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Periodic and coordinated gene expression between a diazotroph and its diatom host


In the surface ocean, light fuels photosynthetic carbon fixation of phytoplankton, playing a critical role in ecosystem processes including carbon export to the deep sea. In oligotrophic oceans, diatom–diazotroph associations (DDAs) play a keystone role in ecosystem function because diazotrophs can provide otherwise scarce biologically available nitrogen to the diatom host, fueling growth and subsequent carbon sequestration. Despite their importance, relatively little is known about the nature of these associations in situ. Here we used metatranscriptomic sequencing of surface samples from the North Pacific Subtropical Gyre (NPSG) to reconstruct patterns of gene expression for the diazotrophic symbiont Richelia and we examined how these patterns were integrated with those of the diatom host over day–night transitions. Richelia exhibited significant diel signals for genes related to photosynthesis, N2 fixation, and resource acquisition, among other processes. N2 fixation genes were significantly co-expressed with host nitrogen uptake and metabolism, as well as potential genes involved in carbon transport, which may underpin the exchange of nitrogen and carbon within this association. Patterns of expression suggested cell division was integrated between the host and symbiont across the diel cycle. Collectively these data suggest that symbiont–host physiological ecology is strongly interconnected in the NPSG.

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  1. 1.

    Carr M-E, Friedrichs MAM, Schmeltz M, Noguchi Aita M, Antoine D, Arrigo KR, et al. A comparison of global estimates of marine primary production from ocean color. Deep Sea Res Part 2. 2006;53:741–70.

  2. 2.

    Karl DM, Church MJ, Dore JE, Letelier RM, Mahaffey C. Predictable and efficient carbon sequestration in the North Pacific Ocean supported by symbiotic nitrogen fixation. Proc Natl Acad Sci USA. 2012;109:1842–9.

  3. 3.

    Dore JE, Letelier RM, Church MJ, Lukas R, Karl DM. Summer phytoplankton blooms in the oligotrophic North Pacific Subtropical Gyre: historical perspective and recent observations. Prog Oceanogr. 2008;76:2–38.

  4. 4.

    Villareal TA, Adornato L, Wilson C, Schoenbaechler CA. Summer blooms of diatom-diazotroph assemblages and surface chlorophyll in the North Pacific gyre: a disconnect. J Geophys Res Oceans. 2011; 116:

  5. 5.

    Wilson C Late Summer chlorophyll blooms in the oligotrophic North Pacific Subtropical Gyre. Geophys Res Lett. 2003; 30:

  6. 6.

    Foster RA, Kuypers MM, Vagner T, Paerl RW, Musat N, Zehr JP. Nitrogen fixation and transfer in open ocean diatom-cyanobacterial symbioses. ISME J. 2011;5:1484–93.

  7. 7.

    Kitajima S, Furuya K, Hashihama F, Takeda S, Kanda J. Latitudinal distribution of diazotrophs and their nitrogen fixation in the tropical and subtropical western North Pacific. Limnol Oceanogr. 2009;54:537–47.

  8. 8.

    Carpenter EJ, Montoya JP, Burns J, Mulholland MR, Subramaniam A, Capone DG. Extensive bloom of a N2-fixing diatom/cyanobacterial association in the tropical Atlantic Ocean. Mar Ecol Prog Ser. 1999;185:273–83.

  9. 9.

    Böttjer D, Dore JE, Karl DM, Letelier RM, Mahaffey C, Wilson ST, et al. Temporal variability of nitrogen fixation and particulate nitrogen export at Station ALOHA. Limnol Oceanogr. 2017;62:200–16.

  10. 10.

    Villareal TA, Pilskaln CH, Montoya JP, Dennett M. Upward nitrate transport by phytoplankton in oceanic waters: balancing nutrient budgets in oligotrophic seas. PeerJ. 2014;2:e302.

  11. 11.

    Villareal TA. Laboratory culture and preliminary characterization of the nitrogen-fixing Rhizosolenia-Richelia symbiosis. Mar Ecol. 1990;11:117–32.

  12. 12.

