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Deep proteogenomics of a unicellular cyanobacterium

ABSTRACT: Cyanobacteria are photoautotrophs that profoundly impact the biogeochemical cycles on Earth. Due to their photosynthetic lifestyle that includes the fixation of atmospheric CO2, they are of increasing interest for a sustainable economy. Knowledge of protein expression and regulation is key for understanding of the cyanobacterial metabolism; however, proteome studies in cyanobacteria are still limited and cover only a fraction of the theoretical proteome. Here, we performed a proteogenomic analysis of 628 LC-MS/MS measurements for the unicellular model cyanobacterium Synechocystis sp. PCC 6803 to characterize the expressed (phospho)proteome, re-annotate known and discover potential novel open reading frames (ORFs). By mapping extensive shotgun MS proteomics data generated by the SCyCode consortium onto a six-frame translation of the Synechocystis genome, we re-annotated 96 start sites and discovered 103 novel open reading frames (ORFs). Through re-analysis of previously published multi-omics datasets, we confirmed 48 re-annotated or novel ORFs with high confidence. Our study resulted in the largest reported proteome and phosphoproteome dataset for Synechocystis, covering expression of about 80% of the theoretical proteome and 642 O-phosphorylation events under various cultivation conditions, such as nitrogen or carbon limitation. This dataset will serve as a resource providing dedicated information on condition-dependent protein expression and phosphorylation.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Exploris 480, Q Exactive HF, LTQ Orbitrap Elite

ORGANISM(S): Synechocystis Sp. Pcc 6803 Substr. Pcc-p

SUBMITTER: Nicolas Nalpas

LAB HEAD: Boris Macek

PROVIDER: PXD033243 | Pride | 2024-01-05

REPOSITORIES: Pride

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Deep proteogenomics of a unicellular cyanobacterium

Project description:Cyanobacteria are photoautotrophs that profoundly impact the biogeochemical cycles on Earth. Due to their photosynthetic lifestyle that includes the fixation of atmospheric CO2, they are of increasing interest for a sustainable economy. Knowledge of protein expression and regulation is key for understanding of the cyanobacterial metabolism; however, proteome studies in cyanobacteria are still limited and cover only a fraction of the theoretical proteome. Here, we performed a proteogenomic analysis of 628 LC-MS/MS measurements for the unicellular model cyanobacterium Synechocystis sp. PCC 6803 to characterize the expressed (phospho)proteome, re-annotate known and discover potential novel open reading frames (ORFs). By mapping extensive shotgun MS proteomics data generated by the SCyCode consortium onto a six-frame translation of the Synechocystis genome, we re-annotated 96 start sites and discovered 103 novel open reading frames (ORFs). Through re-analysis of previously published multi-omics datasets, we confirmed 48 re-annotated or novel ORFs with high confidence. Our study resulted in the largest reported proteome and phosphoproteome dataset for Synechocystis, covering expression of about 80% of the theoretical proteome and 642 O-phosphorylation events under various cultivation conditions, such as nitrogen or carbon limitation. This dataset will serve as a resource providing dedicated information on condition-dependent protein expression and phosphorylation.

2024-01-05 | PXD033290 | Pride

Alterations in the CO2 availability induce alterations in the phospho-proteome of the cyanobacterium Synechocystis sp. PCC 6803

Project description:Cyanobacteria are the only prokaryotes that perform plant-like oxygenic photosynthesis. They evolved an inorganic carbon-concentrating mechanism to adapt to low CO2 conditions. Quantitative phospho-proteomics was applied to analyze regulatory features during the acclimation to low CO2 conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Overall, more than 2500 proteins were quantified, equivalent to approximately 70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the non-correlating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phospho-proteins harboring over 200 site-specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phospho-proteins with significantly reduced phosphorylation levels at lower CO2, whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding spkC mutant was characterized and showed decreased ability to acclimate to low CO2 conditions. Possible phosphorylation targets of SpkC including a BCT1 subunit were identified by phospho-proteomics. Collectively, our study highlights the importance of post-transcriptional regulation of protein abundances as well as post-translational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria.

2021-07-14 | PXD022283 | Pride

The proteome of Synechocystis sp. PCC 6803 cells grown under light-activated heterotrophic growth

Project description:Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Besides being able to grow photoautotrophically, some cyanobacteria are also capable to grow photoheterotrophically, where they use reduced organic compounds as carbon source, or even completely heterotrophically by using reduced organic compounds as carbon and energy source. The well characterized cyanobacterium Synechocystis sp. PCC 6803 can grow in darkness under light-activated heterotrophic growth (LAHG) conditions by using glucose as carbon and energy source. In the present work, we combined pre-fractioning of Synechocystis cellular membranes with a global proteome and lipidome analysis, to shift the analytical focus towards the rearrangement of the internal thylakoid membrane system observed in Synechocystis cells under LAHG conditions.

