In most algae, NO3- assimilation is tightly controlled and is often inhibited by the presence of NH4+. In the marine, non-colonial, non-diazotrophic cyanobacterium Synechococcus UTEX 2380, NO3- assimilation is sensitive to NH4+ only when N does not limit growth. We sequenced the genome of Synechococcus UTEX 2380, studied the genetic organization of the nitrate assimilation related (NAR) genes, and investigated expression and kinetics of the main NAR enzymes, under N or light limitation. We found that Synechococcus UTEX 2380 is a β-cyanobacterium with a full complement of N uptake and assimilation genes and NAR regulatory elements. The nitrate reductase of our strain showed biphasic kinetics, previously observed only in freshwater or soil diazotrophic Synechococcus strains. Nitrite reductase and glutamine synthetase showed little response to our growth treatments, and their activity was usually much higher than that of nitrate reductase. NH4+ insensitivity of NAR genes may be associated with the stimulation of the binding of the regulator NtcA to NAR gene promoters by the high 2-oxoglutarate concentrations produced under N limitation. NH4+ sensitivity in energy-limited cells fits with the fact that, under these conditions, the use of NH4+ rather than NO3- decreases N-assimilation cost, whereas it would exacerbate N shortage under N limitation.

CMNS Cell Biology and Molecular Genetics 2107 Bioscience Research Building University of Maryland College Park MD 20742 4407 USA

Dipartimento di Scienze della Vita e dell'Ambiente Università Politecnica delle Marche Ancona 60131 Italy

Division of Plant Sciences University of Dundee at the James Hutton Institute Invergowrie Dundee DD2 5 DA UK

Institute of Microbiology ASCR Algatech Trebon Czech Republic

National Research Council Institute of Marine Science Venezia Italy

Plant Functional Biology and Climate Change Cluster University of Technology Sydney Ultimo NSW 2007 Australia

School of Biological Sciences University of Western Australia 35 Stirling Highway Crawley WA 6009 Australia

STU UNIVPM Joint Algal Research Center Marine Biology Institute Shantou University Shantou Guangdong 515063 China

Zobrazit více v PubMed

Aichi M, Maeda S-I, Ichikawa K, Omata T.. 2004. Nitrite-responsive activation of the nitrate assimilation operon in cyanobacteria plays an essential role in up-regulation of nitrate assimilation activities under nitrate-limited growth conditions. Journal of Bacteriology 186, 3224–3229. PubMed PMC

Aichi M, Omata T.. 1997. Involvement of NtcB, a LysR family transcription factor, in nitrite activation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp. strain PCC 7942. Journal of Bacteriology 179, 4671–4675. PubMed PMC

Aichi M, Takatani N, Omata T.. 2001. Role of NtcB in activation of nitrate assimilation genes in the cyanobacterium Synechocystis sp. strain PCC 6803. Journal of Bacteriology 183, 5840–5847. PubMed PMC

Aichi M, Yoshihara S, Yamashita M, Maeda S, Nagai K, Omata T.. 2006. Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133. Bioscience, Biotechnology, and Biochemistry 70, 2682–2689. PubMed

Alfonso M, Perewoska I, Kirilovsky D.. 2001. Redox control of ntcA gene expression in Synechocystis sp. PCC 6803. Nitrogen availability and electron transport regulate the levels of the NtcA protein. Plant Physiology 125, 969–981. PubMed PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ.. 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410. PubMed

Ashida H, Saito Y, Kojima C, Kobayashi K, Ogasawara N, Yokota A.. 2003. A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO. Science 302, 286–290. PubMed

Badger MR, Bek EJ.. 2008. Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. Journal of Experimental Botany 59, 1525–1541. PubMed

Bird C, Wyman M.. 2003. Nitrate/nitrite assimilation system of the marine picoplanktonic cyanobacterium Synechococcus sp. strain WH 8103: effect of nitrogen source and availability on gene expression. Applied and Environmental Microbiology 69, 7009–7018. PubMed PMC

Boetzer M, Pirovano W.. 2012. Toward almost closed genomes with GapFiller. Genome Biology 13, R56. PubMed PMC

