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Tracking nitrogen allocation to proteome biosynthesis in a marine microbial community

Nature Microbiology volume 8, pages 498–509 (2023)Cite this article

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Abstract

Microbial growth in many environments is limited by nitrogen availability, yet there is limited understanding of how complex communities compete for and allocate this resource. Here we develop a broadly applicable approach to track biosynthetic incorporation of 15N-labelled nitrogen substrates into microbial community proteomes, enabling quantification of protein turnover and N allocation to specific cellular functions in individual taxa. Application to oligotrophic ocean surface water identifies taxa-specific substrate preferences and a distinct subset of protein functions undergoing active biosynthesis. The cyanobacterium Prochlorococcus is the most effective competitor for acquisition of ammonium and urea and shifts its proteomic allocation of N over the day/night cycle. Our approach reveals that infrastructure and protein-turnover functions comprise substantial biosynthetic demand for N in Prochlorococcus and a range of other microbial taxa. The direct interrogation of the proteomic underpinnings of N limitation with 15N-tracking proteomics illuminates how nutrient stress differentially influences metabolism in co-existing microbes.

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Fig. 1: Proteomic tracking of nitrogen assimilation.
Fig. 2: Nitrogen substrate use by taxon and coarse functional type.
Fig. 3: Net synthesis versus turnover of proteins.
Fig. 4: Proteome allocation by Prochlorococcus.
Fig. 5: Proteome allocation by Pelagibacter and eukaryotic phytoplankton.

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Data availability

Proteomic mass spectral data are available through ProteomeXchange via the MassIVE repository (massive.ucsd.edu) under accession MSV000089118 and via the PRIDE repository (ebi.ac.uk/pride) under accession PXD038614. FASTA sequence databases used for peptide identification are available via Mendeley Data at data.mendeley.com/datasets/7226j6cpp3/1.

Code availability

Code for the cPIE isotope-tracking proteomics analysis pipeline is available at github.com/waldbauerlab.

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Acknowledgements

We are grateful to: the Hawaii Ocean Time-series programme (supported by US National Science Foundation award no. 1756517), especially T. Clemente; the chief scientist of HOT-311, F. Santiago-Mandujano; the marine technicians of the University of Hawaii Ocean Technology Group; and the captain and crew of R/V Kilo Moana for making these experiments possible. Thanks to L. Zhang for maintenance of the biogeochemical proteomics facility at UChicago and members of the Waldbauer and Coleman labs for helpful discussions and support. We are also grateful to L. Kelly for comments that improved the manuscript. This work was supported by the Simons Foundation (grant no. 402971 to J.R.W.).

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Author notes

  1. Amy E. Zimmerman

    Present address: Biological Sciences Division, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA

  2. Justin C. Podowski

    Present address: Division of Data Science and Learning, Argonne National Laboratory, Argonne, IL, USA

  3. Gwendolyn E. Gallagher

    Present address: New York Sea Grant, Stony Brook University, Stony Brook, NY, USA

Authors and Affiliations

  1. Department of the Geophysical Sciences, University of Chicago, Chicago, IL, USA

    Amy E. Zimmerman, Justin C. Podowski, Gwendolyn E. Gallagher, Maureen L. Coleman & Jacob R. Waldbauer

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Contributions

Conceived and designed the experiments: A.E.Z. and J.R.W. Performed the experiments: A.E.Z., G.E.G. and J.R.W. Analysed the data: A.E.Z., J.C.P., M.L.C. and J.R.W. Contributed materials/analysis tools: A.E.Z., J.C.P., M.L.C. and J.R.W. Wrote the paper: A.E.Z., M.L.C. and J.R.W.

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Correspondence to Jacob R. Waldbauer.

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Extended data

Extended Data Fig. 1 cPIE 15N-tracking proteomics pipeline.

Workflow for peptide atom% 15N determination with cPIE (classified Peptide Isotope Enrichment) pipeline.

Extended Data Fig. 2 Peptide 15N incorporation rates.

Histogram of 15N incorporation rates from peptide timecourses (gray; total n = 9062) and fitted t-distribution (solid black line) used to determine peptide enrichment thresholds. Using the t-distribution as a null, the false discovery rate for 15N-enriched peptides (incorporation rate >0.898 atom% / h; n = 862) is constrained to 4.76%.

