Cyanobacteria of the genus Trichodesmium provide about 80 Tg of fixed nitrogen to the surface ocean per year and contribute to marine biogeochemistry, including the sequestration of carbon dioxide. Trichodesmium fixes nitrogen in the daylight, despite the incompatibility of the nitrogenase enzyme with oxygen produced during photosynthesis. While the mechanisms protecting nitrogenase remain unclear, all proposed strategies require considerable resource investment. Here we identify a crucial benefit of daytime nitrogen fixation in Trichodesmium spp. that may counteract these costs. We analysed diel proteomes of cultured and field populations of Trichodesmium in comparison with the marine diazotroph Crocosphaera watsonii WH8501, which fixes nitrogen at night. Trichodesmium’s proteome is extraordinarily dynamic and demonstrates simultaneous photosynthesis and nitrogen fixation, resulting in balanced particulate organic carbon and particulate organic nitrogen production. Unlike Crocosphaera, which produces large quantities of glycogen as an energy store for nitrogenase, proteomic evidence is consistent with the idea that Trichodesmium reduces the need to produce glycogen by supplying energy directly to nitrogenase via soluble ferredoxin charged by the photosynthesis protein PsaC. This minimizes ballast associated with glycogen, reducing cell density and decreasing sinking velocity, thus supporting Trichodesmium’s niche as a buoyant, high-light-adapted colony forming cyanobacterium. To occupy its niche of simultaneous nitrogen fixation and photosynthesis, Trichodesmium appears to be a conspicuous consumer of iron, and has therefore developed unique iron-acquisition strategies, including the use of iron-rich dust. Particle capture by buoyant Trichodesmium colonies may increase the residence time and degradation of mineral iron in the euphotic zone. These findings describe how cellular biochemistry defines and reinforces the ecological and biogeochemical function of these keystone marine diazotrophs.
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Nitrogen fixation rates in the Guinea Dome and the equatorial upwelling regions in the Atlantic Ocean
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The mass spectrometry proteomics data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD016332 and https://doi.org/10.6019/PXD016332 (laboratory experiments) and identifier PXD027796 and https://doi.org/10.6019/PXD027796 (field data). The processed proteomic data are also available at the Biological and Chemical Oceanography Data Management Office (BCO-DMO) (https://www.bco-dmo.org/dataset/783873). Source data are provided for main text Figs. 1–5 and Extended Data Figs. 1–10. Source data are provided with this paper.
Fully reproducible code for sinking velocity calculations, statistics and plotting is available at https://github.com/naheld/Held2020_TrichoDiel.
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This work was supported by NSF Graduate Research Fellowship grant 1122274 (N.A.H.), Gordon and Betty Moore Foundation grant GBMF-3782 (M.A.S.), National Science Foundation grants OCE-1657766, OCE-1850719 and OCE-1924554 (M.A.S.), National Institutes of Health grant GM135709-02 (M.A.S.), and the Woods Hole Oceanographic Institution Ocean Ventures Fund (N.A.H.). N.A.H. was additionally supported by Principles of Microbial Ecosystems collaboration of the Simons Foundation (grant ID 542379). We thank the scientific staff and crew of the AT39-05/Tricolim research expedition, particularly chief scientist D. Hutchins, and the JC150/Ziploc expedition, particularly chief scientist C. Mahaffey. Special thanks to B. White.
The authors declare no competing interests.
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a) Average total spectral counts (peptide to spectrum matches) with error bar representing +/- one standard deviation, at each time point. Each data point is also shown individually as black scatter points. Yellow and indigo bars indicate the light and dark periods, respectively. Total spectral counts were relatively uniform and do not vary systematically throughout the diel cycle, implying consistency in the proteome analyses. b) Total protein content in the culture shown with error bar representing +/- one standard deviation, for biological duplicates after protein precipitation and purification, measured by a colorimetric assay. Higher protein abundances at night may suggest nighttime cell growth. Again, each data point is also shown individually as black scatter points. Yellow and indigo bars indicate the light and dark periods, respectively.
Extended Data Fig. 2 Dynamics of the entire proteome of Trichodesmium erythraeum sp. IMS101 over the diel cycle.
The dynamic range of the normalized spectral count data can be observed, as well as fluctuations in protein abundance occurring throughout the experiment.
