Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation

Abstract

Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

Keywords: LTQ-Orbitrap; Synechocystis sp. PCC 6803; cyanobacteria; nitrogen starvation; peptide dimethylation labeling; phosphoproteome.

Figure 1

Schematic representation of the major metabolic processes in nitrogen supplied cells (left) and nitrogen-starved cells (right). Due to the lack of combined nitrogen sources, glutamine synthetase (GS) cannot produce glutamine, and in consequence, glutamate synthase (GOGAT) reaction becomes substrate-limited, leading to increased 2-OG levels. Anabolic reactions toward nitrogen containing metabolites are tuned down and growth is arrested. The reduction equivalents generated by the light reactions of photosynthesis are now redirected toward glycogen synthesis, whose cellular abundance increases dramatically (Joseph et al., 2014).

Figure 2

Immunoblot validation of P_II protein and phosphorylation dynamics. The change in protein abundance (SDS PAGE) and phosphorylation (Clear native PAGE) of the N-regulatory P_II protein between nitrate- (NO⁻₃) or ammonia-grown (NH⁺₄) and 24h h nitrogen starved cells (-N_24h) from both experiments is shown on the left panel. P⁰_II represents the unphosphorylated trimeric complex and P^1–3_II represent P _II trimers with one, two and three phosphorylated subunits. The AtpB protein serves as loading control for SDS and Clear native PAGE. Corresponding MS acquired protein and phosphorylation event ratios in log₂ scale, relative to the NO⁻₃ are shown on the right panel.

Figure 3

Distribution of quantified phosphorylation events. Ratio distributions of quantified phosphorylation events from nitrogen starved cells to nitrate grown cells (-N/NO⁻₃) and nitrogen starved cells to ammonia grown cells (-N/NH⁺₄) from both experiments are shown. Down-regulated phosphorylation events under nitrogen starvation (threshold 0.5) are colored red, static phosphorylation events are colored green and up-regulated phosphorylation events (threshold 2.0) are colored blue. Classified InterPro terms from phosphoproteins, identified at least twice for each experiment and ratio are listed below the corresponding experimental conditions -N/NO⁻₃ or -N/NH⁺₄. Classified proteins of each indicated InterPro term are listed in Supplementary information 6.

Figure 4

Site specific distribution of significantly regulated phosphorylation events. Log₂ transformed ratio distributions of quantified phosphorylation events from nitrogen starved cells in comparison to nitrate grown cells (-N/NO⁻₃) and nitrogen starved cells to ammonia grown cells (-N/NH⁺₄), relative to the Log₁₀ transformed phosphopeptide intensities are shown. Significantly regulated phosphorylation events from the respective experiments are displayed as stars (in red) and the protein ID and the localized phosphorylation site or phosphopeptide span is indicated.