Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Botany Department Federal University of Santa Catarina Campus de Trindade Florianópolis Brazil

Centre for Ecological Research Balaton Limnological Institute Tihany Hungary

Department Experimental Limnology Leibniz Institute of Freshwater Ecology and Inland Fisheries Stechlin Germany

Department of Biology Mount Allison University Sackville NB Canada

Department of Earth Sciences Utrecht University Utrecht Netherlands

Department of Ecology Berlin Institute of Technology Berlin Germany

Department of Microbiology Oregon State University Corvallis OR United States

Global Change Research Institute Czech Academy of Sciences Brno Czechia

Institute of Microbiology Czech Academy of Sciences Centre Algatech Třeboň Czechia

Institute of Parasitology Czech Academy of Sciences Biology Centre České Budějovice Czechia

Laboratoire de Biologie des Organismes et Ecosystèmes Aquatiques FRE 2030 Muséum National d'Histoire Naturelle CNRS IRD Sorbonne Université Université de Caen Normandie Normandie Université Esplanade de la Paix France

Max Planck Institute for Marine Microbiology Bremen Germany

NIOZ Royal Netherlands Institute for Sea Research and Utrecht University Den Burg Netherlands

Sorbonne Université CNRS Laboratoire d'Océanographie de Villefranche Villefranche sur mer France

Sorbonne Université CNRS Laboratoire d'Océanographie Microbienne Banyuls sur mer France

STU UNIVPM Joint Algal Research Center Marine Biology Institute College of Sciences Shantou University Shantou China

Universidade Federal de São Carlos São Carlos Brazil

University of Technology Sydney Climate Change Cluster Faculty of Science Ultimo NSW Australia

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Ackermann M. (2015). A functional perspective on phenotypic heterogeneity in microorganisms. Nat. Rev. Microbiol. 13 497–508. 10.1038/nrmicro3491 PubMed DOI

Aryal U. K., Stöckel J., Krovvidi R. K., Gritsenko M. A., Monroe M. E., Moore R. J., et al. (2011). Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC 51142 across light-dark diurnal cycles. BMC Syst. Biol. 5:194. 10.1186/1752-0509-5-194 PubMed DOI PMC

Bach L. T., Taucher J. (2019). CO2 effects on diatoms: a synthesis of more than a decade of ocean acidification experiments with natural communities. Ocean Sci. 15 1159–1175. 10.5194/os-15-1159-2019 DOI

Bernstein H. C., Charania M. A., McClure R. S., Sadler N. C., Melnicki M. R., Hill E. A., et al. (2015). Multi-omic dynamics associate oxygenic photosynthesis with nitrogenase-mediated H2 production in Cyanothece sp. ATCC 51142. Sci. Rep. 5:16004. 10.1038/srep16004 PubMed DOI PMC

Berthelot H., Duhamel S., L’Helguen S., Maguer J.-F., Wang S., Cetinić I., et al. (2019). NanoSIMS single cell analyses reveal the contrasting nitrogen sources for small phytoplankton. ISME J. 13 651–662. 10.1038/s41396-018-0285-288 PubMed DOI PMC

Blake W. J., Kærn M., Cantor C. R., Collins J. J. (2003). Noise in eukaryotic gene expression. Nature 422 633–637. 10.1038/nature01546 PubMed DOI

Bonnet S., Grosso O., Moutin T. (2011). Planktonic dinitrogen fixation along a longitudinal gradient across the Mediterranean Sea during the stratified period (BOUM cruise). Biogeosciences 8 2257–2267. 10.5194/bg-8-2257-2011 DOI

Burnat M., Picossi S., Valladares A., Herrero A., Flores E. (2019). Catabolic pathway of arginine in anabaena involves a novel bifunctional enzyme that produces proline from arginine. Mol. Microbiol. 111 883–897. 10.1111/mmi.14203 PubMed DOI

