Biogenesis of the photosynthetic apparatus requires complicated molecular machinery, individual components of which are either poorly characterized or unknown. The BtpA protein has been described as a factor required for the stability of photosystem I (PSI) in cyanobacteria; however, how the BtpA stabilized PSI remains unexplained. To clarify the role of BtpA, we constructed and characterized the btpA-null mutant (ΔbtpA) in the cyanobacterium Synechocystis sp. PCC 6803. The mutant contained only c. 1% of chlorophyll and nearly no thylakoid membranes. However, this strain, growing only in the presence of glucose, was genetically unstable and readily generated suppressor mutations that restore the photoautotrophy. Two suppressor mutations were mapped into the hemA gene encoding glutamyl-tRNA reductase (GluTR) - the first enzyme of tetrapyrrole biosynthesis. Indeed, the GluTR was not detectable in the ΔbtpA mutant and the suppressor mutations restored biosynthesis of tetrapyrroles and photoautotrophy by increased GluTR expression or by improved GluTR stability/processivity. We further demonstrated that GluTR associates with a large BtpA oligomer and that BtpA is required for the stability of GluTR. Our results show that the BtpA protein is involved in the biogenesis of photosystems at the level of regulation of tetrapyrrole biosynthesis.

Biology Centre of the Czech Academy of Sciences České Budějovice 370 05 Czech Republic

Department of Biological and Environmental Science Nanoscience Centre University of Jyväskylä Jyväskylä 40014 Finland

Faculty of Science University of South Bohemia České Budějovice 370 05 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Centre Algatech Třeboň 379 01 Czech Republic

Wicking Dementia Research and Education Centre University of Tasmania Hobart Tas 7005 Australia

Zobrazit více v PubMed

Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2: 8.

Balasubramanian R, Shen G, Bryant DA, Golbeck JH. 2006. Regulatory roles for IscA and SufA in iron homeostasis and redox stress responses in the cyanobacterium Synechococcus sp. strain PCC 7002. Journal of Bacteriology 188: 3182-3191.

Bartsevich VV, Pakrasi HB. 1997. Molecular identification of a novel protein that regulates biogenesis of photosystem I, a membrane protein complex. Journal of Biological Chemistry 272: 6382-6387.

Bryant DA, Hunter CN, Warren MJ. 2020. Biosynthesis of the modified tetrapyrroles-the pigments of life. Journal of Biological Chemistry 295: 6888-6925.

Brzezowski P, Richter AS, Grimm B. 2015. Regulation and function of tetrapyrrole biosynthesis in plants and algae. Biochimica et Biophysica Acta-Bioenergetics 1847: 968-985.

Bučinská L, Kiss E, Koník P, Knoppová J, Komenda J, Sobotka R. 2018. The ribosome-bound protein Pam68 promotes insertion of chlorophyll into the CP47 subunit of photosystem II. Plant Physiology 176: 2931-2942.

Chen GE, Hitchcock A, Mareš J, Gong Y, Tichý M, Pilný J, Kovářová L, Zdvihalová B, Xu J, Hunter CN et al. 2021. Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria. Proceedings of the National Academy of Sciences, USA 118: e2024633118.

Curran TD, Abacha F, Hibberd SP, Rolfe MD, Lacey MM, Green J. 2017. Identification of new members of the Escherichia coli K-12 MG1655 SlyA regulon. Microbiology 163: 400-409.

Czarnecki O, Hedtke B, Melzer M, Rothbart M, Richter A, Schroter Y, Pfannschmidt T, Grimm B. 2011. An Arabidopsis GluTR binding protein mediates spatial separation of 5-aminolevulinic acid synthesis in chloroplasts. Plant Cell 23: 4476-4491.

Dobáková M, Sobotka R, Tichý M, Komenda J. 2009. Psb28 protein is involved in the biogenesis of the photosystem II inner antenna CP47 (PsbB) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiology 149: 1076-1086.

Ermakova-Gerdes S, Vermaas WFJ. 1999. Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding. Journal of Biological Chemistry 274: 30540-30549.

Fang Y, Zhao S, Zhang F, Zhao A, Zhang W, Zhang M, Liu L. 2016. The Arabidopsis glutamyl-tRNA reductase (GluTR) forms a ternary complex with FLU and GluTR-binding protein. Scientific Reports 6: 19756.

