Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2 Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2 The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 μmol m-2 s-1) than photosynthesis and that both are similarly inhibited by light intensities of >500 μmol m-2 s-1 This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface.IMPORTANCECrocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

Collaborative Mass Spectrometry Innovation Center Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego San Diego California USA

Daniel K Inouye Center for Microbial Oceanography Research and Education University of Hawaii Honolulu Hawaii USA

Department of Earth Atmospheric and Planetary Sciences Massachusetts Institute of Technology Cambridge Massachusetts USA

Institute of Microbiology The Czech Academy of Sciences Třeboň Czech Republic

Marine Chemistry and Geochemistry Department and Biology Department Woods Hole Oceanographic Institution Woods Hole Massachusetts USA

School of Oceanography University of Washington Seattle Washington USA

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mSphere. 2020 Feb 5;5(1):e00075-20. doi: 10.1128/mSphere.00075-20. PubMed

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Thomas WH. 1970. On nitrogen deficiency in tropical pacific oceanic phytoplankton: photosynthetic parameters in poor and rich water. Limnol Oceanogr 15:380–385. doi:10.4319/lo.1970.15.3.0380. DOI

Thomas WH, Owen RW. 1971. Estimating phytoplankton production from ammonium and chlorophyll concentrations in nutrient-poor water of the eastern tropical Pacific Ocean. Fish Bull 69:87–92.

Ryther JH, Dunstan WM. 1971. Nitrogen, phosphorus, and eutrophication in the coastal marine environment. Science 171:1008–1013. doi:10.1126/science.171.3975.1008. PubMed DOI

Caperon J, Meyer J. 1972. Nitrogen-limited growth of marine phytoplankton. I. Changes in population characteristics with steady-state growth rate. Deep Sea Res 19:601–618. doi:10.1016/0011-7471(72)90089-7. DOI

Falkowski PG. 1997. Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean. Nature 387:272–275. doi:10.1038/387272a0. DOI

Gruber N. 2004. The dynamics of the marine nitrogen cycle and its influence on atmospheric CO2 variations In Follows M, Oguz T (ed), The ocean carbon cycle and climate. Kluwer Academic, Dordrecht, Netherlands.

Gruber N, Galloway JN. 2008. An Earth-system perspective of the global nitrogen cycle. Nature 451:293–296. doi:10.1038/nature06592. PubMed DOI

Thompson AW, Zehr JP. 2013. Cellular interactions: lessons from the nitrogen-fixing cyanobacteria. J Phycol 49:1024–1035. doi:10.1111/jpy.12117. PubMed DOI

Montoya JP, Holl CM, Zehr JP, Hansen A, Villareal TA, Capone DG. 2004. High rates of N2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean. Nature 430:1027–1031. doi:10.1038/nature02824. PubMed DOI

Falcón LI, Carpenter EJ, Cipriano F, Bergman B, Capone DG. 2004. N2 fixation by unicellular bacterioplankton from the Atlantic and Pacific oceans: phylogeny and in situ rates. Appl Environ Microbiol 70:765–770. doi:10.1128/aem.70.2.765-770.2004. PubMed DOI PMC

Dyhrman ST, Haley ST. 2006. Phosphorus scavenging in the unicellular marine diazotroph Crocosphaera watsonii. Appl Environ Microbiol 72:1452–1458. doi:10.1128/AEM.72.2.1452-1458.2006. PubMed DOI PMC

Moisander PH, Beinart RA, Hewson I, White AE, Johnson KS, Carlson CA, Montoya JP, Zehr JP. 2010. Unicellular cyanobacterial distributions broaden the oceanic N2 fixation domain. Science 327:1512–1514. doi:10.1126/science.1185468. PubMed DOI

Church MJ, Mahaffey C, Letelier RM, Lukas R, Zehr JP, Karl DM. 2009. Physical forcing of nitrogen fixation and diazotroph community structure in the North Pacific subtropical gyre. Global Biogeochem Cycles 23:GB2020. doi:10.1029/2008GB003418. DOI