Caudron F., Barral Y. (2013). A super-assembly of Whi3 encodes memory of deceptive encounters by single cells during yeast courtship. Cell 155 1244–1257. 10.1016/j.cell.2013.10.046 PubMed DOI

Červený J., Sinetova M. A., Valledor L., Sherman L. A., Nedbal L. (2013). Ultradian metabolic rhythm in the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. Proc. Natl. Acad. Sci. U S A. 110 13210–13215. 10.1073/pnas.1301171110 PubMed DOI PMC

Colón-López M. S., Sherman L. A. (1998). Transcriptional and translational regulation of photosystem I and II genes in light-dark- and continuous-light-grown cultures of the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142. J. Bacteriol. 180 519–526. 10.1128/jb.180.3.519-526.1998 PubMed DOI PMC

Coplen T. B. (2011). Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Commun. Mass Spectrom. 25 2538–2560. 10.1002/rcm.5129 PubMed DOI

Damodaran S. P., Eberhard S., Boitard L., Rodriguez J. G., Wang Y., Bremond N., et al. (2015). A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii. PLoS One 10:e0118987. 10.1371/journal.pone.0118987 PubMed DOI PMC

Dekaezemacker J., Bonnet S. (2011). Sensitivity of N 2 fixation to combined nitrogen forms ( DOI

Deschamps P., Colleoni C., Nakamura Y., Suzuki E., Putaux J.-L., Buleon A., et al. (2008). Metabolic symbiosis and the birth of the plant kingdom. Mol. Biol. Evol. 25 536–548. 10.1093/molbev/msn053 PubMed DOI

Dron A., Rabouille S., Claquin P., Chang P., Raimbault V., Talec A., et al. (2012). Light:dark (12:12 h) quantification of carbohydrate fluxes in Crocosphaera watsonii. Aquat. Microb. Ecol. 68 43–55. 10.3354/ame01600 DOI

Dron A., Rabouille S., Claquin P., Talec A., Raimbault V., Sciandra A. (2013). Photoperiod length paces the temporal orchestration of cell cycle and carbon-nitrogen metabolism in Crocosphaera watsonii. Environ. Microbiol. 15 3292–3304. 10.1111/1462-2920.12163 PubMed DOI

Eichner M., Kranz S. A., Rost B. (2014). Combined effects of different CO2 levels and N sources on the diazotrophic cyanobacterium Trichodesmium. Physiol. Plant. 152 316–330. 10.1111/ppl.12172 PubMed DOI PMC

Elowitz M. B., Levine A. J., Siggia E. D., Swain P. S. (2002). Stochastic gene expression in a single cell. Science 297 1183–1186. 10.1126/science.1070919 PubMed DOI

Flores E., Arévalo S., Burnat M. (2019). Cyanophycin and arginine metabolism in cyanobacteria. Algal. Res. 42:101577 10.1016/j.algal.2019.101577 DOI

Foster R. A., Sztejrenszus S., Kuypers M. M. M. (2013). Measuring carbon and N2 fixation in field populations of colonial and free-living unicellular cyanobacteria using nanometer-scale secondary ion mass spectrometry1. J. Phycol. 49 502–516. 10.1111/jpy.12057 PubMed DOI

Gallon J. R. (1992). Reconciling the incompatible: N2 fixation And O2. New Phytol. 122 571–609. 10.1111/j.1469-8137.1992.tb00087.x DOI

Großkopf T., LaRoche J. (2012). Direct and indirect costs of dinitrogen fixation in Crocosphaera watsonii WH8501 and possible implications for the nitrogen cycle. Front. Microbiol. 3:236. 10.3389/fmicb.2012.00236 PubMed DOI PMC

Holl C. M., Montoya J. P. (2005). Interactions between nitrate uptake and nitrogen fixation in continuous cultures of the marine diazotroph Trichodesmium (Cyanobacteria). J. Phycol. 41 1178–1183. 10.1111/j.1529-8817.2005.00146.x DOI