Bolger AM, Lohse M, Usadel B.. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. PubMed PMC

Bruland KW, Lohan MC, Aguilar-Islas AM, Smith GJ, Sohst B, Baptista A.. 2008. Factors influencing the chemistry of the near-field Columbia River plume: Nitrate, silicic acid, dissolved Fe, and dissolved Mn. Journal of Geophysical Research: Oceans 113, doi:10.1029/2007JC004702. DOI

Collos Y, Harrison PJ.. 2014. Acclimation and toxicity of high ammonium concentrations to unicellular algae. Marine Pollution Bulletin 80, 8–23. PubMed

Dai G, Deblois CP, Liu S, Juneau P, Qiu B.. 2008. Differential sensitivity of five cyanobacterial strains to ammonium toxicity and its inhibitory mechanism on the photosynthesis of rice-field cyanobacterium Ge–Xian–Mi (Nostoc). Aquatic Toxicology 89, 113–121. PubMed

Delwiche CF, Palmer JD.. 1996. Rampant horizontal transfer and duplication of Rubisco genes in eubacteria and plastids. Molecular Biology and Evolution 13, 873–882. PubMed

Dortch Q. 1990. The interaction between ammonium and nitrate uptake in phytoplankton. Marine Ecology Progress Series 61, 183–201.

Espinosa J, Forchhammer K, Burillo S, Contreras A.. 2006. Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA. Molecular Microbiology 61, 457–469. PubMed

Espinosa J, Forchhammer K, Contreras A.. 2007. Role of the Synechococcus PCC 7942 nitrogen regulator protein PipX in NtcA-controlled processes. Microbiology 153, 711–718. PubMed

Espinosa J, Rodríguez-Mateos F, Salinas P, Lanza VF, Dixon R, de la Cruz F, Contreras A.. 2014. PipX, the coactivator of NtcA, is a global regulator in cyanobacteria. Proceedings of the National Academy of Sciences, USA 111, E2423–E2430. PubMed PMC

Esteves-Ferreira AA, Inaba M, Fort A, Araújo WL, Sulpice R.. 2018. Nitrogen metabolism in cyanobacteria: metabolic and molecular control, growth consequences and biotechnological applications. Critical Reviews in Microbiology 44, 541–560. PubMed

Fanesi A, Raven JA, Giordano M.. 2014. Growth rate affects the responses of the green alga Tetraselmis suecica to external perturbations. Plant, Cell and Environment 37, 512–519. PubMed

Fernandez E, Galvan A.. 2008. Nitrate assimilation in Chlamydomonas. Eukaryotic Cell 7, 555–559. PubMed PMC

Forcada-Nadal A, Llácer JL, Contreras A, Marco-Marín C, Rubio V.. 2018. The PII-NAGK-PipX-NtcA regulatory axis of cyanobacteria: a tale of changing partners, allosteric effectors and non-covalent interactions. Frontiers in Molecular Biosciences 5, 91. PubMed PMC

Forchhammer K. 2008. PII signal transducers: novel functional and structural insights. Trends in Microbiology 16, 65–72. PubMed

Forchhammer K, de Marsac NT.. 1995. Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942. Journal of Bacteriology 177, 2033–2040. PubMed PMC

Forchhammer K, Selim KA.. 2020. Carbon/nitrogen homeostasis control in cyanobacteria. FEMS Microbiology Reviews 44, 33–53. PubMed PMC

Gawronski JD, Benson DR.. 2004. Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection. Analytical Biochemistry 327, 114–118. PubMed

Giordano M. 2013. Homeostasis: an underestimated focal point of ecology and evolution. Plant Science 211, 92–101. PubMed

Giordano M, Raven JA.. 2014. Nitrogen and sulfur assimilation in plants and algae. Aquatic Botany 118, 45–61.