Webb EA, Ehrenreich IM, Brown SL, Valois FW, Waterbury JB. 2009. Phenotypic and genotypic characterization of multiple strains of the diazotrophic cyanobacterium, Crocosphaera watsonii, isolated from the open ocean. Environ Microbiol 11:338–348. doi:10.1111/j.1462-2920.2008.01771.x. PubMed DOI

Fu FX, Yu E, Garcia NS, Gale J, Luo Y, Webb EA, Hutchins DA. 2014. Differing responses of marine N2 fixers to warming and consequences for future diazotroph community structure. Aquat Microb Ecol 72:33–46. doi:10.3354/ame01683. DOI

Gallon JR. 1981. The oxygen sensitivity of nitrogenase: a problem for biochemists and micro-organisms. Trends Biochem Sci 6:19–23. doi:10.1016/0968-0004(81)90008-6. DOI

Wang ZC, Burns A, Watt GD. 1985. Complex formation and O2 sensitivity of Azotobacter vinelandii nitrogenase and its component proteins. Biochemistry 24:214–221. doi:10.1021/bi00322a031. PubMed DOI

Inomura K, Bragg J, Follows MJ. 2017. A quantitative analysis of the direct and indirect costs of nitrogen fixation: a model based on Azotobacter vinelandii. ISME J 11:166–175. doi:10.1038/ismej.2016.97. PubMed DOI PMC

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

Shi T, Ilikchyan I, Rabouille S, Zehr JP. 2010. Genome-wide analysis of diel gene expression in the unicellular N2-fixing cyanobacterium Crocosphaera watsonii WH 8501. ISME J 4:621–632. doi:10.1038/ismej.2009.148. PubMed DOI

Saito MA, Bertrand EM, Dutkiewicz S, Bulygin VV, Moran DM, Monteiro FM, Follows MJ, Valois FW, Waterbury JB. 2011. Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii. Proc Natl Acad Sci 108:2184–2189. doi:10.1073/pnas.1006943108. PubMed DOI PMC

Wilson ST, Aylward FO, Ribalet F, Barone B, Casey JR, Connell PE, Eppley JM, Ferrón S, Fitzsimmons JN, Hayes CT, Romano AE, Turk-Kubo KA, Vislova A, Armbrust EV, Caron DA, Church MJ, Zehr JP, Karl DM, Delong EF. 2017. Coordinated regulation of growth, activity and transcription in natural populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera. Nat Microbiol 2. doi:10.1038/nmicrobiol.2017.118. PubMed DOI

Garcia HE, Locarnini RA, Boyer TP, Antonov JI, Baranova OK, Zweng MM, Reagan JR, Johnson DR. 2013. Dissolved oxygen, apparent oxygen utilization, and oxygen saturation In Levitus S, Mishonov A (ed), World Ocean Atlas 2013, vol 3 NOAA, Silver Spring, MD.

Robertson JE, Watson AJ, Langdon C, Ling RD, Wood JW. 1993. Diurnal variation in surface pCO2 and O2 at 60°N, 20°W in the North Atlantic. Deep Res Part II 40:409–422. doi:10.1016/0967-0645(93)90024-H. 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. doi:10.3389/fmicb.2012.00236. PubMed DOI PMC

Grimaud GM, Rabouille S, Dron A, Sciandra A, Bernard O. 2014. Modelling the dynamics of carbon—nitrogen metabolism in the unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501, under variable light regimes. Ecol Modell 291:121–133. doi:10.1016/j.ecolmodel.2014.07.016. DOI

Dixon R, Kahn D. 2004. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2:621–631. doi:10.1038/nrmicro954. PubMed DOI

Capone DG. 1988. Benthic nitrogen fixation, p 85–123. In Blackburn TH, Sorensen J (ed), Nitrogen cycling in coastal marine environments. John Wiley and Sons, New York, NY.