Inomura K., Deutsch C., Wilson S. T., Masuda T., Lawrenz E., Bučinská L., et al. (2019). Quantifying oxygen management and temperature and light dependencies of nitrogen fixation by Crocosphaera watsonii. mSphere 4:e00531-19 10.1128/msphere.00531-519 PubMed DOI PMC

Karl D., Michaels A., Bergman B., Capone D., Carpenter E., Letelier R., et al. (2002). “Dinitrogen fixation in the world’s oceans,” in The Nitrogen Cycle at Regional to Global Scales, eds Boyer E. W., Howarth R. W. (Dordrecht: Springer; ), 10.1007/978-94-017-3405-9_2 DOI

Lee H., Rhee S. (2020). Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena. J. Biol. Chem. 295 5751–5760. 10.1074/jbc.RA120.012768 PubMed DOI PMC

Li H., Sherman D. M., Bao S., Sherman L. A. (2001). Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria. Arch. Microbiol. 176 9–18. 10.1007/s002030100281 PubMed DOI

Mareš J., Johansen J. R., Hauer T., Zima J., Ventura S., Cuzman O., et al. (2019). Taxonomic resolution of the genus Cyanothece (Chroococcales, Cyanobacteria), with a treatment on Gloeothece and three new genera, Crocosphaera, Rippkaea, and Zehria. J. Phycol. 55 578–610. 10.1111/jpy.12853 PubMed DOI

Masuda T., Furuya K., Kodama T., Takeda S., Harrison P. J. (2013). Ammonium uptake and dinitrogen fixation by the unicellular nanocyanobacterium Crocosphaera watsonii in nitrogen-limited continuous cultures. Limnol. Oceanogr. 58 2029–2036. 10.4319/lo.2013.58.6.2029 DOI

Masuda T., Inomura K., Takahata N., Shiozaki T., Sano Y., Deutsch C., et al. (2020). Heterogeneous nitrogen fixation rates confer energetic advantage and expanded ecological niche of unicellular diazotroph populations. Commun. Biol. 3:172 10.1038/s42003-020-0894-894 PubMed DOI PMC

Messineo L. (1966). Modification of the Sakaguchi reaction: spectrophotometric determination of arginine in proteins without previous hydrolysis. Arch. Biochem. Biophys. 117 534–540. 10.1016/0003-9861(66)90094-90094 DOI

Mitsui A., Kumazawa S., Takahashi A., Ikemoto H., Cao S., Arai T. (1986). Strategy by which nitrogen-fixing unicellular cyanobacteria grow photoautotrophically. Nature 323 720–722. 10.1038/323720a0 DOI

Mohr W., Intermaggio M. P., LaRoche J. (2010). Diel rhythm of nitrogen and carbon metabolism in the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii WH8501. Environ. Microbiol. 12 412–421. 10.1111/j.1462-2920.2009.02078.x PubMed DOI

Mohr W., Vagner T., Kuypers M. M. M., Ackermann M., LaRoche J. (2013). Resolution of conflicting signals at the single-cell level in the regulation of cyanobacterial photosynthesis and nitrogen fixation. PLoS One 8:e66060. 10.1371/journal.pone.0066060 PubMed DOI PMC

Montoya J. P., Holl C. M., Zehr J. P., Hansen A., Villareal T. A., Capone D. G. (2004). High rates of N2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean. Nature 430 1027–1031. 10.1038/nature02824 PubMed DOI

Mulholland M. R., Ohki K., Capone D. G. (2001). Nutrient controls on nitrogen uptake and metabolism by natural populations and cultures of Trichodesmium (Cyanobacteria). J. Phycol. 37 1001–1009. 10.1046/j.1529-8817.2001.00080.x DOI

Nassoury N., Fritz L., Morse D. (2001). Circadian changes in ribulose-1,5-bisphosphate carboxylase/oxygenase distribution inside individual chloroplasts can account for the rhythm in dinoflagellate carbon fixation. Plant Cell 13 923–934. 10.2307/3871349 PubMed DOI PMC