Herrero A, Flores E.. 2018. Genetic responses to carbon and nitrogen availability in Anabaena. Environmental Microbiology 21, 1–17. PubMed

Herrero A, Flores E, Guerrero MG.. 1981. Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719. Journal of Bacteriology 145, 175–180. PubMed PMC

Herrero A, Muro-Pastor AM, Flores E.. 2001. Nitrogen control in cyanobacteria. Journal of Bacteriology 183, 411–425. PubMed PMC

Jiang F, Wisén S, Widersten M, Bergman B, Mannervik B.. 2000. Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library. Journal of Molecular Biology 301, 783–793. PubMed

Kaffes A, Thoms S, Trimborn S, Rost B, Langer G, Richter KU, Köhler A, Norici A, Giordano M.. 2010. Carbon and nitrogen fluxes in the marine coccolithophore Emiliania huxleyi grown under different nitrate concentrations. Journal of Experimental Marine Biology and Ecology 393, 1–8.

Kobayashi M, Rodríguez R, Lara C, Omata T.. 1997. Involvement of the C-terminal domain of an ATP-binding subunit in the regulation of the ABC-type nitrate/nitrite transporter of the cyanobacterium Synechococcus sp. strain PCC 7942. Journal of Biological Chemistry 272, 27197–27201. PubMed

Kobayashi M, Takatani N, Tanigawa M, Omata T.. 2005. Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC 6803. Journal of Bacteriology 187, 498–506. PubMed PMC

Lara C, Romero JM, Guerrero MG.. 1987. Regulated nitrate transport in the cyanobacterium Anacystis nidulans. Journal of Bacteriology 169, 4376–4378. PubMed PMC

Lee H, Erickson L.. 1987. Theoretical and experimental yields for photoautotrophic, mixotrophic, and photoheterotrophic growth. Biotechnology and Bioengineering 29, 476–481. PubMed

Lee HM, Flores E, Herrero A, Houmard J, Tandeau de Marsac N.. 1998. A role for the signal transduction protein PII in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Letters 427, 291–295. PubMed

L’Helguen S, Maguer J-F, Caradec J.. 2008. Inhibition kinetics of nitrate uptake by ammonium in size-fractionated oceanic phytoplankton communities: implications for new production and f-ratio estimates. Journal of Plankton Research 30, 1179–1188.

Llácer JL, Espinosa J, Castells MA, Contreras A, Forchhammer K, Rubio V.. 2010. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII. Proceedings of the National Academy of Sciences, USA 107, 15397–15402. PubMed PMC

Ludwig M, Bryant DA.. 2012. Acclimation of the global transcriptome of the cyanobacterium Synechococcus sp. strain PCC 7002 to nutrient limitations and different nitrogen sources. Frontiers in Microbiology 145, 145. PubMed PMC

Luque I, Flores E, Herrero A.. 1994. Molecular mechanism for the operation of nitrogen control in cyanobacteria. The EMBO Journal 13, 2862–2869. PubMed PMC

Luque I, Vázquez-Bermúdez MF, Paz-Yepes J, Flores E, Herrero A.. 2004. In vivo activity of the nitrogen control transcription factor NtcA is subjected to metabolic regulation in Synechococcus sp. strain PCC 7942. FEMS Microbiology Letters 236, 47–52. PubMed

Maeda S-I, Aoba R, Nishino Y, Omata T.. 2019. A novel bacterial nitrate transporter composed of small transmembrane proteins. Plant and Cell Physiology 60, 2180–2192. PubMed

Maeda S-I, Kawaguchi Y, Ohe T-A, Omata T.. 1998. cis-Acting sequences required for NtcB-dependent, nitrite-responsive positive regulation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp. strain PCC 7942. Journal of Bacteriology 180, 4080–4088. PubMed PMC

Marqués S, Florencio FJ, Candau P.. 1989. Ammonia assimilating enzymes from cyanobacteria: In situ and in vitro assay using high-performance liquid chromatography. Analytical Biochemistry 180, 152–157. PubMed

Martin-Nieto J, Flores E, Herrero A.. 1992. Biphasic kinetic behavior of nitrate reductase from heterocystous, nitrogen-fixing cyanobacteria. Plant Physiology 100, 157–163. PubMed PMC

