Biological nitrogen fixation is an energy-intensive process that contributes significantly toward supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria, light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems relies on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remain inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors larger amounts of PBS in its heterocysts, and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains large amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities, the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities. IMPORTANCE The function of phycobilisomes, the large antenna protein complexes in heterocysts has long been debated. This study provides direct evidence of the involvement of these proteins in supporting nitrogenase activity in Anabaena 33047, a heterocystous cyanobacterium that has an affinity for high light intensities. This strain was previously known to be recalcitrant to genetic manipulation and, hence, despite its many appealing traits, remained largely unexplored. We developed a genetic modification system for this strain and generated a ΔnblA mutant that exhibited resistance to phycobilisome degradation upon nitrogen starvation. Physiological characterization of the strain indicated that PBS degradation is not essential for acclimation to nitrogen deficiency and retention of PBS is advantageous for nitrogenase function.

Department of Biology Washington University St Louis Missouri USA

Photon Systems Instruments Drasov Czech Republic

State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan China

See more in PubMed

Kumar K, Mella-Herrera RA, Golden JW. 2010. Cyanobacterial heterocysts. Cold Spring Harb Perspect Biol 2:a000315. doi:10.1101/cshperspect.a000315. PubMed DOI PMC

Haselkorn R. 1978. Heterocysts. Annu Rev Plant Physiol 29:319–344. doi:10.1146/annurev.pp.29.060178.001535. DOI

Magnuson A. 2019. Heterocyst thylakoid bioenergetics. Life (Basel) 9:13. doi:10.3390/life9010013. PubMed DOI PMC

Zheng L, Li Y, Li X, Zhong Q, Li N, Zhang K, Zhang Y, Chu H, Ma C, Li G, Zhao J, Gao N. 2019. Structural and functional insights into the tetrameric photosystem I from heterocyst-forming cyanobacteria. Nat Plants 5:1087–1097. doi:10.1038/s41477-019-0525-6. PubMed DOI

Ow SY, Noirel J, Cardona T, Taton A, Lindblad P, Stensjo K, Wright PC. 2009. Quantitative overview of N2 fixation in Nostoc punctiforme ATCC 29133 through cellular enrichments and iTRAQ shotgun proteomics. J Proteome Res 8:187–198. doi:10.1021/pr800285v. PubMed DOI

Ow SY, Cardona T, Taton A, Magnuson A, Lindblad P, Stensjo K, Wright PC. 2008. Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags. J Proteome Res 7:1615–1628. doi:10.1021/pr700604v. PubMed DOI

Cardona T, Magnuson A. 2010. Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme. Biochim Biophys Acta 1797:425–433. doi:10.1016/j.bbabio.2009.12.014. PubMed DOI

Alberte RS, Tel-Or E, Packer L, Thornber JP. 1980. Functional organisation of the photo-synthetic apparatus in heterocysts of nitrogen-fixing cyanobacteria. Nature 284:481–483. doi:10.1038/284481a0. DOI

Bogorad L. 1975. Phycobiliproteins and complementary chromatic adaptation. Annu Rev Plant Physiol 26:369–401. doi:10.1146/annurev.pp.26.060175.002101. DOI

Richaud C, Zabulon G, Joder A, Thomas J-C. 2001. Nitrogen or sulfur starvation differentially affects phycobilisome degradation and expression of the nblA gene in Synechocystis strain PCC 6803. J Bacteriol 183:2989–2994. doi:10.1128/JB.183.10.2989-2994.2001. PubMed DOI PMC

Grossman ARSR, Bhaya D, Dolganov N. 1998. Phycobilisome degradation and responses of cyanobacteria to nutrient limitation and high light. In G G. (ed), Photosynthesis: mechanisms and effects. doi:10.1007/978-94-011-3953-3_669. Springer, Dordrecht. DOI

Collier JL, Grossman AR. 1994. A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria. EMBO J 13:1039–1047. doi:10.1002/j.1460-2075.1994.tb06352.x. PubMed DOI PMC

Grossman AR, Schaefer MR, Chiang GG, Collier JL. 1993. The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiol Rev 57:725–749. doi:10.1128/mr.57.3.725-749.1993. PubMed DOI PMC

Grossman AR, Bhaya D, He Q. 2001. Tracking the light environment by cyanobacteria and the dynamic nature of light harvesting. J Biol Chem 276:11449–11452. doi:10.1074/jbc.R100003200. PubMed DOI

Schwarz R, Forchhammer K. 2005. Acclimation of unicellular cyanobacteria to macronutrient deficiency: emergence of a complex network of cellular responses. Microbiology (Reading) 151:2503–2514. doi:10.1099/mic.0.27883-0. PubMed DOI

Moreno J, Vargas MÁ, Rodrı x, Guez H, Rivas J, Guerrero MG. 2003. Outdoor cultivation of a nitrogen-fixing marine cyanobacterium, Anabaena sp. ATCC 33047. Biomol Eng 20:191–197. doi:10.1016/S1389-0344(03)00051-0. PubMed DOI

Kumar A, Tabita FR, Van Baalen C. 1982. Isolation and characterization of heterocysts from Anabaena sp. strain CA. Arch Microbiol 133:103–109. doi:10.1007/BF00413520. DOI

