLLGLNFPADDIPPHARE LFLKLRVRSVVDVNNQQIGQSPVVKTGEFDICYRPVDPCHVEYLTAMGVKSSLVTPILHQEKLWGLLVSHHSQPRQVTED EIEVVQLVVDQLSIAIAHSSLLSQAREKAEREAIVNRVASLLHSLPTIALQSALEATVAAFNGSGGRLCMLKLYICGQQP LIEQYNVWQEHYKGCEYDVWAISDIYQISSLRSLQAAFGETKRSILIIPLHYRQQGYLSIFRDEIDTETLWAGHFDPDER QSFPRQSFAVWRESKQAQARQWTVEEIELAREIGKQFASAIQQYELY joined_4269 (Chlorogloeopsis fritschii PCC9212) LQENLMRRIINRIRLSLDLEEILTTTVAELRSFLGTDRVMIYKFNVDDSGQVIAESINDNQLPLLGLNFPADDIPPHARE LFLKLRVRSVVDVNNQQIGQSPVVKTGEFDICYRPVDPCHVEYLTAMGVKSSLVTPILHQEKLWGLLVSHHSQPRQVTED EIEVVQLVVDQLSIAIAHSSLLSQAREKAEREAIVNRVASLLHSLPTIALQSALEATVAAFNGSGGRLCMLKLYICGQQP LIEQYNVWQEHYKGCEYDVWAISDIYQISSLRSLQAAFGETKRSILIIPLHYRQQGYLSIFRDEIDTETLWAGHFDPDER QSFPRQSFAVWRESKQAQARQWTVEEIELAREIGKQFASAIQQYELY FisPCC7414_5518 (Fischerella muscicola PCC 7414) LQKDLLRRLTNRIRRSLKLQEILTTTVAEVRSFLGTDRVMIYKFHDDESGQVIAESINDHNLPLLGLNFPADDIPPDARE LFIKSRVRSVIDLDHQQIGQSIVLDTGEVDIHYRPVDPCHIEYLKAMGVKSSIVVPIIHQDQLWGLLVSHHSLSRSVNEY EIEVLQMVVEQLSVAIAHSDLLTQARTQAERESIVNRIATLLHSLPAIALQPALETAVTAFGGAGGRLCVVKLYTYGQQP FLEQYSVWQEYYKSGEYDVWGISDIYETPGLRTLQPAFAATKRSMLMIPLQYRQQGYLSIFRNEIDTEKLWAGQFDPDSR QQYPRLSFEVWRESKKAQAHKWTLAEIELAREIGKQFASAIQQYRLY PCC7521_282 (Fischerella thermalis PCC 7521) LQKDLLRRLTNRIRRSLELQEILTTTVAEVRSFLGTERVMIYKFHDDDSGQVIAESINDQKLPLLGLNFPADDIPSHARE LFIKSRVRSVVDLDNQQIGHSIVLDTGEVDIRYRPVDPCHVEYLKAMGVKSTIVVPIIHQDKLWGLLVSHHSLSRSVSEY

