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Dynamic diel proteome and daytime nitrogenase activity supports buoyancy in the cyanobacterium Trichodesmium

Abstract

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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Fig. 1: Dynamics of the Trichodesmium proteome in comparison to Crocosphaera.
Fig. 2: Nitrogenase concentration and activity over the diel cycle.
Fig. 3: Temporal dynamics of biomass composition, nitrogen fixation rates, and associated proteins in Trichodesmium.
Fig. 4: A glycogen synthesis protein emerges as a hub in the Trichodesmium proteome.
Fig. 5: Buoyancy properties of Trichodesmium and other phytoplankton.
Fig. 6: Summary of physiological properties and behaviors that are reinforced by daytime nitrogen fixation.

Data availability

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. 15 and Extended Data Figs. 110. Source data are provided with this paper.

Code availability

Fully reproducible code for sinking velocity calculations, statistics and plotting is available at https://github.com/naheld/Held2020_TrichoDiel.

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

N.A.H., J.B.W. and M.A.S. conceived and designed the experiment. N.A.H., R.M.K, D.M.M. and F.W.V. performed the experiments and analyses. M.R.M. and N.A.H. performed mass spectrometry analyses. M.J., N.A.H. and M.A.S. developed sinking velocity calculations. K.M.S. performed synchrotron element map analyses. All authors contributed to the writing and editing of the manuscript.

Corresponding author

Correspondence to Mak A. Saito.

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The authors declare no competing interests.

Peer review information

Nature Microbiology thanks Ilka Axmann, David Karl, Adam Kustka and Marc Strous for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Total spectral counts and average protein content across the diel cycle.

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.

Source data

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.

Source data

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.

Source data

Extended Data Fig. 4 Proteome dynamics of field Trichodesmium population over the diel cycle.

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.

Source data

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.

Source data

Extended Data Fig. 6 POC content versus total protein spectral counts.

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.

Source data

Extended Data Fig. 7 Glycogen content of Trichodesmium populations sampled in situ by depth.

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.

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Extended Data Fig. 8 Glycogen content of Trichodesmium colonies separated by morphology.

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.

Source data

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.

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Supplementary information

Supplementary Information

Supplementary Discussion, Table 1, Box 1 and Figs. 1–5.

Reporting Summary

Supplementary Table

Supplementary Tables 2–9.

Peer Review File

Source data

Source Data Fig. 1

Diel proteome data of Trichodesmium (main laboratory experiment) and Crocosphaera with annotated KO modules.

Source Data Fig. 2

Quantitative data for nitrogenase proteins and specific activity data for NifH.

Source Data Fig. 3

Selected protein and physiological data from main laboratory experiment.

Source Data Fig. 4

Spearmann correlation statistics for significant positive and negative correlations between protein pairs, and selected protein data used to generate the network.

Source Data Fig. 5

Modelling input and output data; Trichodesmium and Crocosphaera glycogen content over the diel cycle.

Source Data Extended Data Fig. 1

Total spectral counts and total protein content for samples in main laboratory experiment.

Source Data Extended Data Fig. 2

Global proteome data for main laboratory experiment.

Source Data Extended Data Fig. 3

Global proteome data organized into KO modules for separate replicate replicate experiment.

Source Data Extended Data Fig. 4

Global proteome data organized into KO modules for field samples.

Source Data Extended Data Fig. 5

NifH specific activity for Crocosphaera watsonii.

Source Data Extended Data Fig. 6

Total spectrum counts and POC content for samples in main laboratory experiment.

Source Data Extended Data Fig. 7

Glycogen content data for field samples at different depths.

Source Data Extended Data Fig. 8

Glycogen content for field samples separated by morphology.

Source Data Extended Data Fig. 10

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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