Misra HS, Mahajan SK. 2000. Excitation energy transfer from phycobilisomes to photosystems: a phenomenon associated with the temporal separation of photosynthesis and nitrogen fixation in a cyanobacterium, Plectonema boryanum. Biochim Biophys Acta 1459:139–147. doi:10.1016/S0005-2728(00)00123-7. PubMed DOI

Watanabe M, Semchonok DA, Webber-Birungi MT, Ehira S, Kondo K, Narikawa R, Ohmori M, Boekema EJ, Ikeuchi M. 2014. Attachment of phycobilisomes in an antenna-photosystem I supercomplex of cyanobacteria. Proc Natl Acad Sci USA 111:2512–2517. doi:10.1073/pnas.1320599111. PubMed DOI PMC

Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP. 1997. Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179:1998–2005. doi:10.1128/jb.179.6.1998-2005.1997. PubMed DOI PMC

Elhai J, Wolk CP. 1988. Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167:747–754. doi:10.1016/0076-6879(88)67086-8. PubMed DOI

Trebst A. 2007. Inhibitors in the functional dissection of the photosynthetic electron transport system. Photosynth Res 92:217–224. doi:10.1007/s11120-007-9213-x. PubMed DOI

Joliot P, Joliot A. 2005. Quantification of cyclic and linear flows in plants. Proc Natl Acad Sci USA 102:4913–4918. doi:10.1073/pnas.0501268102. PubMed DOI PMC

Berla BM, Saha R, Maranas CD, Pakrasi HB. 2015. Cyanobacterial alkanes modulate photosynthetic cyclic electron flow to assist growth under cold stress. Sci Rep 5:14894. doi:10.1038/srep14894. PubMed DOI PMC

Marathe A, Kallas T. 2012. Cyclic electron transfer pathways in Syncechococcus sp. PCC 7002 cyanobacteria during photosynthesis at high light intensity Master of Science - Biology thesis, The University of Wisconsin Oshkosh.

Dines M, Sendersky E, David L, Schwarz R, Adir N. 2008. Structural, functional, and mutational analysis of the NblA protein provides insight into possible modes of interaction with the phycobilisome. J Biol Chem 283:30330–30340. doi:10.1074/jbc.M804241200. PubMed DOI PMC

Ungerer J, Pakrasi HB. 2016. Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria. Sci Rep 6:39681. doi:10.1038/srep39681. PubMed DOI PMC

Yu J, Liberton M, Cliften PF, Head RD, Jacobs JM, Smith RD, Koppenaal DW, Brand JJ, Pakrasi HB. 2015. Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Sci Rep 5:8132. doi:10.1038/srep08132. PubMed DOI PMC

Baier K, Lehmann H, Stephan DP, Lockau W. 2004. NblA is essential for phycobilisome degradation in Anabaena sp. strain PCC 7120 but not for development of functional heterocysts. Microbiology (Reading) 150:2739–2749. doi:10.1099/mic.0.27153-0. PubMed DOI

Peterson RB, Dolan E, Calvert HE, Ke B. 1981. Energy transfer from phycobiliproteins to photosystem I in vegetative cells and heterocysts of Anabaena variabilis. Biochim Biophys Acta 634:237–248. doi:10.1016/0005-2728(81)90142-0. PubMed DOI

Almon H, Böhme H. 1982. Photophosphorylation in isolated heterocysts from the blue-green alga Nostoc muscorum. Biochim Biophys Acta (BBA) - Bioenergetics 679:279–286. doi:10.1016/0005-2728(82)90298-5. PubMed DOI

Janaki S, Wolk CP. 1982. Synthesis of nitrogenase by isolated heterocysts. Biochim Biophys Acta (BBA) - Gene Structure and Expression 696:187–192. doi:10.1016/0167-4781(82)90027-6. PubMed DOI

Tel-Or E, Stewart WD. 1976. Photosynthetic electron transport, ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica. Biochim Biophys Acta 423:189–195. doi:10.1016/0005-2728(76)90177-8. PubMed DOI

Miyake C, Horiguchi S, Makino A, Shinzaki Y, Yamamoto H, Tomizawa K-i. 2005. Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves. Plant Cell Physiol 46:1819–1830. doi:10.1093/pcp/pci197. PubMed DOI

Huang W, Yang Y-J, Hu H, Zhang S-B. 2015. Different roles of cyclic electron flow around photosystem I under sub-saturating and saturating light intensities in tobacco leaves. Front Plant Sci 6:923. doi:10.3389/fpls.2015.00923. PubMed DOI PMC

Stacey G, Van Baalen C, Tabita FR. 1977. Isolation and characterization of a marine Anabaena sp. capable of rapid growth on molecular nitrogen. Arch Microbiol 114:197–201. doi:10.1007/BF00446862. DOI

Pfeffer S, Brown RM. 2016. Complete genome sequence of the cyanobacterium Anabaena sp. Genome Announc 4:e00809-16. doi:10.1128/genomeA.00809-16. PubMed DOI PMC

Bandyopadhyay A, Stockel J, Min H, Sherman LA, Pakrasi HB. 2010. High rates of photobiological H2 production by a cyanobacterium under aerobic conditions. Nat Commun 1:139. doi:10.1038/ncomms1139. PubMed DOI