14

EIEVLQMVVEQLSVAIAHSDLLTQARAQAERETIVNRIATLLHSLPAIALQPALETAVTAFGGCGGRLCVVKIYTYGQQP FLEQYSVWQEYYKSGEYDVWAISDIYATPGLRTLQPAFAVTKRSMLMIPLQYRQQGYLSIFCNEIDTEKLWAGQFDPDKR QKYPRLSFEVWRQSKKAQARKWTLAEIELAREIGKQFASAIQQYILY Nos7107_2053 (Nostoc sp. PCC 71070 WQESLLRRITNRIRQTLELEEIIATTTAEVRYLLGTDRVMIYQFHTDGSGQVVGESIYEQRLPLLGLNFPADDIPPHARE LFIKSKVRSIVNLELQQIGHSPLLDTGELDIRYRPVDPCHAEYLTAMGVKSSVVAPIFHQEQLWGLLVSHNSQGHSISEY ELAAMQMVVDQLSVAIAQSNLLTQARAKAERESVINRIATLLHSLPTIVLQPALEATVAAFKGSGGRLCIIQVYTCGQQP FMEQYSVWQQHYESKVYDVWAINDIYQTSNLRTLQLAFQPTKRSLLMIPLYYRQQGYLSIFRDEIDTETLWAGQIDRDRR QRFPRLSFDIWKESKTAQAQQWTATEIELAREIAKHFASAIQQYELY Npun_R4776 (Nostoc punctiforme) FEESLLRRITNRIRRSLELEEIITVTTAEVRSLLKTDRVMIYKFHADGNGQVIAESIYNNRLPLLGLNFPADDIPLSARE LFLKLRVRSVVNVDTQEIGQIHLLDNGETEIRYRSVDSCHIEYLTAMGVKSSVVAPILYQDQLWGLLVSHNSEARLISEY ELEAVQMVVEQLSVAIAQSSLLTQVRKTAERETIINRIATLLHSLPTIVLQPALEAAIAAFNGVGGRLCIIKLFICGQQP LIEQYSIWQEHYKSGKYDVWAISDLYNSPDLRSLQVAFQPTKRGILTIPLQYRQQGYLSIFRNEVDTETLWAGQYDSDQR QLYPRRSFEVWRESKKAQAQKWTVEEIELARDIGKHFASAIQQYELQ Anacy_0015 (Anabaena cylindrica PCC 7122) -QETLLRRITNCIRQSLELKDIITATTAEVQSLLGTERVMIYKFHADGSGQVIAESIYENRIPLLGLNFPADDIPLDAREM FIKSRVRSVVNVDSRQISQSPQLETGEIEILYRSVDPCHVEYLTAMGVKSSVVAPIIYEDRLWGLLVSHHSEARDVAEHE LEAMQMVVEQLSVAIAQSHLLTQARAKAEREAIINRIATLLHSLPTIVLQPALEATVAAFNGVGGRLCIFQLYVCGQQPL LEQYSVWQEHYKSGQYNIWAIPDIYQDSNLRIVQLAFKPTKRSILMIPLHYRQEGYLSIFRNEIDTETLWAGKFDPDNRQ LYPRLSFDLWSESKQGQAQKWTDEEIELAREISNHFASAIQQYELY Cal7507_1566 (Calothrix sp. PCC 7507) LQESLLRRITNRIRQTLELEDIITTTTAEMRSLLGTDRVMIYKFHADGSGQVITESIDNDKLPLLGLNFPADDIPPQARE LFIKSQVRSVVNVETQQIGQSPLLETGQLDIHYRAVDPCHLEYLTAMGVKSSLVAPILCQKELWGLLVSHHSESHEILED