    Wilson ST, Aylward FO, Ribalet F, Barone B, Casey JR, Connell PE, et al. Coordinated regulation of growth, activity and transcription in natural populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera. Nat Microbiol. 2017;2:17118.

  13. 13.

    Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010.

  14. 14.

    Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.

  15. 15.

    Alexander H, Jenkins BD, Rynearson TA, Dyhrman ST. Metatranscriptome analyses indicate resource partitioning between diatoms in the field. Proc Natl Acad Sci USA. 2015;112:E2182–90.

  16. 16.

    Foster RA, Goebel NL, Zehr JP. Isolation of Calothrix rhizosoleniae (Cyanobacteria) Strain Sc01 from Chaetoceros (Bacillariophyta) Spp. diatoms of the Subtropical North Pacific Ocean. J Phycol. 2010;46:1028–37.

  17. 17.

    Hilton JA. Ecology and evolution of diatom-associated cyanobacteria through genetic analyses. Dissertation. University of California: Santa Cruz, CA; 2014.

  18. 18.

    Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26:589–95.

  19. 19.

    Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015;31:166–9.

  20. 20.

    Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, et al. From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 2006;34:D354–7. Database issue

  21. 21.

    Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007;35:W182–5. Web Server issue

  22. 22.

    Thaben PF, Westermark PO. Detecting rhythms in time series with RAIN. J Biol Rhythms. 2014;29:391–400.

  23. 23.

    Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

  24. 24.

    Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B. 1995;57:289–300.

  25. 25.

    Aylward FO, Eppley JM, Smith JM, Chavez FP, Scholin CA, DeLong EF. Microbial community transcriptional networks are conserved in three domains at ocean basin scales. Proc Natl Acad Sci USA. 2015;112:5443–8.

  26. 26.

    Ottesen EA, Young CR, Gifford SM, Eppley JM, Marin R 3rd, Schuster SC, et al. Ocean microbes. Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages. Science. 2014;345:207–12.

  27. 27.

    Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinform. 2008;9:559.

  28. 28.

    Kenkel CD, Matz MV. Gene expression plasticity as a mechanism of coral adaptation to a variable environment. Nat Ecol Evol. 2016;1:14.

  29. 29.

    Rose NH, Seneca FO, Palumbi SR. Gene networks in the wild: identifying transcriptional modules that mediate coral resistance to experimental heat stress. Genome Biol Evol. 2015;8:243–52.

  30. 30.

    Grote A, Voronin D, Ding T, Twaddle A, Unnasch TR, Lustigman S, et al. Defining Brugia malayi and Wolbachia symbiosis by stage-specific dual RNA-seq. PLoS Negl Trop Dis. 2017;11:e0005357.

  31. 31.

    Waldbauer JR, Rodrigue S, Coleman ML, Chisholm SW. Transcriptome and proteome dynamics of a light-dark synchronized bacterial cell cycle. PLoS One. 2012;7:e43432.

  32. 32.

    Church MJ, Short CM, Jenkins BD, Karl DM, Zehr JP. Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean. Appl Environ Microbiol. 2005;71:5362–70.

  33. 33.

    Follett CL, White AE, Wilson ST, Follows MJ. Nitrogen fixation rates diagnosed from diurnal changes in elemental stoichiometry. Limnol Oceanogr. 2018.

  34. 34.

    Shi T, Ilikchyan I, Rabouille S, Zehr JP. Genome-wide analysis of diel gene expression in the unicellular N2-fixing cyanobacterium Crocosphaera watsonii WH 8501. ISME J. 2010;4:621–32.

  35. 35.

    Stockel J, Welsh EA, Liberton M, Kunnvakkam R, Aurora R, Pakrasi HB. Global transcriptomic analysis of Cyanothece 51142 reveals robust diurnal oscillation of central metabolic processes. Proc Natl Acad Sci USA. 2008;105:6156–61.

  36. 36.

    Frischkorn KR, Haley ST, Dyhrman ST. Coordinated gene expression between Trichodesmium and its microbiome over day-night cycles in the North Pacific Subtropical Gyre. ISME J. 2018;12:997–1007.

