Quantitative Proteomics Shows Extensive Remodeling Induced by Nitrogen Limitation in Prochlorococcusmarinus SS120

Abstract

Prochlorococcus requires the capability to accommodate to environmental changes in order to proliferate in oligotrophic oceans, in particular regarding nitrogen availability. A precise knowledge of the composition and changes in the proteome can yield fundamental insights into such a response. Here we report a detailed proteome analysis of the important model cyanobacterium Prochlorococcus marinus SS120 after treatment with azaserine, an inhibitor of ferredoxin-dependent glutamate synthase (GOGAT), to simulate extreme nitrogen starvation. In total, 1,072 proteins, corresponding to 57% of the theoretical proteome, were identified-the maximum proteome coverage obtained for any Prochlorococcus strain thus far. Spectral intensity, calibrated quantification by the Hi3 method, was obtained for 1,007 proteins. Statistically significant changes (P value of <0.05) were observed for 408 proteins, with the majority of proteins (92.4%) downregulated after 8 h of treatment. There was a strong decrease in ribosomal proteins upon azaserine addition, while many transporters were increased. The regulatory proteins P_II and PipX were decreased, and the global nitrogen regulator NtcA was upregulated. Furthermore, our data for Prochlorococcus indicate that NtcA also participates in the regulation of photosynthesis. Prochlorococcus responds to the lack of nitrogen by slowing down translation, while inducing photosynthetic cyclic electron flow and biosynthesis of proteins involved in nitrogen uptake and assimilation. IMPORTANCEProchlorococcus is the most abundant photosynthetic organism on Earth, contributing significantly to global primary production and playing a prominent role in biogeochemical cycles. Here we study the effects of extreme nitrogen limitation, a feature of the oligotrophic oceans inhabited by this organism. Quantitative proteomics allowed an accurate quantification of the Prochlorococcus proteome, finding three main responses to nitrogen limitation: upregulation of nitrogen assimilation-related proteins, including transporters; downregulation of ribosome proteins; and induction of the photosystem II cyclic electron flow. This suggests that nitrogen limitation affects a range of metabolic processes far wider than initially believed, with the ultimate goal of saving nitrogen and maximizing the nitrogen uptake and assimilation capabilities of the cell.

Keywords: marine cyanobacteria; nitrogen limitation; nitrogen metabolism; prochlorococcus; quantitative proteomics.

FIG 1

Effect of azaserine on cultures of Prochlorococcus marinus SS120. (A) Intracellular 2-OG concentration. Aliquots were taken at different times. The control culture (no addition) is shown by blue bars. Azaserine (100 µM) was added to cultures at time zero, and the values for these cultures are indicated by the red bars. (B) GS activity after 8-h treatment. (C) ICDH activity after 8-h treatment. The graphs represent the data from three independent biological replicates. Values are means plus standard deviation (error bars). Mean values that are significantly different (P ≤ 0.01) by Student’s t test are indicated by two asterisks. (D) Western blotting using anti-GS antibodies. (E) Western blotting using anti-ICDH antibodies. Densitometry results from the obtained bands are shown below the blots. One hundred percent corresponds to the intensity for the control situation. 75 and 48 indicate molecular mass in kilodaltons.

FIG 2

Proteome discovery. Metabolic diagram showing the pathways where the identified proteins are involved. We mapped KEGG identifiers for 396 proteins out of the 1,072 proteins identified. The diagram was made by using Pathview package in R software to highlight proteins with known function on top of the standard KEGG metabolic pathway scheme for Prochlorococcus SS120.

FIG 3

Analysis of the quantitative proteomic results. (A) Volcano plot showing the proteins. The proteins were indicated in color as follows: blue, fold change of <2, P value of <0.05; red, fold change of >2, P value of <0.05; yellow, fold change of >2, P value of >0.05. (B) Enrichment analysis. The 377 proteins significantly downregulated obtained by Progenesis were used for enrichment analysis in String 10 using default parameters. CoA, coenzyme A.

FIG 4

Outline of the cyclic electron flow around photosystem II in Prochlorococcus strain SS120. Orange arrows depict the cyclic electron flow, enhanced under N limitation. Blue arrows show the linear electron flow. Abbreviations: PQ, plastoquinones; PSII RC, photosystem II reaction center; cyt, cytochrome; PC, plastocyanine; PSI, photosystem I; Q_A, A plastoquinone.

FIG 5

Effect of azaserine addition on gene expression in Prochlorococcus SS120. Azaserine (100 µM) was added to cultures. Cells were collected after 8 h, and gene expression was measured by qRT-PCR. Data are the average values for six independent biological replicates. Error bars correspond to standard deviations. Mean values that are significantly different by Student’s t test are indicated by asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01.