EIEVVQMVVDQLSIAIAQSSLLTQARKKANREATINRIATLLHALPTIVLQPALEATVAAFGGSGGRLCIIRLYTCGHQP LIEQSNVWQEHYQSGEYDVWAISDIYQTSNMRTVQVAFEATKRCILMIPLQYRQEGYLSIFRNGIDTETLWAGRFDSDQR QLYPRQSFELWRETKTAQVQKWTDEEIEMSREIGKHFASAIQQYELY Mic7113_5715 (Microcoleus sp. PCC 7113) MYGGLLHGITSRIRQSLELQEILTATVTEVRSFLQTDRVKVYRFHPDESGQVVAESIAIDRLPLLGLNFPAGDIPPQARE MFVKAHQRSIVDVAAQQIILSSNPRSIVDDILQRPVDSCHLDYLTAMGVLSSLVVPILHQEHLWGLLVSHHSQPRAFKEE ELQLVQLIADQLSIAIAQSELLTQARKQVRRETLINQITHLLHSPRNMPLQLVLEQIVRAASGSGGRLYLRTLYTCGTQP LLEENSFWQPLMQPESLHLQIIPDLYEDPQLESLTQAFRPTPRGLLVMPLYHGSEGCLTIFRNAIEIETLWAGYWDKDQR QQRPRASFEVWREYQKSQPPQWTREDIELIESLSLHLAMAIMQNRLY Chro_4230 (Chroococcidiopsis thermalis PCC 7203) PRNSLLNRITNRIHQSLELQQILSVAVQEIRAFLQIDRVKVYKFHPDGTGQVIAEAIAAKRLPLLGLHFPADDIPAHARE MFVKIGARSIVDVPQQQISLSYLPQTTGDEILQRPVDPCHVEYLTAMGVQSSLVIPILHDCQLWGLLACHHAQPKTFRQE QLDVVRLVVEHLAIAISQSQLLTQARERAIRQELINKISTLLHSPHSIQLQIALESIVNSVDGSGGRLYLTEIYTDKAQP MLENAPFWQQLMAGVQHGYVAIANLYQAPELLEVAPAFQTTNRSLFVMPLHYGSEGCLTIFRDAIDTDITWAGRFDPDER ISRVRHSFEAWRELKRGQSKEWTSSEIELVQALANHLSLAVMQHRLH WP_017718168 (Oscillatoria sp. PCC 10802) AEGMLLNRMTNRIRQSLELHEILSATVAEVRSFLSTDRVKVYRFERDGSGQVIAESVYQNRLPLQGLHFPATDIPPQIRE MFVKARVRSIVDVPAQRITLSPSPETTGSEILQRPVDPCHIEYLTAMGVNSSLVVPILHGKELWGLLVSHHVQPKTFSAS DLQIVQMVADQVSVAIAHSALLSSTRSQARREALINQISTLLHAPLNIQLQGVLEKIVAAVGGAGGRLYLPELYTTGKLL WLEYHPFWQQLMAIAAAPVRAIDDLYQEPRLAQVAFAFQQTPRGMLLMPLRYSQQGCLTLFRSEIDTDIFWAGRFDRDER NAKVRHSFEAWRELKYGCAHPWTEEEIELVEGAGTHLSMAVMQNRLY UYC_01803 (Chlorogloeopsis fritschii PCC 6912) LHKDFLHRITNRIKRSLELQEILAATVTELREILSTDRVKVYRFDMDGNGEVIAESIYNQRLPLLGLHFPANDIPEESRQ MYLLARQRTIVDVSSGRIGLSRLPETGKPNFQYRRVDPCHLEYLKAMGVQSSLIVPIVDKPRLWGLLVSHHSKPRTFLKR