Knapp AN. 2012. The sensitivity of marine N2 fixation to dissolved inorganic nitrogen. Front Microbiol 3:374. doi:10.3389/fmicb.2012.00374. PubMed DOI PMC

Li WKW. 1980. Temperature adaptation in phytoplankton: cellular and photosynthetic characteristics, p 259–279. In Falkowski PG. (ed), Primary productivity in the sea. Plenum Press, New York, NY.

Geider RJ, Macintyre HL, Kana TM. 1997. Dynamic model of phytoplankton growth and acclimation: responses of the balanced growth rate and the chlorophyll a: carbon ratio to light, nutrient-limitation and temperature. Mar Ecol Prog Ser 148:187–200. doi:10.3354/meps148187. DOI

Cullen JJ. 1990. On models of growth and photosynthesis in phytoplankton. Deep Res 37:667–683. doi:10.1016/0198-0149(90)90097-F. DOI

Geider RJ, Macintyre HL, Kana TM. 1998. A dynamic regulatory model of phytoplanktonic acclimation to light, nutrients, and temperature. Limnol Oceanogr 43:679–694. doi:10.4319/lo.1998.43.4.0679. DOI

Dron A, Rabouille S, Claquin P, Roy B, Talec A, Sciandra A. 2012. Light-dark (12:12) cycle of carbon and nitrogen metabolism in Crocosphaera watsonii WH8501: relation to the cell cycle. Environ Microbiol 14:967–981. doi:10.1111/j.1462-2920.2011.02675.x. PubMed DOI

Pennebaker K, Mackey KRM, Smith RM, Williams SB, Zehr JP. 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. doi:10.1111/j.1462-2920.2010.02144.x. PubMed DOI PMC

Zehr JP. 2011. Nitrogen fixation by marine cyanobacteria. Trends Microbiol 19:162–173. doi:10.1016/j.tim.2010.12.004. PubMed DOI

Sohm JA, Edwards BR, Wilson BG, Webb EA. 2011. Constitutive extracellular polysaccharide (EPS) production by specific isolates of Crocosphaera watsonii. Front Microbiol 2:229. doi:10.3389/fmicb.2011.00229. PubMed DOI PMC

Inomura K, Bragg J, Riemann L, Follows MJ. 2018. A quantitative model of nitrogen fixation in the presence of ammonium. PLoS One 13:e0208282. doi:10.1371/journal.pone.0208282. PubMed DOI PMC

Inomura K, Wilson ST, Deutsch C. 2019. Mechanistic model for the coexistence of nitrogen fixation and photosynthesis in marine Trichodesmium. mSystems 4:e00210-19. doi:10.1128/mSystems.00210-19. PubMed DOI PMC

MacDougall JDB, McCabe M. 1967. Diffusion coefficient of oxygen through tissues. Nature 215:1173–1174. doi:10.1038/2151173a0. PubMed DOI

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

Vermaas WF. 2001. Photosynthesis and respiration in cyanobacteria, p 245–251. In Encyclopedia of life sciences. Macmillan, London, United Kingdom. doi:10.1038/npg.els.0001670. DOI

Sabra W, Zeng AP, Lünsdorf H, Deckwer WD. 2000. Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol 66:4037–4044. doi:10.1128/aem.66.9.4037-4044.2000. PubMed DOI PMC

Wang D, Xu A, Elmerich C, Ma LZ. 2017. Biofilm formation enables free-living nitrogen-fixing rhizobacteria to fix nitrogen under aerobic conditions. ISME J 11:1602–1613. doi:10.1038/ismej.2017.30. PubMed DOI PMC

Cornejo-Castillo FM, Zehr JP. 2019. Hopanoid lipids may facilitate aerobic nitrogen fixation in the ocean. Proc Natl Acad Sci U S A 116:18269–18271. doi:10.1073/pnas.1908165116. PubMed DOI PMC