Flores E, Arévalo S, Burnat M. 2019. Cyanophycin and arginine metabolism in cyanobacteria. Algal Research 42: 101577.

de la Fuente A, Martin JF, Rodriguez-Garcia A, Liras P. 2004. Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine. Journal of Bacteriology 186: 6501-6507.

Hanaichi T, Sato T, Iwamoto T, Malavasi-Yamashiro J, Hoshino M, Mizuno N. 1986. A stable lead by modification of Sato's method. Journal of Electron Microscopy 35: 304-306.

Heinz S, Liauw P, Nickelsen J, Nowaczyk M. 2016. Analysis of photosystem II biogenesis in cyanobacteria. Biochimica et Biophysica Acta-Bioenergetics 1857: 274-287.

Herranen M, Battchikova N, Zhang PP, Graf A, Sirpio S, Paakkarinen V, Aro EM. 2004. Towards functional proteomics of membrane protein complexes in Synechocystis sp PCC 6803. Plant Physiology 134: 470-481.

Hollingshead S, Kopečná J, Jackson PJ, Canniffe DP, Davison PA, Dickman MJ, Sobotka R, Hunter CN. 2012. Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803. Journal of Biological Chemistry 287: 27823-27833.

Hou Z, Pang X, Hedtke B, Grimm B. 2021. In vivo functional analysis of the structural domains of FLUORESCENT (FLU). The Plant Journal 107: 360-376.

Hou Z, Yang Y, Hedtke B, Grimm B. 2019. Fluorescence in blue light (FLU) is involved in inactivation and localization of glutamyl-tRNA reductase during light exposure. The Plant Journal 97: 517-529.

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772-780.

Keseler IM, Collado-Vides J, Gama-Castro S, Ingraham J, Paley S, Paulsen IT, Peralta-Gil M, Karp PD. 2005. EcoCyc: a comprehensive database resource for Escherichia coli. Nucleic Acids Research 33(Database issue): D334-D337.

Knoppová J, Sobotka R, Tichý M, Yu J, Koník P, Halada P, Nixon PJ, Komenda J. 2014. Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis. Plant Cell 26: 1200-1212.

Komenda J, Sobotka R. 2019. Chlorophyll-binding subunits of photosystem I and II: Biosynthesis, chlorophyll incorporation and assembly. In: Grimm B, ed. Metabolism, structure and function of plant tetrapyrroles: Control mechanisms of chlorophyll biosynthesis and analysis of chlorophyll-binding proteins. Amsterdam, the Netherlands: Elsevier, 195-223.

Kopečná J, Pilný J, Krynická V, Tomčala A, Kis M, Gombos Z, Komenda J, Sobotka R. 2015. Lack of phosphatidylglycerol inhibits chlorophyll biosynthesis at multiple sites and limits chlorophyllide reutilization in the cyanobacterium Synechocystis 6803. Plant Physiology 169: 1307-1317.

Kopf M, Klähn S, Scholz I, Matthiessen JKF, Hess WR, Voß B. 2014. Comparative analysis of the primary transcriptome of Synechocystis sp. PCC 6803. DNA Research 21: 527-539.

Koskela MM, Skotnicová P, Kiss É, Sobotka R. 2020. Purification of protein-complexes from the cyanobacterium Synechocystis sp. PCC 6803 using FLAG-affinity chromatography. Bio-Protocol 10: e3616.

Lee J, Lee HJ, Shin MK, Ryu WS. 2004. Versatile PCR-mediated insertion or deletion mutagenesis. BioTechniques 36: 398-400.

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408.

Marc F, Weigel P, Legrain C, Glansdorff N, Sakanyan V. 2001. An invariant threonine is involved in self-catalyzed cleavage of the precursor protein for ornithine acetyltransferase. Journal of Biological Chemistry 276: 25404-25410.

Mareš J, Strunecký O, Bučinská L, Wiedermannová J. 2019. Evolutionary patterns of thylakoid architecture in cyanobacteria. Frontiers in Microbiology 10: 277.

Morimoto K, Nishio K, Nakai M. 2002. Identification of a novel prokaryotic HEAT-repeats-containing protein which interacts with a cyanobacterial IscA homolog. FEBS Letters 519(1-3): 123-127.