EIRMVQQVADQLSLAIAVNNLLAQNRAEQKRGAIINRISILLNTVPEIQLSAALKETITALGGVGGRLHISQLYTWGDQP LLEKHSAWQNWITQCHGNVLAIQDLYKQPQFEAIAPFFQSTRRGILVIPLHYRQKGSLSIFRHEFDTEILWAGCGEENQR QRLPQFSFAAWREQKKEQALEWKPEETSLAEGLYHHFSIAIQQQLIY joined_1122 (Chlorogloeopsis fritschii PCC 9212) LHKDFLHRITNRIKRSLELQEILAATVTELREILSTDRVKVYRFDMDGNGEVIAESIYNQRLPLLGLHFPANDIPEESRQ MYLLARQRTIVDVSSGRIGLSRLPETGKPNFQYRRVDPCHLEYLKAMGVQSSLIVPIVDKPRLWGLLVSHHSKPRTFLKR EIRMVQQVADQLSLAIAVNNLLAQNRAEQKRGAIINRISILLNTVPEIQLSAALKETITALGGVGGRLHISQLYTWGDQP LLEKHSAWQNWITQCHGNVLAIQDLYKQPQFEAIAPFFQSTRRGILVIPLHYRQKGSLSIFRHEFDTEILWAGCGEENQR QRLPQFSFAAWREQKKEQALEWKPEETSLAEGLYHHFSIAIQQQLIY Cal7507_4151 (Calothrix sp. PCC 7507) WQEELLHGMTSRIRRSLELSEILTATVAEARSFLGTDRVMVYRFNADTSGEVIAESIYEQRLPLLGLNFPGDDIPRVARE MFLQFRQRSIVDVANGKIGLFPLPETGEFNIYYRQVDPCHIEYLSSMGVQSSLVMPIVHKPELWGLLVSHHSQPRTIVEK EVRVLQQVVDQVEIAIAQSNLLSETRTQQQQEATINQITTLLHKLPTIQLQEALETTVVALNGVGGRLYIKELYTWGDQP IIEQHPVWQNWMTQSPGSVWAITDLKQEPYLRVLALAFQSTQQGLLVIPLYYRQNGVLSIFRAKVDTEILWAGRCDQNRR QRLPQLSFEVWREQIKGQAPKWQPKDISLAQALCYHFSMAIQQQQMY Npun_R5313 (Nostoc punctiforme) LQESLLHRMIKQIRRSLDLQEILTTTVTEVRSFLRADRVKVYRFDTSGSGEVIAESIHNERLPLLGLRFPVHDIPEAARE MFLLAGQRSIVDVANHKIGLSPLTETGKHNIYYRKVDPCHIQYLKAMGVQSSLVVPILDKPKLWGLLVSHQSKPRKILKR EIKVLQQVADQVAIAIAQSNLLTAALTQQKQEATINRVTTLLHKLPTIQLQGAIEEVITAFSGVGGRLYIRELYTWGDQP IIEQHPTWQNWMTECPGNIWATSDLYKEPHLRVLALAFRSTQRGLMVIPLHYREQGVLSIFRAEFETEILWAGRCEQNRR QLLPQLSFEIWREQKKGLAPEWKPEDMTLAQALYDHFSMAIQQQQIY RfpA (CYJSC1_DRAFT_64120) LFALLLHRITRRIRQSLELEEILASTVAEVRSFLGTDRVKIYRFHGDGSGEVVAEAIRDQRLPLLHHNFPAGDIPREARE LFLSVRQRSIVNVSTQQIGISPFPGSMM- DIRFRKVDPCHVEYLTAMGVKSSLVVPILAEERLWGLLVSHHATPRQLGERELHLVQLIADQLEIAIAQSNLLSQTRLRA