2015-03-03 | PXD000980 | Pride

A systematic survey of the light-dependent protein quality control in a model cyanobacterium

Project description:We conducted a large-scale identification of protein degradation under light or dark condition in the model cyanobacterium Synechocystis PCC 6803 (Hereafter referred as Synechocystis) using the isobaric labeling based quantitative proteomics technique. This is the first discovery about redox-regulated protein degradation in cyanobacteria. Our results provide the first comprehensive catalog of the light-dependent protein degradation events, and would act as an important resource in future study toward understanding light-regulated processes and protein quality control mechanism in cyanobacteria.

2021-11-10 | PXD023181 | Pride

Developmental Dynamics of RNA Translation in the Human Brain

Project description:The precise regulation of gene expression is fundamental to neurodevelopment, plasticity, and cognitive function. While several studies have profiled transcription in the developing human brain, there is a gap in our understanding of accompanying translational regulation. We performed ribosome profiling on 73 human prenatal and adult cortex samples. We characterized the translational regulation of annotated open reading frames (ORFs) and identified thousands of previously unknown translation events, including small ORFs that give rise to human- and/or brain-specific microproteins, many of which we independently verified using proteomics. Ribosome profiling in stem cell-derived human neuronal cultures corroborated these findings and revealed that several neuronal activity-induced non-coding RNAs encode previously undescribed microproteins. Physicochemical analysis of brain microproteins identified a class of proteins that contain arginine-glycine-glycine (RGG) repeats and thus may be regulators of RNA metabolism. This resource expands the known translational landscape of the human brain and illuminates previously unknown brain-specific protein products.

2022-09-29 | PXD035950 | Pride

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

Project description: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 signalling, 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 towards the unbiased 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 labelling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2,382 proteins and 183 phosphorylation events and quantifying 2,111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 hours. Among the proteins with increased phosphorylation, the PII signalling 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.

2016-05-09 | PXD001831 | Pride

REPARATION: Ribosome Profiling Assisted (Re-) Annotation of Bacterial genomes - PART 2

Project description:Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated techniques depend heavily on sequence context and often underestimate the complexity of the proteome. We developed REPARATION (RibosomeE Profiling Assisted (Re-)AnnotaTION), a de novo algorithm that takes advantage of experimental evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation. Ribo-seq next generation sequencing technique that provides a genome-wide snapshot of the position translating ribosome along an mRNA at the time of the experiment. REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds to screen for spurious ORFs based on a growth curve model. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel ORFs including variants of previously annotated and novel small ORFs (<71 codons). Our predictions were supported by matching mass spectrometry (MS) proteomics data and sequence conservation analysis. REPARATION is unique in that it makes use of experimental Ribo-seq data to perform de novo ORF delineation in bacterial genomes, and thus can identify putative coding ORFs irrespective of the sequence context of the reading frame.

2017-08-03 | PXD005901 | Pride

DNA-protein affinity precipitation to identify proteins interacting with the atpT promoter in Synechocystis sp. PCC 6803

Project description:The small protein AtpΘ (encoded by gene atpT) identified in cyanobacteria becomes maximum expressed during low-energy conditions such as during darkness. In the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803), AtpΘ was demonstrated to inhibit the ATP hydrolysis activity of the F0F1 ATP synthase. Here, the regulation of atpT gene expression was studied in greater detail. The darkness-induced activation of the atpT promoter indicated the existence of regulatory factors. To identify such factors, DNA-protein affinity precipitation was performed using biotinylated DNA fragments. These fragments contained either the promoter-5’UTR (PatpT66-5’UTR) as bait or, as negative control, only the 5’UTR (atpT 5’UTR). Each DNA fragment was incubated with total protein samples isolated from exponential phase Synechocystis 6803 which had been kept in the light or 12 h in darkness prior to protein preparation. The resulting protein samples were then analyzed by mass spectrometry.

2022-04-14 | PXD027801 | Pride

Proximity labeling facilitates defining neighborhood proteome of photosystem II oxygen evolution complex in a model cyanobacterium

Project description:APEX2-based proximity labeling coupled with mass spectrometry have great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach it is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis, and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO.

2023-02-17 | PXD036113 | Pride

Chlorosis as developmental program in cyanobacteria: The proteomic fundament for surviving and awakening

Project description:Cyanobacteria that do not fix nitrogen adapt to prolonged periods of nitrogen deprivation in a chlorotic state where cell growth and metabolism is arrested. Upon nutrient availability, the cells return back to vegetative growth within 2-3 days. This resuscitation process is highly orchestrated and relies on the stepwise re-installation and activation of essential cellular structures and functions. We investigate transition into chlorosis and return to vegetative life as a simple model of a cellular developmental process that represents a fundamental survival strategy in biology. In the present study, we describe the process through quantitative proteomics and phosphoproteomics analysis of long-term nitrogen-starved Synechocystis sp. PCC 6803 cells during different time points of resuscitation. Intriguingly, substantial O phosphorylation of proteins involved in photosynthesis was detected in the chlorotic state, including hyperphosphorylation of the remaining phycobiliproteins. We provide evidence that hyperphosphorylation of the terminal rod linker CpcD increases the lifespan of phycobiliproteins during chlorosis.

2018-06-28 | PXD008027 | Pride

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