Phycobilisome breakdown effector NblD is required to maintain the cellular amino acid composition during nitrogen starvation

Abstract

Small proteins are critically involved in the acclimation response of photosynthetic cyanobacteria to nitrogen starvation. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the arginine catabolism pathway and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite co-regulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply. Importance Cyanobacteria play important roles in the global carbon and nitrogen cycles. In their natural environment, these organisms are exposed to fluctuating nutrient conditions. Nitrogen starvation induces a coordinated nitrogen-saving program that includes the breakdown of nitrogen-rich photosynthetic pigments, particularly phycobiliproteins. The small protein NblD was recently identified as an effector of phycobilisome breakdown in cyanobacteria. In this study, we demonstrate that the NblD-mediated degradation of phycobiliproteins is needed to sustain cellular pools of soluble amino acids and other crucial metabolites. The essential role played by NblD in metabolic homeostasis explains why genes encoding this small protein are conserved in almost all members of cyanobacterial radiation.

FIG 1

Response of the Synechocystis 6803 WT and ΔnblD mutant during the acclimation to N starvation. (A) Phenotype under nitrogen-replete (+N) conditions and after 24 h of nitrogen depletion (−N). (B) Spectrometric measurements of cultures normalized to OD₇₅₀.

FIG 2

(A) Total soluble amino acid content and (B) total soluble metabolite content (nonproteinogenic amino acids and important intermediates) of the wild type and ΔnblD cultures in nitrogen-repleted (+N) and nitrogen-depleted BG11 (−N) medium after 3 h and 24 h. For each time point and strain, triplicates were measured, and significance (*, P < 0.05) was calculated using a t test; n.s., not significant. The raw data are given in Table S2.

FIG 3

Development of amino acid contents (in mM for Glu and μM for all other) in the WT and ΔnblD mutant in nitrogen-replete (+N) and nitrogen-deplete (−N) medium after 3 h and 24 h. Amino acids are grouped according to the observed differences between wild type and mutant as follows: difference already observed in +N conditions; no difference between strains; early differences (after 3 h); later differences (24 h). Significance was calculated with a two-sample t test with unequal variance (Welch’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001) for the development in each strain and between the strains at corresponding time points (see Table S4 for details).

FIG 4

Phycobilisome individual amino acid (aa) composition (%) plotted against their increase or decrease observed in relation to the initial values measured in nitrogen sufficient conditions. (A and C) WT after 3 h of N limitation. (B and D) WT after 24 h of N limitation. (E and F) ΔnblD mutant after 3 and 24 h. Phycobilisome composition was calculated based on the structure description by Arteni et al. (57), assuming a common complex with 6 rods each with 3 hexameric phycocyanin discs and a tricylindrical allophycocyanin core. A line shows low correlation between the variables, and a poor R-squared value indicates a high variation of the data in relation to this line.

FIG 5

Comparison of metabolite content (in mM for Glu, citrate, 3PGA, malate, and 2-OG, and in μM for all others) in WT and ΔnblD deletion mutant in nitrogen-replete (+N) and nitrogen-deplete (−N) medium after 3 h and 24 h shown alongside their biosynthetic pathways. Succinate was below the detection limit (b.d.) in the ΔnblD sample under nitrogen-replete conditions. Significance was calculated with the two-sample t test with unequal variance (Welch’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001) for the development in each strain and between the strains at corresponding time points (details in Table S4).