  37. 37.

    Wiig JA, Rebelein JG, Hu Y. Nitrogenase complex. eLS: John Wiley & Sons, Ltd, Chichester. 2014 []

  38. 38.

    Zehr JP, Turner PJ. Nitrogen fixation: Nitrogenase genes and gene expression. Methods Microbiol. 2001;30:271–86.

  39. 39.

    Borthakur D, Basche M, Buikema WJ, Borthakur PB, Haselkorn R. Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC 7120. Mol General Genet Manage. 1990;221:227–34.

  40. 40.

    Flaherty BL, Nieuwerburgh FV, Head SR, Golden JW. Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combinded-nitrogen deprivation. BMC Genom. 2011;12:332.

  41. 41.

    Kushige H, Kugenuma H, Matsuoka M, Ehira S, Ohmori M, Iwasaki H. Genome-wide and heterocyst-specific circadian gene expression in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 2013;195:1276–84.

  42. 42.

    Peterson RB, Wolk CP. Localization of an upatke hydrogenase in Anabaena. Plant Physiol. 1978;61:688–91.

  43. 43.

    Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wunschiers R, Lindblad P. Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol Rev. 2002;66:1–20.

  44. 44.

    Zhang X, Sherman DM, Sherman LA. The uptake hydrogenase in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain PCC 7822 protects nitrogenase from oxygen toxicity. J Bacteriol. 2014;196:840–9.

  45. 45.

    Bothe H, Schmitz O, Yates MG, Newton WE. Nitrogen fixation and hydrogen metabolism in cyanobacteria. Microbiol Mol Biol Rev. 2010;74:529–51.

  46. 46.

    Wilson ST, Tozzi S, Foster RA, Ilikchyan I, Kolber ZS, Zehr JP, et al. Hydrogen cycling by the unicellular marine diazotroph Crocosphaera watsonii strain WH8501. Appl Environ Microbiol. 2010;76:6797–803.

  47. 47.

    McGary K, Nudler E. RNA polymerase and the ribosome: the close relationship. Curr Opin Microbiol. 2013;16:112–7.

  48. 48.

    Follett CL, Dutkiewicz S, Karl DM, Inomura K, Follows MJ. Seasonal resource conditions favor a summertime increase in North Pacific diatom-diazotroph associations. ISME J. 2018;12:1543–57.

  49. 49.

    Mills MM, Ridame C, Davey M, La Roche J, Geider RJ. Iron and phosphorus co-limit nitrogen fixation in the eastern tropical North Atlantic. Nature. 2004;429:292–4.

  50. 50.

    Bes MT, Hernandez JA, Peleato ML, Fillat MF. Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter. FEMS Microbiol Lett. 2001;194:187–92.

  51. 51.

    Ghassemian M, Straus NA. Fur regulates the expression of iron-stress genes in the cyanobacterium Synechococcus sp. strain PCC 7942. Microbiology. 1996;142:1469–76.

  52. 52.

    Thompson DK, Beliaev AS, Giometti CS, Tollaksen SL, Khare T, Lies DP, et al. Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress. Appl Environ Microbiol. 2002;68:881–92.

  53. 53.

    Pi H, Helmann JD. Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis. Proc Natl Acad Sci USA. 2017;114:12785–90.

  54. 54.

    Escolar L, Perez-Martin J, de Lorenzo V. Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol. 1999;181:6223–9.

  55. 55.

    Fillat MF, Peleato ML, Razquin P, Gómez-Moreno C. Effects of iron-deficiency in photosynthetic electron transport and nitrogen fixation in the cyanobacterium Anabaena: flavodoxin induction as adaptive response. In: Abadia J, editor. Iron nutrition in soils and plants. Kluwer Academic Publishers. Dordrecht, the Netherlands. 1995;315–21.

  56. 56.

    Saito MA, Bertrand EM, Dutkiewicz S, Bulygin VV, Moran DM, Monteiro FM, et al. Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii. Proc Natl Acad Sci USA. 2011;108:2184–9.

  57. 57.