Pennebaker K., Mackey K. R. M., Smith R. M., Williams S. B., Zehr J. P. (2010). Diel cycling of DNA staining and nifH gene regulation in the unicellular cyanobacterium Crocosphaera watsonii strain WH 8501 (Cyanophyta). Environ. Microbiol. 12 1001–1010. 10.1111/j.1462-2920.2010.02144.x PubMed DOI PMC

Polerecky L., Adam B., Milucka J., Musat N., Vagner T., Kuypers M. M. M. (2012). Look@NanoSIMS - a tool for the analysis of nanoSIMS data in environmental microbiology. Environ. Microbiol. 14 1009–1023. 10.1111/j.1462-2920.2011.02681.x PubMed DOI

Polerecky L., Eichner M., Masuda T., Zavřel T., Rabouille S., Campbell D. A., et al. Calculation and Interpretation of Substrate Assimilation Rates in Microbial Cells Based on Isotopic Composition Data Obtained by Nanosims. (In Revision) PubMed PMC

Provasoli L., McLaughlin J. J. A., Droop M. R. (1957). The development of artificial media for marine algae. Arch. Mikrobiol. 25 392–428. 10.1007/BF00446694 PubMed DOI

Rabouille S., Claquin P. (2016). Photosystem-II shutdown evolved with Nitrogen fixation in the unicellular diazotroph Crocosphaera watsonii. Environ. Microbiol. 18 477–485. 10.1111/1462-2920.13157 PubMed DOI

Rabouille S., Campbell D. A., Masuda T., Zavřel T., Bernát G., Polerecky L., et al. Electron and Biomass Dynamics of Cyanothece Under Interacting Nitrogen and Carbon Limitations. (In Revision) PubMed PMC

Rabouille S., Van de Waal D. B., Matthijs H. C. P., Huisman J. (2014). Nitrogen fixation and respiratory electron transport in the cyanobacterium Cyanothece under different light/dark cycles. FEMS Microbiol. Ecol. 87 630–638. 10.1111/1574-6941.12251 PubMed DOI

Raj A., van Oudenaarden A. (2008). Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135 216–226. 10.1016/j.cell.2008.09.050 PubMed DOI PMC

Raser J. M., O’Shea E. K. (2013). Noise in gene expression: origins, consequences, and control. Science 309 2010–2013. 10.1126/science.1105891 PubMed DOI PMC

Reddy K. J., Haskell J. B., Sherman D. M., Sherman L. A. (1993). Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece. J. Bacteriol. 175 1284–1292. 10.1128/jb.175.5.1284-1292.1993 PubMed DOI PMC

Rippka R. (1988). Isolation and purification of cyanobacteria. Methods Enzymol. 167 3–27. 10.1016/0076-6879(88)67004-67002 PubMed DOI

Sanchez A., Choubey S., Kondev J. (2013). Regulation of noise in gene expression. Annu. Rev. Biophys. 42 469–491. 10.1146/annurev-biophys-083012-130401 PubMed DOI

Schneegurt M. A., Sherman D. M., Nayar S., Sherman L. A. (1994). Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142. J. Bacteriol. 176 1586–1597. 10.1128/jb.176.6.1586-1597.1994 PubMed DOI PMC

Schreiber F., Littmann S., Lavik G., Escrig S., Meibom A., Kuypers M. M. M., et al. (2016). Phenotypic heterogeneity driven by nutrient limitation promotes growth in fluctuating environments. Nat. Microbiol. 1:16055. 10.1038/nmicrobiol.2016.55 PubMed DOI

Sherman L. A., Meunier P., Colón-López M. S. (1998). Diurnal rhythms in metabolism: a day in the life of a unicellular, diazotrophic cyanobacterium. Photosynth. Res. 58 25–42. 10.1023/A:1006137605802 DOI

Short S. M., Zehr J. P. (2007). Nitrogenase gene expression in the Chesapeake Bay Estuary. Environ. Microbiol. 9 1591–1596. 10.1111/j.1462-2920.2007.01258.x PubMed DOI