Extended Data Fig. 3 Proteome dynamics of separate replicate laboratory experiment over the diel cycle.
a) Clustered heatmap of a replicate diel experiment conducted one year prior to the main experiment, under the same experimental conditions. Protein abundances were summed for each KO module and normalized across each row. b) Dynamics of the proteome clusters over the diel cycle, with each KO module represented as a line and colored based on the clustering in panel (A). Rapid oscillations of the proteome and clustering of the nitrogenase/nitrogen metabolism proteins with the photosystems are similar in the main experiment. Yellow and dark purple bars indicate the light and dark periods, respectively.
a) Clustered heatmap of the proteome of a field Trichodesmium population sampled in situ over a diel cycle. Protein abundances were summed for each KO module and normalized across each row. b) Dynamics of the proteome clusters over the diel cycle, with each KO module represented as a line and colored based on the clustering in panel (A). Though the sampling was lower resolution than in the laboratory experiments, the rapid oscillations of the proteome are reproduced. Yellow and dark purple bars indicate the light and dark periods, respectively.
Extended Data Fig. 5 In vivo specific activity of the nitrogenase NifH protein over the diel cycle for Crocosphaera watsonii.
In vivo specific activity of the nitrogenase NifH protein (nmol ethyelene produced per min per mg NifH) over the diel cycle for Crocosphaera watsonii34. Unlike in Trichodesmium which exhibits significant variability in nitrogenase activity throughout the diel cycle, in Crocosphaera nitrogenase is either not present or highly present and very active.
POC content versus total protein spectral counts in the main laboratory experiment. These are weakly correlated suggesting that POC content is driven mainly by carbohydrate content, not protein abundance.
The populations were sampled on August 7, 2017 at 31°W 22°N in the early morning. Error bars are standard deviations of the mean value of the biological triplicates, and corresponding data points are plotted in grey circles. For each depth, n = 3 samples collected from replicate phytoplankton net sampling events, n = 2 samples for depth = 160 m.
Glycogen content of Trichodesmium colonies sampled in situ at the surface and separated by morphology. The populations were sampled from the surface on March 10, 2018 at 65 22.420 °W 17 0.284 °N and separated by morphology at the time of picking.
Extended Data Fig. 9 Synchrotron-based element maps used to determine mass of particulate iron associated with a puff-type colony.
Synchrotron-based element maps used to determine mass of particulate iron associated with a puff-type colony, data originally collected as in Held et al., 202020. The left image is the X-ray fluorescence-based concentration, the middle image represents pixels with sufficiently high Fe to be considered a particle, and the right image is the product of the left and middle images. The total particulate Fe was determined as the area integrated Fe of the right image. The scale bar represents 180 microns. As detailed in Held et al., 2021, five Trichodesmium colonies of differing morphologies and degrees of particle association were examined in this way. These images are representative of a Trichodesmium colony with average-to-high particle loading.
Extended Data Fig. 10 Calibration curves for 15N labeled standard peptides used for absolute quantitation of the nitrogenase proteins.
Precursor ion intensities were linearly correlated with analyzed peptide concentrations between 0-10 fmol μL−1.
Diel proteome data of Trichodesmium (main laboratory experiment) and Crocosphaera with annotated KO modules.
Quantitative data for nitrogenase proteins and specific activity data for NifH.
Selected protein and physiological data from main laboratory experiment.
Spearmann correlation statistics for significant positive and negative correlations between protein pairs, and selected protein data used to generate the network.
Modelling input and output data; Trichodesmium and Crocosphaera glycogen content over the diel cycle.
Total spectral counts and total protein content for samples in main laboratory experiment.
Global proteome data for main laboratory experiment.
Global proteome data organized into KO modules for separate replicate replicate experiment.
Global proteome data organized into KO modules for field samples.
NifH specific activity for Crocosphaera watsonii.
Total spectrum counts and POC content for samples in main laboratory experiment.
Glycogen content data for field samples at different depths.
Glycogen content for field samples separated by morphology.
Calibration data for 15N-labelled peptide standards.
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Held, N.A., Waterbury, J.B., Webb, E.A. et al. Dynamic diel proteome and daytime nitrogenase activity supports buoyancy in the cyanobacterium Trichodesmium. Nat Microbiol 7, 300–311 (2022). https://doi.org/10.1038/s41564-021-01028-1
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