    Pereira N, Shilova IN, Zehr JP. Molecular markers define progressing stages of phosphorus limitation in the nitrogen-fixing cyanobacterium, Crocosphaera. J Phycol. 2016;52:274–82.

  58. 58.

    Dyhrman ST, Haley ST. Phosphorus scavenging in the unicellular marine diazotroph Crocosphaera watsonii. Appl Environ Microbiol. 2006;72:1452–8.

  59. 59.

    Harke MJ, Berry DL, Ammerman JW, Gobler CJ. Molecular response of the bloom-forming cyanobacterium, Microcystis aeruginosa, to phosphorus limitation. Microb Ecol. 2012;63:188–98.

  60. 60.

    Scanlan DJ, Silman NJ, Donald KM, Wilson WH, Carr NG, Joint I, et al. An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton. Appl Environ Microbiol. 1997;63:2411–20.

  61. 61.

    Boyle EA, Huested SS, Jones SP. On the distribution of copper, nickel, and cadmium in the surface waters of the North Atlantic and North Pacific Ocean. J Geophys Res Oceans. 1981; 86.

  62. 62.

    Huertas MJ, Lopez-Maury L, Giner-Lamia J, Sanchez-Riego AM, Florencio FJ. Metals in cyanobacteria: analysis of the copper, nickel, cobalt and arsenic homeostasis mechanisms. Life (Basel). 2014;4:865–86.

  63. 63.

    Rodriguez IB, Ho TY. Diel nitrogen fixation pattern of Trichodesmium: the interactive control of light and Ni. Sci Rep. 2014;4:4445.

  64. 64.

    Bruland KW. Oceanographic distributions of cadmium, zinc, nickel, and copper in the North Pacific. Earth Planet Sci Lett. 1980;47:176–98.

  65. 65.

    Twining BS, Baines SB. The trace metal composition of marine phytoplankton. Annu Rev Mar Sci. 2013;5:191–215.

  66. 66.

    Markov D, Naryshkina T, Mustaev A, Severinov K. A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly. Genes Dev. 1999;13:2439–48.

  67. 67.

    Borukhov S, Goldfarb A. Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly. Protein Expr Purif. 1993;4:503–11.

  68. 68.

    Lavaud J, Materna AC, Sturm S, Vugrinec S, Kroth PG. Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching. PLoS One. 2012;7:e36806.

  69. 69.

    Ashworth J, Coesel S, Lee A, Armbrust EV, Orellana MV, Baliga NS. Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana. Proc Natl Acad Sci USA. 2013;110:7518–23.

  70. 70.

    Sorek M, Diaz-Almeyda EM, Medina M, Levy O. Circadian clocks in symbiotic corals: the duet between Symbiodinium algae and their coral host. Mar Genomics. 2014;14:47–57.

  71. 71.

    Wier AM, Nyholm SV, Mandel MJ, Massengo-Tiasse RP, Schaefer AL, Koroleva I, et al. Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis. Proc Natl Acad Sci USA. 2010;107:2259–64.

  72. 72.

    Pao SS, Paulsen IT, Saier MH Jr. Major facilitator superfamily. Microbiol Mol Biol Rev. 1998;62:1–34.

  73. 73.

    Collos Y, Berges JA. Nitrogen metabolism in phytoplankton. In: Duarte CM, editor. Marine ecology: Encyclopedia of Life Support Systems (EOLSS), Paris, France. 2002;1–18.

  74. 74.

    Alexander H, Rouco M, Haley ST, Wilson ST, Karl DM, Dyhrman ST. Functional group-specific traits drive phytoplankton dynamics in the oligotrophic ocean. Proc Natl Acad Sci USA. 2015;112:E5972–9.

  75. 75.

    Udvardi MK, Day DA. Metabolite transport across symbiotic membranes of legume nodules. Annu Rev Plant Physiol Plant Mol Biol. 1997;48:493–523.

  76. 76.

    Mus F, Crook MB, Garcia K, Garcia Costas A, Geddes BA, Kouri ED, et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Appl Environ Microbiol. 2016;82:3698–710.

  77. 77.