15

HQEQTINQIATLLHAAPQVPLQTVLEQTVIALQANGGRLYLPQLFTTGPQPHLEQHPHWQTWLEMESSNLWAIADLYRAV MPSDLAIAFLTVQRGLLIVRLYYQQQGYLSLFRGEIDFERIWAGRIDNDPRQLRPRRSFETWRELKQGQSHPWSTADQEL VRALADHFAMAIHQHLLY WP_009769091 (Oscillatoriales sp. JSC-12) PLEMLLHRMMNRIRRSLELPEILAGTVAEVRSFLGTDRVKVYRFHPDGSGEVVAESIHQKRLPLLGQRFPADDIPVQARE LFLKARQRSIVDVSAQRIGVSPLPETGKADIRFRPVDPCHVDYLMAMGVQSSLVVPILHRDHLWGLLVSHHATPRQITKR ELQVVQLVADQVSIAIAQATLLDQTRTRAEQEATINRVSWLLHSMTEMQLQQALEQTVGALKGTGGRLFIAQLFTVGEQP KVETHPQWQSWLETQVENVWAIADVLHSGMPSDLALSFQTARCSLLVVRLQYRQQGYLSVFRPAIDIETLWARRVDDDPR QLRPRQSFAAWLELKSDQSQEWKINELQLAQALGKHFAMALHQSGLY Ple7327_1873 (Pleurocapsa sp. PCC 7327) RLAILLHRMTNRIRQSLELSEILSATVAEVRDFLETDRVMVYRFDGDGSGEVVAESVRKMRLPLLGQHFPAQDIPEEIRE LYLKARLRSIVDVTSQQIGLSPLPETGEPDIRFRPVDPCHAEYLTAMGVKSSLVIPILHGERLWGLLVSHHSLPRRMTER ELQVVQLVADQVSIAIAHAILLEGTRLQARQEATINRVATLLHSMTEMQVQQALEQTVSALEGVGGRVCI- QRFVCGKQPPIEEHPDWQAWLDKEESEVWAIADLHQALLPSDLATAFLAAGRGVLIIRLQYRQQGYLSIFREAIDTEIFW AGRPDDAPRNLRPRQSFETWRELKQGQARSWTSSEIELARSLANHFAMAIHQYELY UYC_02206 (Chlorogloeopsis fritschii PCC 6912) ELEVLLYRMMNRIRQSVELPAILSVTVAEIRGFLETDRVKIYRFDTDGSGEVVAESVNEERLPLLGQHFPAEDIPSEARE LFLLARQRSIVDVAAQQIGLSPVPEAEESDIRFRAVDSCHVKYLTSMGVQSSLVVPILHREHLWGLLVSHHSLPRQVTEQ ELQVVQLVADQLSIAIAQAILLEQSRLQAQQEATINRVAKLLHSMTEMQLQQALELTVSALQGAGGRICITQLFTTGKQP LLEEHPSWREWLRMEGKQVWAVDDLYQAALQSDLAQMFLGAGRSLLIIGLQYRQHGYLSIFRDEIDIETIWARRPDNDQR HLYPIISFETWRELKRGQAQPWQATEIELAQALASHFAMAIHQYQLY joined_1488 (Chlorogloeopsis fritschii PCC 9212) ELEVLLYRMMNRIRQSVELPAILSVTVAEIRGFLETDRVKIYRFDTDGSGEVVAESVNEERLPLLGQHFPAEDIPSEARE LFLLARQRSIVDVAAQQIGLSPVPEAEESDIRFRAVDSCHVKYLTSMGVQSSLVVPILHREHLWGLLVSHHSLPRQVTEQ ELQVVQLVADQLSIAIAQAILLEQSRLQAQQEATINRVAKLLHSMTEMQLQQALELTVSALQGAGGRICITQLFTTGKQP LLEEHPSWREWLRMEGKQVWAVDDLYQAALQSDLAQMFLGAGRSLLIIGLQYRQHGYLSIFRDEIDIETIWARRPDNDQR HLYPIISFETWRELKRGQAQPWQATEIELAQALASHFAMAIHQYQLY WP_009453706 (Fischerella sp. JSC-11) ELEVLLHRIMNRIRQSLELPAILSITVAEIRGFLETDRVKIYRFDTDGSGEVVAESVDKERLPLLGQHFPAQDIPSEARE LFLLARQRSIVDVGTQQIGLSPAPEADESDIRFRSVDPCHVEYLTSMGVQSSLVVPILHREQLWGLLVSHHSLPRQITEQ ELQVVQLVADQLSIAIAQSTLLEQSRLQAQQEATINRVAKLLHSMTQMQVQQALELTVSALQGAGGRICIAQLFTTGKQP PLEEHPSWREWLRMEESPVWVVNDLYQAALQSDLAQAFLKSGRTLLIVRLQYRQQGYLSIFRNEIDVETIWARRPDDDER HLSPIKSFEVWRELKRGQVQEWKTTEIELAQALASHFAIAIHQYQLY FisPCC7414_4902 (Fischerella muscicola PCC 7414) ELEVLLHRMMNRIRQSLELPAILSITVAEIRGFLETDRVKIYRFDTDGSGEVVAESVDQERLPLLGQHFPAQDIPQEVRE LFLLARQRSIVDVATQQIGLSPGPEADEFDMRFRSVDPCHVEYLTSMGVQSSLVVPILHCQQLWGLLVSHHSLPRQVTER