Characterization of chlorophyll f synthase heterologously produced in Synechococcus sp. PCC 7002
In diverse terrestrial cyanobacteria, Far-Red Light Photoacclimation (FaRLiP) promotes extensive remodeling of the photosynthetic apparatus, including photosystems (PS)I and PSII and the cores of phycobilisomes, and is accompanied by the concomitant biosynthesis of chlorophyll (Chl) d and Chl f. Chl f synthase, encoded by chlF, is a highly divergent paralog of psbA; heterologous expression of chlF from Chlorogloeopsis fritscii PCC 9212 led to the light-dependent production of Chl f in Synechococcus sp. PCC 7002 (Ho et al., Science 353, aaf9178 (2016)). In the studies reported here, expression of the chlF gene from Fischerella thermalis PCC 7521 in the heterologous system led to enhanced synthesis of Chl f. N-terminally [His]10-tagged ChlF7521 was purified and identified by immunoblotting and tryptic-peptide mass fingerprinting. As predicted from its sequence similarity to PsbA, ChlF bound Chl a and pheophytin a at a ratio of ~ 3–4:1, bound β-carotene and zeaxanthin, and was inhibited in vivo by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Cross-linking studies and the absence of copurifying proteins indicated that ChlF forms homodimers. Flash photolysis of ChlF produced a Chl a triplet that decayed with a lifetime (1/e) of ~ 817 µs and that could be attributed to intersystem crossing by EPR spectroscopy at 90 K. When the chlF7521 gene was expressed in a strain in which the psbD1 and psbD2 genes had been deleted, significantly more Chl f was produced, and Chl f levels could be further enhanced by specific growth-light conditions. Chl f synthesized in Synechococcus sp. PCC 7002 was inserted into trimeric PSI complexes.
KeywordsFaRLiP Chlorophyll Photosynthesis Fischerella thermalis PCC 7521 Cyanobacteria Photosystem I
This work was supported by the National Science Foundation grant MCB-1613022 to D.A.B and J.H.G. This research was also conducted under the auspices of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the DOE, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC 0001035 (D.A.B.). A.v.d.E. acknowledges support from the Natural Science and Engineering Research Council, Canada in the form of a Discovery Grant. D.P.C. was supported by a European Commission Marie Skłodowska-Curie Global Fellowship (660652). The authors thank Yue Lu at Washington University in St. Louis for performing the mass spectrometric analyses.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Allakhverdiev SI, Kreslavski V, Zharmukhamedov SK, Voloshin RA, Korol’kova DV, Tom T, Shen J-R (2016) Chlorophylls d and f and their role in primary photosynthetic processes of cyanobacteria. Biochemistry 81:201–212Google Scholar
- Bryant DA (1991) Cyanobacterial phycobilisomes; progress toward complete structural and functional analysis via molecular genetics. In: Bogorad L, Vasil IK (eds) Cell Culture and somatic cell genetics of plants, Vol 7B, Academic Press, London, pp 257–300Google Scholar
- Ferlez B, Cowgill JB, Dong W, Gisriel C, Lin S, Flores M, Walters K, Cetnar D, Redding KE, Golbeck JH (2016) Thermodynamics of the electron acceptors in Heliobacterium modesticaldum: an exemplar of an early homodimeric type I photosynthetic reaction center. Biochemistry 55:2358–2370CrossRefGoogle Scholar
- Frigaard N-U, Sakuragi Y, Bryant DA (2004) Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation. Meth Mol Biol 274325–274340Google Scholar
- Fujita Y, Murakami A (1987) Regulation of electron transport composition in cyanobacterial photosynthetic system: stoichiometry among photosystem I and II complexes and their light-harvesting antennae and cytochrome b 6/f complex. Plant Cell Physiol 28:1547–1553Google Scholar
- Gingrich JC, Gasparich GE, Sauer K, Bryant DA (1990) Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium Synechococcus sp. PCC 7002. Photosynth Res 24:137–150Google Scholar
- Li Y, Lin Y, Loughlin PC, Chen M (2014) Optimization and effects of different culture conditions on growth of Halomicronema hongdechloris—a filamentous cyanobacterium containing Chl f. Front Plant Sci 5:67Google Scholar
- Manning WM, Strain HH (1943) Chlorophyll d, a green pigment in red algae. J Biol Chem 151:1–19Google Scholar
- Nixon PJ, Trost JT, Diner BA (1992) Role of the carboxy-terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344. Biochemistry 31:10859–10871CrossRefGoogle Scholar
- Pérez AA, Gajewski JP, Ferlez BH, Ludwig M, Baker CS, Golbeck JH, Bryant DA (2016) A Zn++-inducible expression platform for Synechococcus sp. strain PCC 7002 based on the smtA promoter/operator and SmtB repressor. Appl Environ Microbiol 83:e02491–e02416Google Scholar
- Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 278:1–61Google Scholar
- Shen G, Zhao J, Reimer SK, Antonkine ML, Cai Q, Weiland SM, Golbeck JH, Bryant DA (2002) Assembly of Photosystem I: I. Inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity. J Biol Chem 277:20343–20354CrossRefGoogle Scholar
- Shen G, Schluchter WM, Bryant DA (2008) Biogenesis of phycobiliproteins. I. cpcS and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002. Identify a phyocobiliprotein lyase specific for Cys-82/84 Sites of the β-phycocyanin and allophycocyanin subunits. J Biol Chem 283:7503–7512CrossRefGoogle Scholar
- Zhao C, Gan F, Shen G, Bryant DA (2015) RfpA, RfpB, and RfpC are the master control elements of far-red light photoacclimation (FaRLiP). Front Microbiol 6:1303Google Scholar