    Moog D, Rensing SA, Archibald JM, Maier UG, Ullrich KK. Localization and evolution of putative triose phosphate translocators in the diatom Phaeodactylum tricornutum. Genome Biol Evol. 2015;7:2955–69.

  78. 78.

    Mulligan C, Fischer M, Thomas GH. Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea. FEMS Microbiol Rev. 2011;35:68–86.

  79. 79.

    Croft MT, Lawrence AD, Raux-Deery E, Warren MJ, Smith AG. Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature. 2005;438:90–3.

  80. 80.

    Helliwell KE, Lawrence AD, Holzer A, Kudahl UJ, Sasso S, Krautler B, et al. Cyanobacteria and eukaryotic algae use different chemical variants of vitamin B12. Curr Biol. 2016;26:999–1008.

  81. 81.

    Slavoff SA, Chen I, Choi YA, Ting AY. Expanding the substrate tolerance of biotin ligase through exploration of enzymes from diverse species. J Am Chem Soc. 2008;130:1160–2.

  82. 82.

    Cohen NR, Ellis KA, Burns WG, Lampe RH, Schuback N, Johnson Z. et al. Iron and vitamin interactions in marine diatom isolates and natural assemblages of the Northeast Pacific Ocean. Limnol Oceanogr. 2017;62:2076–96.

  83. 83.

    Sundström BG. Observations on Rhizosolenia clevei Ostenfeld (Bacillariophyceae) and Richelia intracellularis Schmidt (Cyanophyceae). Bot Mar. 1984;27:345–55.

  84. 84.

    Villareal TA. Marine nitrogen-fixing diatom-cyanobacteria symbioses. In: Carpenter EJ, Capone DG, Rueter JG, editors. Marine pelagic cyanobacteria: Trichodesmium and other Diazotrophs. Dordrecht: Springer Netherlands; 1992. p. 163–75.

  85. 85.

    Villareal TA. Division cycles in the nitrogen-fixing Rhizosolenia (Bacillariophyceae)-Richelia (Nostocaceae) symbiosis. Eur J Phycol. 1989;24:357–65.

  86. 86.

    Chisolm SW, Azam F, Eppley RW. Silicic acid incorportation in marine diatoms on light:dark cycles: use as an assay for phased cell division. Limnol Oceanogr. 1978;23:518–29.

  87. 87.

    Knight MJ, Senior L, Nancolas B, Ratcliffe S, Curnow P. Direct evidence of the molecular basis for biological silicon transport. Nat Commun. 2016;7:11926.

  88. 88.

    Paulsrud P, Lindblad P. Fasciclin domain proteins are present in Nostoc symbionts of lichens. Appl Environ Microbiol. 2002;68:2036–9.

  89. 89.

    Reynolds WS, Schwarz JA, Weis VM. Symbiosis-enhanced gene expression in cnidarian-algal associations: cloning and characterization of a cDNA, sym32, encoding a possible cell adhesion protein. Comp Biochem Physiol A. 2000;126:33–44.

  90. 90.

    Rodriguez-Navarro DN, Dardanelli MS, Ruiz-Sainz JE. Attachment of bacteria to the roots of higher plants. FEMS Microbiol Lett. 2007;272:127–36.

  91. 91.

    Polovina JJ, Howell EA, Abecassis M. Ocean’s least productive waters are expanding. Geophys Res Lett. 2008; 35.

  92. 92.

    Signorini SR, Franz BA, McClain CR. Chlorophyll variability in the oligotrophic gyres: mechanisms, seasonality and trends. Front Mar Sci. 2015;2:1

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For assistance with field work, the authors thank the operational staff of the Simons Collaboration on Ocean Processes and Ecology (SCOPE) and the captain and crew of the R/V Kilo Moana.


Funding was provided by the Simons Foundation (SCOPE award ID 329108 to STD and JPZ). Computational support was provided by the National Science Foundation under Grant Nos. DBI-1458641 and ABI-1062432 to Indiana University.

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The authors declare that they have no conflict of interest.

Correspondence to Sonya T. Dyhrman.

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