ELQVVQLVADQLSIAIAQSTLLEQSRLQAQQEATINRVAKLLHSMTQMQVQQALELTVSALQGAGGRVCIAQLFTTGKQP PLEEYPSWREWLRMEESPVWVVNDLYQAALQSDLAQAFLKSGRTLLILRLQYRQQGYLSIFRNEIDIETIWARRPDDDER HLSPIKSFEVWRELKRGQVQEWKATEIELAQALASHFAMAIHQYQLY Cal7507_0267 (Calothrix sp. PCC 7507) QLEALLHRMMNRIRQSLELPAILSGTVAEVRIFLGTDRVKVYRFDADASGEVVAESVDCDRLPLLGQHFPAEDIPLAARE LFLRARQRSIVDVAAQQIGVSPLPETTESDIRFRAVDPCHVEYLTSMGVQSSLVVPILHRDQLWGLLVSHHSSPREVRER ELQVVQLVADQLSIAIAQNTLLEQTRAQADQEATINRVAKLLHSMTEMQLQQALELTISALQSAGGRIYIAQVFSCGDWG LFEEHPGWQQWLNIEGKQVWAITDLYQAALQSDLAQAFFDVGRSLLVICLQYRQQGYLTIFRHEIDTETLWARRTDDDLR HQRPVQSFEVWRELKQSQAQEWKTAEIELAQALGSHFAMAIHQYELY Chro_1028 (Chroococcidiopsis thermalis PCC 7203) SLEVLLHRMMNRIRQSLELPAILSGTVAEVRAFLGTDRVMVYRFDLDGSGEVVAESVQEERLPLLGQHFPAEDIPPLARE LFLKARQRSIVDVAAQEIGSSSLPVTGVSDIRFRPVDPCHVEYLTSMGVQSSLVVPILHRDRLWGLLVSHHSLPRQVTDR ELQVVQLVADQLSIAIAQSTLLEQTRIQAQQEATINRVAKLLHSMTEMQLQQALELTVSALGGTGGRVYIAQLLTCGEQP YLEEYSSWRSWLDAQDRCTWAIADWHGLEIPPELALAWFPVRRGLLIVRLQYRQQGYLSVFRREIEIETLWARRPDDDPR HTRPIQSFETWRELKQGQAQAWTSAEIELAQALGSHFALAIQQYELY WP_017714992 (Oscillatoria sp. PCC10802) LQEVLLHRMTNRIRQFLELQEILAATVAEVRSFLKTDRVMVYRFEADGSGEVIAESVNNHRLPMLGLHFPADDIPLEARE LYVRARARTIVDVNSQQIGMSPLAQTGNPDIRYRPVDSCHAAYLKAMGVQSSVVVPAIHQNELWGLLVSHHSEPQFIFDQ DLQLLQEVADQVSVALAHSIMLSQVRAQQAREATINRIATLLHAQPTIQLQAALQATVAAFEGAGGRLYICELVTCGDQP QLEEHPLWGQGLAAGSGGIWAITDIYKDAQFLLLAPAFQPTRRGLLAVPLHYRQQGFLSVFREEIETETLWAGQFDTSAK QQRPRESFQGWRELKKGQSREWVREEIELAQAIGYQFSMAIQQYLLY WP_008051387 (Arthrospira sp. PCC8005) LHQVLLRRIISRIRQSLELPEILKATVAEVRSFLGTDRIMIYRFDQDASGEVVAESIYDDRLPLLGLHFPADDIPQEARD RYVKLRQRTIVNVNSGTISISPLPETGEPEIKSRSVDPCHISYLTAMGVQSSVVVPILYSNQLWGLLVSHHSEPRETSPL ELQILQLVVDQMTIAIGQADMLAKTRKQAMLEAAINKVASLLHEQPTIQLQAALEATVEDFQGSGGRLYIPEVFYCGEQP IMEEHPLWQKWLREGRGNIWAITDLYRESELRIFTPAFQATARGLLVFPIQYRHNGCLTIFRNEIDTETIWAGRFDPSLK QMMPRQSFEVWRELKQGQAQPWTADEIRLGQALGKHFSMAIQQYMLY WP_023064756 (Lyngbya aestuarii) SEQHLLYRITKRISQSLELEEILTATVAEVRAFLETDRVMIYQFSADASGQVMAESIYENHLPLKGLHFPADDIPTAARE MFRTLGQRSIVNIATGQIGWSPVTDETENEINYRSVDPCHIAYLKAMGVQSSLVIPILHNRQLWGLLVSHHAEPWEISES QLQFVQLIVDQLSIAIKQSTLLSYTRQQASREATINQIASLLHQQPTIQLQDALEATVQALEGVGGRLYISEVWTCGEQP ILEEHPLWQQWIRMGESESWAIPDLYKEPLFRVLSPAFQATKRGLLILTIRYCQKAVLSIFRNEIDTEILWAGRFESDMK QILPRQSFEIWRELKKGQAQAWTEAERALAEGLKAQFAMAIQQYLLY WP_009784365 (Lyngbya sp. PCC8106) SEQHLLYRITKRISQSLELEEILTATVAEVRAFLETDRVMIYQFSADDSGQVMAESIDENHLSLKGLHFPADDIPTTARE MFRTLGQRSIVNIATGQIGWSPITDETENEINYRSVDPCHIAYLKAMGVQSSLVIPIFHNRQLWGLLVSHHAEPWEISES QLQLIQMIVDQLSIAIKQSTLLSHTRQQARREATINQVASLLHQQPTIQLQEALEATVKALEGIGGRLYISEVWSCGEQP