Massouras A, Hens K, Gubelmann C, Uplekar S, Decouttere F, Rougemont J, Cole ST, Deplancke B.. 2010. Primer-initiated sequence synthesis to detect and assemble structural variants. Nature Methods 7, 485–486. PubMed

Mérida A, Candau P, Florencio FJ.. 1991. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium. Journal of Bacteriology 173, 4095–4100. PubMed PMC

Moore KR, Magnabosco C, Momper LM, Gold DA, Bosak T, Fournier GP.. 2019. An expanded ribosomal phylogeny of cyanobacteria supports a deep placement of plastids. Frontiers in Microbiology 10, 1612. PubMed PMC

Moore LR, Post AF, Rocap G, Chisholm SW.. 2002. Utilization of different nitrogen sources by the marine cyanobacteria Prochlorococcus and Synechococcus. Limnology and Oceanography 47, 989–996.

Muro-Pastor MI, Reyes JC, Florencio FJ.. 2005. Ammonium assimilation in cyanobacteria. Photosynthesis Research 83, 135–150. PubMed

Nicholas D, Nason A.. 1957. Determination of nitrate and nitrite. Methods in Enzymology 3, 981–984.

Nissen P, Martín-Nieto J.. 1998. ‘Multimodal’ kinetics: Cyanobacterial nitrate reductase and other enzyme, transport and binding systems. Physiologia Plantarum 104, 503–511.

Nitschmann WH, Peschek GA.. 1986. Oxidative phosphorylation and energy buffering in cyanobacteria. Journal of Bacteriology 168, 1205–1211. PubMed PMC

Noctor G, Foyer CH.. 1998. A re-evaluation of the ATP: NADPH budget during C3 photosynthesis: a contribution from nitrate assimilation and its associated respiratory activity?. Journal of Experimental Botany 49, 1895–1908.

Oaks A, Stulen I, Jones K, Winspear MJ, Misra S, Boesel IL.. 1980. Enzymes of nitrogen assimilation in maize roots. Planta 148, 477–484. PubMed

Ohashi Y, Shi W, Takatani N, Aichi M, Maeda S, Watanabe S, Yoshikawa H, Omata T.. 2011. Regulation of nitrate assimilation in cyanobacteria. Journal of Experimental Botany 62, 1411–1424. PubMed

Paerl RW, Tozzi S, Kolber ZS, Zehr JP.. 2012. Variation in the abundance of Synechococcus sp. cc9311 narB mRNA relative to changes in light, nitrogen growth conditions and nitrate assimilation. Journal of Phycology 48, 1028–1039. PubMed

Peterson GL. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry 83, 346–356. PubMed

Rae BD, Long BM, Badger MR, Price GD.. 2013. Functions, compositions, and evolution of the two types of carboxysomes: Polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiology and Molecular Biology Reviews 77, 357–379. PubMed PMC

Rose TM, Henikoff JG, Henikoff S.. 2003. CODEHOP (COnsensus-DEgenerate hybrid oligonucleotide primer) PCR primer design. Nucleic Acids Research 31, 3763–3766. PubMed PMC

Ruan Z, Giordano M.. 2017. The use of NH4+ rather than NO3 − affects cell stoichiometry, C allocation, photosynthesis and growth in the cyanobacterium Synechococcus sp. UTEX LB 2380, only when energy is limiting. Plant, Cell and Environment 40, 227–236. PubMed

Ruan Z, Prášil O, Giordano M.. 2018. The phycobilisomes of Synechococcus sp. are constructed to minimize nitrogen use in nitrogen-limited cells and to maximize energy capture in energy-limited cells. Environmental and Experimental Botany 150, 152–160.