Morimoto K, Sato S, Tabata S, Nakai M. 2003. A HEAT-repeats containing protein, IaiH, stabilizes the iron-sulfur cluster bound to the cyanobacterial IscA homologue, IscA2. Journal of Biochemistry 134: 211-217.

Nagano N, Hutchinson EG, Thornton JM. 1999. Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels. Protein Science 8: 2072-2084.

Nellaepalli S, Ozawa S-I, Kuroda H, Takahashi Y. 2018. The photosystem I assembly apparatus consisting of Ycf3-Y3IP1 and Ycf4 modules. Nature Communications 9: 2439.

Phillips JD, Bergonia HA, Reilly CA, Franklin MR, Kushner JP. 2007. A porphomethene inhibitor of uroporphyrinogen decarboxylase causes porphyria cutanea tarda. Proceedings of the National Academy of Sciences, USA 104: 5079-5084.

Pilný J, Kopečná J, Noda J, Sobotka R. 2015. Detection and quantification of heme and chlorophyll precursors using a High Performance Liquid Chromatography (HPLC) system equipped with two fluorescence detectors. Bio-Protocol 5: e1390.

Pinto FL, Thapper A, Sontheim W, Lindblad P. 2009. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. BMC Molecular Biology 10: 79.

Richter AS, Banse C, Grimm B. 2019. The GluTR-binding protein is the heme-binding factor for feedback control of glutamyl-tRNA reductase. eLife 13: e46300.

Roose JL, Frankel LK, Bricker TM. 2014. The PsbP domain protein 1 functions in the assembly of lumenal domains in photosystem I. Journal of Biological Chemistry 289: 23776-23785.

Schwabe TM, Gloddek K, Schluesener D, Kruip J. 2003. Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences 786: 45-59.

Shen J, Williams-Carrier R, Barkan A. 2017. PSA3, a potein on the sromal fce of the thylakoid membrane, promotes Photosystem I accumulation in cooperation with the assembly factor PYG7. Plant Physiology 174: 1850-1862.

Skotnicová P, Staleva-Musto H, Kuznetsova V, Bína D, Konert MM, Lu S, Polívka T, Sobotka R. 2021. Plant LHC-like proteins show robust folding and static non-photochemical quenching. Nature Communications 12: 6890.

Sobotka R, Komenda J, Bumba L, Tichý M. 2005. Photosystem II assembly in CP47 mutant of Synechocystis sp PCC 6803 is dependent on the level of chlorophyll precursors regulated by ferrochelatase. Journal of Biological Chemistry 280: 31595-31602.

Sobotka R, McLean S, Žuberová M, Hunter CN, Tichý M. 2008. The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC 6803. Journal of Bacteriology 190: 2086-2095.

Tichý M, Bečková M, Kopečná J, Noda J, Sobotka R, Komenda J. 2016. Strain of Synechocystis PCC 6803 with aberrant assembly of photosystem II contains tandem duplication of a large chromosomal region. Frontiers in Plant Science 7: 648.

Vavilin D, Brune DC, Vermaas W. 2005. N-15-labeling to detennine chlorophyll synthesis and degradation in Synechocystis sp PCC 6803 strains lacking one or both photosystems. Biochimica et Biophysica Acta-Bioenergetics 1708: 91-101.

Wang P, Grimm B. 2021. Connecting chlorophyll metabolism with accumulation of the photosynthetic apparatus. Trends in Plant Science 26: 484-495.

Zak E, Norling B, Andersson B, Pakrasi HB. 1999. Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803. European Journal of Biochemistry 261: 311-316.

Zak E, Pakrasi HB. 2000. The BtpA protein stabilizes the reaction center proteins of photosystem I in the cyanobacterium Synechocystis sp PCC 6803 at low temperature. Plant Physiology 123: 215-222.

Zhao A, Fang Y, Chen X, Zhao S, Dong W, Lin Y, Gong W, Liu L. 2014. Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein. Proceedings of the National Academy of Sciences, USA 111: 6630-6635.

Cyanophycinase is required for heterotrophy in cyanobacteria

High-light-inducible proteins control associations between chlorophyll synthase and the Photosystem II biogenesis factor Ycf39

Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium

The biogenesis and maintenance of PSII: Recent advances and current challenges