Chen Y-B, Zehr JP, Mellon M. 1996. Growth and nitrogen fixation of the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. IMS 101 in defined media: evidence for a circadian rhythm. J Phycol 32:916–923. doi:10.1111/j.0022-3646.1996.00916.x. DOI

Nedbal L, Trtílek M, Červený J, Komárek O, Pakrasi HB. 2008. A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics. Biotechnol Bioeng 100:902–910. doi:10.1002/bit.21833. PubMed DOI

Sato T. 1968. A modified method for lead staining of thin sections. J Electron Microsc (Tokyo) 17:158–159. PubMed

Abràmoff MD, Magalhães PJ, Ram SJ. 2004. Image processing with ImageJ. Biophotonics Int 11:36–42.

Capone DG, Montoya JP. 2001. Nitrogen fixation and denitrification. Methods Microbiol 30:501–515. doi:10.1016/S0580-9517(01)30060-0. DOI

Breitbarth E, Mills MM, Friedrichs G, Laroche J. 2004. The Bunsen gas solubility coefficient of ethylene as a function of temperature and salinity and its importance for nitrogen. Limnol Oceanogr Methods 2:282–288. doi:10.4319/lom.2004.2.282. DOI

Masuda T, Bernát G, Bečková M, Kotabová E, Lawrenz E, Lukeš M, Komenda J, Komenda J, Prášil O. 2018. Diel regulation of photosynthetic activity in the oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501. Environ Microbiol 20:546–560. doi:10.1111/1462-2920.13963. PubMed DOI

Kolber ZS, Prášil O, Falkowski PG. 1998. Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols. Biochim Biophys Acta 1367:88–106. doi:10.1016/s0005-2728(98)00135-2. PubMed DOI

Suggett DJ, Prášil O, Borowitzka MA. 2010. Chlorophyll a fluorescence in aquatic sciences: methods and applications. Springer, Dordrecht, Netherlands.

Oxborough K, Moore CM, Suggett DJ, Lawson T, Chan HG, Geider RJ. 2012. Direct estimation of functional PSII reaction center concentration and PSII electron flux on a volume basis: a new approach to the analysis of fast repetition rate fluorometry (FRRf) data. Limnol Oceanogr Methods 10:142–154. doi:10.4319/lom.2012.10.142. DOI

Silsbe GM, Oxborough K, Suggett DJ, Forster RM, Ihnken S, Komárek O, Lawrenz E, Prášil O, Röttgers R, Šicner M, Simis SGH, Van Dijk MA, Kromkamp JC. 2015. Toward autonomous measurements of photosynthetic electron transport rates: an evaluation of active fluorescence-based measurements of photochemistry. Limnol Oceanogr Methods 13:138–155.

Eilers PHC, Peeters J. 1988. A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton. Ecol Modell 42:199–215. doi:10.1016/0304-3800(88)90057-9. DOI

Silsbe GM, Kromkamp JC. 2012. Modeling the irradiance dependency of the quantum efficiency of photosynthesis. Limnol Oceanogr Methods 10:645–652. doi:10.4319/lom.2012.10.645. DOI

Bühler T, Sann R, Monter U, Dingier C, Kuhla J, Oelze J. 1987. Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii. Arch Microbiol 148:247–251. doi:10.1007/BF00414820. DOI

Benson BB, Krause D. 1984. The concentration and isotopic fractionation of oxygen dissolved in freshwater and seawater in equilibrium with the atmosphere. Limnol Oceanogr 29:620–632. doi:10.4319/lo.1984.29.3.0620. DOI

Fernandez AC, Phillies G. 1983. Temperature dependence of the diffusion coefficient of polystyrene latex spheres. Biopolymers 22:593–595. doi:10.1002/bip.360220203. DOI

Kestin J, Sokolov M, Wakeham WA. 1978. Viscosity of liquid water in the range −8°C to 150°C. J Phys Chem Ref Data 7:941–948. doi:10.1063/1.555581. DOI

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