Extended Data Fig. 3 Taxonomic composition of inoculum and incubation proteomes.

Proteome taxonomic composition in initial inocula and after 11 hours incubation with added substrates or no-amendment control. Only peptides associated with complete 15N incorporation rate timecourses are included (n = 1605). Similar trends were observed when considering intensity, number of spectra, or number of distinct peptides (cf. Extended Data Fig. 4A & B).

Extended Data Fig. 4 15N incorporation and data quantity.

(A) and (B) Taxonomic composition of 15N-enriched peptides in each N amendment, characterized by either MS/MS spectra (A) or MS1 signal intensity (B) associated with detected 15N-enriched peptides. (C) and (D) Relationship between 15N incorporation rate and quantity of MS data for peptide timecourses (n = 9062), expressed as either total MS/MS spectra (C) or total MS1 signal intensity (D). Dotted and dashed horizonal lines indicate the lower bound of incorporation rate for 15N-enriched peptides and the upper bound for non-enriched peptides, respectively; intermediate peptides fall in between the two lines. Datapoints are colored by taxon as in (A) and (B).

Extended Data Fig. 5 Peptide 15N incorporation categories by N amendment and incubation period.

Total peptide timecourses detected and 15N enrichment categories by nitrogen amendment and incubation period. Fraction of enriched peptides in the ammonium amendment increased from 21.4% in the day/dusk incubation to 26.6% in the night/dawn incubation, while enriched peptides in the urea amendment increased from 14.3% in day/dusk to 24.3% during night/dawn; changes in the other amendments between the two incubation periods were negligible.

Extended Data Fig. 6 Distribution of peptide 15N enrichment rates by taxon.

Histograms of peptide 15N enrichment rates by taxon in the 15N-ammonium (A) and -urea (B) treatments. Dashed lines indicate the threshold enrichment rate (>0.898 atom% h−1) for 15N-enriched peptides.

Extended Data Fig. 7 Prochlorococcus 15N proteome allocation across N substrate additions.

Prochlorococcus 15N allocation to proteome functional categories across all substrate amendments (n = 2850 timecourses from 469 distinct peptides).

Extended Data Fig. 8 Prochlorococcus proteome expression changes across N substrate additions.

Changes in Prochlorococcus peptide abundances during the incubations across all natural-abundance substrate amendments (n = 880 timecourses from 143 distinct peptides). In contrast to the 15N incorporation patterns (Extended Data Fig. 7), peptide abundance changes (including day/night differences) were consistent across all amendments. This uniformity of protein expression patterns, irrespective of whether an amended substrate was being incorporated or not, is further evidence of the absence of a fertilization effect in these experiments and the representativeness of these results for in situ activity.

Extended Data Fig. 9 Pelagibacter transporter 15N incorporation.

Incorporation of 15N into subgroups of transporters in Pelagibacter (n = 1583 timecourses from 258 distinct peptides). No subgroup of transporters appeared more highly labeled than others in either experiment.

Extended Data Fig. 10 Turnover of cytoskeletal proteins in Isochrysidales.

Incorporation of 15N (solid lines) and relative abundance change (dashed lines) into four cytoskeleton (actin/tubulin) peptides of Isochrysidales (coccolithophorids) during the day/dusk and night/dawn ammonium-amended incubations. That 15N incorporation is observed despite a decline in abundance of these peptides suggests these proteins are turned over rapidly, likely as part of cytoskeletal remodeling during coccolith export to the cell surface.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1 and 2.

Reporting Summary

Supplementary Data 1

Amino acid sequences, metadata, 15N incorporation rate (atom% h−1), rate of relative abundance change (log2 h−1), and taxonomic and functional annotations for 1,740 peptides with timecourses in at least one natural abundance or 15N nitrogen amendment.

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Zimmerman, A.E., Podowski, J.C., Gallagher, G.E. et al. Tracking nitrogen allocation to proteome biosynthesis in a marine microbial community. Nat Microbiol 8, 498–509 (2023). https://doi.org/10.1038/s41564-022-01303-9

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