Ruan Z, Raven JA, Giordano M.. 2017. In Synechococcus sp. competition for energy between assimilation and acquisition of C and those of N only occurs when growth is light limited. Journal of Experimental Botany 68, 3829–3839. PubMed

Sakamoto T, Bryant DA.. 1999. Nitrate transport and not photoinhibition limits growth of the freshwater cyanobacterium Synechococcus species PCC 6301 at low temperature. Plant Physiology 119, 785–794. PubMed PMC

Sakamoto T, Inoue-Sakamoto K, Bryant DA.. 1999. A novel nitrate/nitrite permease in the marine cyanobacterium Synechococcus sp. strain PCC 7002. Journal of Bacteriology 181, 7363–7372. PubMed PMC

Sakamoto T, Inoue-Sakamoto K, Persson S, Bryant DA.. 2008. Transcription factor NtcB specifically controls the nitrate assimilation genes in the marine cyanobacterium Synechococcus sp. strain PCC 7002. Phycological Research 56, 223–237.

Sánchez-Baracaldo P, Ridgwell A, Raven JA.. 2014. A neoproterozoic transition in the marine nitrogen cycle. Current Biology 24, 652–657. PubMed

Scanlan DJ, Ostrowski M, Mazard S, Dufresne A, Garczarek L, Hess WR, Post AF, Hagemann M, Paulsen I, Partensky F.. 2009. Ecological genomics of marine picocyanobacteria. Microbiology and Molecular Biology Reviews 73, 249–299. PubMed PMC

Scherer S, Häfele U, Krüger GH, Böger P.. 1988. Respiration, cyanide-insensitive oxygen uptake and oxidative phosphorylation in cyanobacteria. Physiologia Plantarum 72, 379–384.

Sekowska A, Ashida H, Danchin A.. 2019. Revisiting the methionine salvage pathway and its paralogues. Microbial Biotechnology 12, 77–97. PubMed PMC

Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J.. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7, 539. PubMed PMC

Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313. PubMed PMC

Tabita FR, Satagopan S, Hanson TE, Kreel NE, Scott SS.. 2008. Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. Journal of Experimental Botany 59, 1515–1524. PubMed

Tanigawa R, Shirokane M, Maeda S-i, Omata T, Tanaka K, Takahashi H.. 2002. Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro. Proceedings of the National Academy of Sciences, USA 99, 4251–4255. PubMed PMC

Wan N, DeLorenzo DM, He L, You L, Immethun CM, Wang G, Baidoo EE, Hollinshead W, Keasling JD, Moon TS.. 2017. Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence. Biotechnology and Bioengineering 114, 1593–1602. PubMed

Wang TH, Chen YH, Huang JY, Liu KC, Ke SC, Chu HA.. 2011. Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N2-fixing cyanobacterium Cyanothece sp. PCC 8801. Plant Physiology and Biochemistry 49, 1369–1376. PubMed

Wang TH, Fu H, Shieh YJ.. 2003. Monomeric NarB is a dual-affinity nitrate reductase, and its activity is regulated differently from that of nitrate uptake in the unicellular diazotrophic cyanobacterium Synechococcus sp. strain RF-1. Journal of Bacteriology 185, 5838–5846. PubMed PMC

Watzer B, Spät P, Neumann N, Koch M, Sobotka R, Macek B, Hennrich O, Forchhammer K.. 2019. The signal transduction protein PII controls ammonium, nitrate and urea uptake in cyanobacteria. Frontiers in Microbiology 10, 1428. PubMed PMC

Wyman M, Bird C.. 2007. Lack of control of nitrite assimilation by ammonium in an oceanic picocyanobacterium, Synechococcus sp. strain WH 8103. Applied and Environmental Microbiology 73, 3028–3033. PubMed PMC

Zhang S, Bryant DA.. 2011. The tricarboxylic acid cycle in cyanobacteria. Science 334, 1551–1553. PubMed

Zhang S, Qian X, Chang S, Dismukes GC, Bryant DA.. 2016. Natural and synthetic variants of the tricarboxylic acid cycle in cyanobacteria: introduction of the GABA shunt into Synechococcus sp. PCC 7002. Frontiers in Microbiology 7, 1972. PubMed PMC

Zhao M-X, Jiang Y-L, He Y-X, Chen Y-F, Teng Y-B, Chen Y, Zhang C-C, Zhou C-Z.. 2010. Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate. Proceedings of the National Academy of Sciences, USA 107, 12487–12492. PubMed PMC