16

ILEEHPLWQHWIKTRDTEVWAITDLYKESLFRVLCPAFVSTKRGILIVSIQYRQKAILSIFRNEIDTEILWAGRFESNLQ QILPRQSFETWRELKKGQAQPWIEEEISLAEGLAAQLAMAIEQYLLY #=GR SS_cons H..HHHHHHHH......HHHHHHHHHHHHH......EEEE........EEEEE......................HHHHH ................E..EEE.....EE........HHHHHHHHH....EEEEEEEEE..EEEEEEEEEE.......HH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH....HHH..HHHHHH.....EEEEEE.EEEEEE... .HH..HHHHHHH........E......................EEEEEE......EEEEE....EEEEEE......X... .........EEEEEE........HHHHHHHHHHHHHHHHHHHHXXXX #=GR SA_cons 03422241136437455134101300340250020000102544532001021254357225012221112512243124 11032112031413533321120631431244124157231410431501000200021954110000011364260334 10300130052013202322245535333403512430452167455320251151014105140000015201103310 7333053014003557165223330244611231267047121000001044631000001134452422011524X421 5361175186463425441633651112012102301431121XXXX Figure S4. Protein sequences and multiple sequence alignment used for maximum-likelihood phylogenetic analysis in PhyML-Structure (49). Sequences for RfpA orthologs encoded as two open reading frames in Fischerella species as a result of sequencing or assembly errors were not included in this analysis.

17

Figure S5. RfpA (and its GAF domain) is photoresponsive to red and far-red light. The absorption spectra of the recombinant GAF domain of knotless phytochrome RfpA after illumination with 645-nm light (blue line) and 700-nm light (red line). The black line shows the difference spectrum for red-absorbing (Pr) form and the far-red-absorbing (Pfr) form.

18

Figure S6. Additional characterization of RfpA. (A) Normalized photochemical difference spectra are shown as (15Z – 15E) for full-length RfpA (red) and for the N-terminally truncated NpR4776-GAF-PHY photosensor (dark blue; truncation indicated on jellybean domain architecture). (B) The RfpA photochemical difference spectrum (blue) is compared to the action spectrum for reverse photoconversion (red). Reverse photoconversion is reported as the ratio of absorbance at 640 nm to absorbance at 710 nm and was assessed by irradiating RfpA to photoequilibrium with a series of bandpass interference filters (40 nm FWHM) at the indicated center wavelengths. A broad range of light wavelengths can drive the forward photoconversion of RfpA into the far-red absorbing form (Pfr), but only far-red light triggers reverse photoconversion to the red-absorbing form (Pr). Symbols for the domain structure depiction are described in the legend for Fig. 1

19

Figure S7. Transmittance properties of the red and green plastic filters used in the experiments described in this study to produce green light (GL) and red light (RL). When combined and used with a tungsten source, these filters mostly transmit wavelengths greater than 690 nm (FRL), although there is a small amount of transmittance between 550 and 600 nm.

20

Figure S8. JSC-1 cells grown in FRL or 710-nm light from LEDs have red-shifted fluorescence emission at 77 K. Low-temperature fluorescence emission spectra of whole cells of JSC-1 grown in white light (WL, black line), green light (GL, green line), 645-nm light (645-RL, red line), far-red light (FRL, blue line), and 710-nm light (710-FRL, blue dotted line). The excitation wavelength was 440 nm, and the spectra were normalized at the emission maximum to facilitate comparison.

21

Figure S9. JSC-1 cells grown in FRL and 710-nm light synthesize three chlorophylls. HPLC elution profile at monitored at 705 nm of pigments extracted from cells producing Chl a, Chl d, and/or Chl f. Pigments were extracted from cells of strain JSC-1 grown in WL (black line), FRL (blue line), 645-nm light (645-RL, red line), or 710-nm light (710-FRL, brown line). Peak 1, Chl d; Peak 2, Chl f; and Peak 3, Chl a.

22

Figure S10. JSC-1 cells grown in FRL and 710-nm light synthesize Chls a, d, and f. In-line absorption spectra for Chls identified in Fig. S8: Chl d (Peak 1, dotted line); Peak 2, Chl f (black line); Peak 3, Chl a (solid gray line). Spectra were normalized at the Qy absorption maximum.