Stöckel J., Welsh E. A., Liberton M., Kunnvakkam R., Aurora R., Pakrasi H. B. (2008). Global transcriptomic analysis of Cyanothece 51142 reveals robust diurnal oscillation of central metabolic processes. Proc. Natl. Acad. Sci. U S A. 105 6156–6161. 10.1073/pnas.0711068105 PubMed DOI PMC

Toepel J., Welsh E., Summerfield T. C., Pakrasi H. B., Sherman L. A. (2008). Differential transcriptional analysis of the cyanobacterium Cyanothece sp. strain ATCC 51142 during light-dark and continuous-light growth. J. Bacteriol. 190 3904–3913. 10.1128/JB.00206-208 PubMed DOI PMC

Tuit C., Waterbury J., Ravizza G. (2004). Diel variation of molybdenum and iron in marine diazotrophic cyanobacteria. Limnol. Oceanogr. 49 978–990. 10.4319/lo.2004.49.4.0978 DOI

Van Baalen C. (1962). Studies on marine blue-green algae. Bot. Mar. 4 129–139. 10.1515/botm.1962.4.1-2.129 DOI

Webb E. A., Ehrenreich I. M., Brown S. L., Valois F. W., Waterbury J. B. (2009). Phenotypic and genotypic characterization of multiple strains of the diazotrophic cyanobacterium, Crocosphaera watsonii, isolated from the open ocean. Environ. Microbiol. 11 338–348. 10.1111/j.1462-2920.2008.01771.x PubMed DOI

Welsh E. A., Liberton M., Stöckel J., Loh T., Elvitigala T., Wang C., et al. (2008). The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. Proc. Natl. Acad. Sci. U S A. 105 15094–15099. 10.1073/pnas.0805418105 PubMed DOI PMC

Wilson S. T., Aylward F. O., Ribalet F., Barone B., Casey J. R., Connell P. E., et al. (2017). Coordinated regulation of growth, activity and transcription in natural populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera. Nat. Microbiol. 2:17118. 10.1038/nmicrobiol.2017.118 PubMed DOI

Zavřel T., Sinetova M. A., Búzová D., Literáková P., Červený J. (2015a). Characterization of a model cyanobacterium Synechocystis sp. PCC 6803 autotrophic growth in a flat-panel photobioreactor. Eng. Life Sci. 15 122–132. 10.1002/elsc.201300165 DOI

Zavřel T., Sinetova M. A., Červený J. (2015b). Measurement of chlorophyll a and carotenoids concentration in Cyanobacteria. Bio-protocol 5:e1467 10.21769/BioProtoc.1467 DOI

Zavřel T., Chmelík D., Sinetova M. A., Červený J. (2018). Spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis. JoVE 139 e58076. 10.3791/58076 PubMed DOI PMC

Zehr J. P. (2011). Nitrogen fixation by marine cyanobacteria. Trends Microbiol. 19, 162–173. PubMed

Zehr J. P., Waterbury J. B., Turner P. J., Montoya J. P., Omoregie E., Steward G. F., et al. (2001). Unicellular cyanobacteria fix N2 in the subtropical north Pacific Ocean. Nature 412 635–638. 10.1038/35088063 PubMed DOI

Zhang H., Liu Y., Nie X., Liu L., Hua Q., Zhao G. P., et al. (2018). The cyanobacterial ornithine-ammonia cycle involves an arginine dihydrolase article. Nat. Chem. Biol. 14 575–581. 10.1038/s41589-018-0038-z PubMed DOI

The balance between photosynthesis and respiration explains the niche differentiation between Crocosphaera and Cyanothece

Calculation and Interpretation of Substrate Assimilation Rates in Microbial Cells Based on Isotopic Composition Data Obtained by nanoSIMS

Quantifying Cyanothece growth under DIC limitation

Electron & Biomass Dynamics of Cyanothece Under Interacting Nitrogen & Carbon Limitations