23

Figure S11. HPLC separation of chlorophylls and pheophytins a, d, and f for mass spectrometry. The structures of Chl a, Chl d, and Chl f esterified with phytol are shown (right).

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Figure S12. Absorption spectra of Chls a, d, and f and the corresponding pheophytins. In-line absorption spectra for Chls and the corresponding pheophytins isolated from JSC-1 cells by reversed-phase HPLC (see Fig. S11).

25

Figure S13. Mass spectrophotometric identification of pheophytin f.

26

Figure S14. MS-MS analysis of pheophytin f. Ions corresponding to major decay products are identified.

27

Figure S15. Mass spectrophotometric identification of pheophytin d. Note that the ions at m/z = ~832.2 and ~919.5 are derived from unknown contaminants and are not derived from any Chl.

28

Figure S16. MS-MS analysis of pheophytin d. Ions corresponding to major decay products are identified.

29

Figure S17. Phycobiliproteins from strain JSC-1 carry phycocyanobilin (PCB) and phycoerythrobilin (PEB) chromophores. Phycobilisome samples were diluted 1:5 with 10.0 M urea, pH 3.0, to produce final conditions of 8.0 M urea at pH 3.0. Absorption spectra in 8.0 M urea, pH 3.0 of phycobilisomes from strain JSC-1 cells grown in 645-nm light (645-RL, red line) and 710-nm light (710-FRL, blue line). The absorption spectrum of denatured PBS from Synechococcus sp. PCC 7002 (7002, black line), which only synthesizes phycobiliproteins carrying phycocyanobilin (60), is shown for comparison.

30

Figure S18. Acclimation of the photosynthetic apparatus of JSC-1 to 710-nm light enhances photosynthetic performance as measured by oxygen evolution. The maximal light-saturated rate of O2 evolution for JSC-1 cells was ~100 µmol O2 (mg Chl)-1 h-1. Cells grown in 645-nm light (645-RL) or 710-nm light (710-FRL) exhibited similar light saturation behavior with white light provided from a halogen source. With far-red actinic light (wavelengths >695 nm) obtained by using a red/green filter combination with the halogen source, the maximum rate of oxygen evolution for cells grown in far-red light was 97 µmol O2 (mg Chl)-1 h-1 but the maximal rate for cells grown in red light was only about 60% of that value (59 µmol O2 (mg Chl)-1 h-1). The points in the far-red light (right) panel without error bars represent single measurements; points with error bars show the average and standard deviations for at least three independent measurements. The sample concentration was 28-30 µg Chl ml-1 for white actinic light and ~8-10 µg Chl ml-1 for far-red actinic light. Measurements were made at 30°C. Far-red actinic light was obtained by using a halogen source with a combination of red and green filters (see Figure S7). As an equipment control, the O2 evolution rate of Synechococcus sp. PCC 7002 with white light was measured at 38°C; these cells had an O2 evolution rate of 381 ± 13 µmol O2 (mg Chl)-1 h-1.

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Table S1. Genes encoding subunits of Photosystem I (PS I), Photosystem II (PS II), and phycobilisomes (PBS) and related proteins in strain JSC-1. The table shows transcript ratios for the listed genes for cells grown in far-red light for 24 hours compared to transcript levels for cells grown in white light. The table also shows those proteins that were identified by tryptic peptide LC-MS/MS analysis in gradient fractions containing PS I, PS II, and PBS from cells grown in red light and far-red light. An “unused peptide” score of 1.3 indicates at least 95% confidence for a positive identification, and a score greater than 2.0 indicates a 99% confidence for a positive identification. The pink (PS I), green (PS II) and blue (PBS) shading identifies the genes (proteins) for core components that are encoded in the 21-gene photosynthesis cluster shown in Fig. S2. Phycobiliproteins were low-abundance contaminants of fractions #1 and #2 containing PS I and PS II or PS I trimers (see Fig. 4). The composition data for PBS are more accurately reflected from the data for isolated PBS from cells grown in 645-nm light or 710-nm light. Table S2. Complete transcription profiling data for transcripts for strain JSC-1 cells grown in white light and far-red light. Cells were grown in white light to an OD750 nm = 0.3 and were then transferred to far-red light for 24 hours prior to isolation of RNA from each condition. For additional details, see SOM, Materials and Methods.

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