Interplay between photosynthetic electron flux and organic carbon sinks in sucrose-excreting Synechocystis sp. PCC 6803 revealed by omics approaches.

BACKGROUND: Advancing the engineering of photosynthesis-based prokaryotic cell factories is important for sustainable chemical production and requires a deep understanding of the interplay between bioenergetic and metabolic pathways. Rearrangements in photosynthetic electron flow to increase the efficient use of the light energy for carbon fixation must be balanced with a strong carbon sink to avoid photoinhibition. In the cyanobacterium Synechocystis sp. PCC 6803, the flavodiiron protein Flv3 functions as an alternative electron acceptor of photosystem I and represents an interesting engineering target for reorganizing electron flow in attempts to enhance photosynthetic CO2 fixation and increase production yield. RESULTS: We have shown that inactivation of Flv3 in engineered sucrose-excreting Synechocystis (S02:Δflv3) induces a transition from photoautotrophic sucrose production to mixotrophic growth sustained by sucrose re-uptake and the formation of intracellular carbon sinks such as glycogen and polyhydroxybutyrate. The growth of S02:Δflv3 exceeds that of the sucrose-producing strain (S02) and demonstrates unforeseen proteomic and metabolomic changes over the course of the nine-day cultivation. In the absence of Flv3, a down-regulation of proteins related to photosynthetic light reactions and CO2 assimilation occurred concomitantly with up-regulation of those related to glycolytic pathways, before any differences in sucrose production between S02 and S02:Δflv3 strains were observed. Over time, increased sucrose degradation in S02:Δflv3 led to the upregulation of respiratory pathway components, such as the plastoquinone reductase complexes NDH-11 and NDH-2 and the terminal respiratory oxidases Cyd and Cox, which transfer electrons to O2. While glycolytic metabolism is significantly up-regulated in S02:Δflv3 to provide energy for the cell, the accumulation of intracellular storage compounds and the increase in respiration serve as indirect sinks for photosynthetic electrons. CONCLUSIONS: Our results show that the presence of strong carbon sink in the engineered sucrose-producing Synechocystis S02 strain, operating under high light, high CO2 and salt stress, cannot compensate for the lack of Flv3 by directly balancing the light transducing source and carbon fixing sink reactions. Instead, the cells immediately sense the imbalance, leading to extensive reprogramming of cellular bioenergetic, metabolic and ion transport pathways that favor mixotrophic growth rather than enhancing photoautotrophic sucrose production.

Cytochrome c M Decreases Photosynthesis under Photomixotrophy in Synechocystis sp. PCC 6803.

Photomixotrophy is a metabolic state that enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we demonstrate, via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual inhibition of QA - reoxidation in wild-type Synechocystis, which largely decreases photosynthesis over 3 d of growth. This process is circumvented by deleting the gene encoding cytochrome c M (CytM), a cryptic c-type heme protein widespread in cyanobacteria. The ΔCytM strain maintained active photosynthesis over the 3-d period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of PSI and PSII. Overall, this resulted in a higher growth rate compared to that of the wild type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localized respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a phenotype similar to that of ΔCytM under photomixotrophy. This, in combination with other physiological data, and in contrast to a previous hypothesis, suggests that CytM does not transfer electrons to these complexes. In summary, our data suggest that CytM may have a regulatory role in photomixotrophy by modulating the photosynthetic capacity of cells.

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO2 step-down.

Photosynthetic cyanobacteria are exposed to rapid changes in light intensity in their natural habitats, as well as in photobioreactors. To understand the effects of such fluctuations on Synechocystis sp. PCC 6803, the global proteome of cells grown under a fluctuating light condition (low background light interrupted with high light pulses) was compared to the proteome of cells grown under constant light with concomitant acclimation of cells to low CO2 level. The untargeted global proteome of Synechocystis sp. PCC 6803 was analyzed by data-dependent acquisition (DDA), which relies on the high mass accuracy and sensitivity of orbitrap-based tandem mass spectrometry. In addition, a targeted selected reaction monitoring (SRM) approach was applied to monitor the proteomic changes in a strain lacking flavodiiron proteins Flv1 and Flv3. This strain is characterized by impaired growth and photosynthetic activity under fluctuating light. An obvious reprogramming of cell metabolism was observed in this study and was compared to a previous transcriptional analysis performed under the same fluctuating light regime. Cyanobacterial responses to fluctuating light correlated at mRNA and protein levels to some extent, but discrepancies indicate that several proteins are post-transcriptionally regulated (affecting observed protein abundances). The data suggest that Synechocystis sp. PCC 6803 maintain higher nitrogen assimilation, serving as an electron valve, for long-term acclimation to fluctuating light upon CO2 step-down. Although Flv1 and Flv3 are known to be crucial for the cells at the onset of illumination, the flavodiiron proteins, as well as components of carbon assimilation pathways, were less abundant under fluctuating light.

Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803.

NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane (TM), unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild-type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light-activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters, but downregulated a few iron homeostasis-related proteins. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that the amount and function of NdbA in the TM has a capacity to modulate the redox signaling of gene expression, but apparently has a major physiological role in maintaining iron homeostasis under LAHG conditions. LC-MS/MS data are available via ProteomeXchange with identifier PXD011671.

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis.

NAD(P)H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a well-characterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact roles of different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (ΔndbC) for a corresponding protein and submitting the mutant to physiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the ΔndbC mutant under light-activated heterotrophic growth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-12 complex, and decreases the expression of several transporters in ambient CO2 growth conditions.

Study of O-Phosphorylation Sites in Proteins Involved in Photosynthesis-Related Processes in Synechocystis sp. Strain PCC 6803: Application of the SRM Approach.

O-Phosphorylation has been shown in photosynthesis-related proteins in a cyanobacterium Synechocystis sp. strain PCC 6803 (thereafter Synechocystis 6803), suggesting that phosphorylation of S, T, and Y residues might be important in photosynthesis-related processes. Investigation of biological roles of these phosphorylation events requires confident knowledge of the phosphorylated sites and prospects for their individual assessment. We performed phosphoproteomic analysis of Synechocystis 6803 using TiO2 enrichment of the phosphopeptides, followed by LC-MS/MS, and discovered 367 phosphorylation sites in 190 proteins participating in various cellular functions. Furthermore, we focused on the large group of phosphoproteins that are involved in light harvesting, photosynthesis-driven electron flow, photoprotection, and CO2 fixation. The SRM approach was applied to verify/improve assignments of phosphorylation sites in these proteins and to investigate possibilities for analysis of phosphopeptide isomers. The SRM assays were designed for peptides comprising 45 phosphorylation sites. The assays contain peptide iRT values and Q1/Q3 transitions comprising those discriminating between phosphopeptide isoforms. The majority of investigated phosphopeptides and phosphorylated isoforms could be individually assessed with the SRM technique. The assays could be potentially used in future quantitative studies to evaluate an extent of phosphorylation in photosynthesis-related proteins in Synechocystis 6803 cells challenged with various environmental stresses.

Changes in Relative Thylakoid Protein Abundance Induced by Fluctuating Light in the Diatom Thalassiosira pseudonana.

One of the hallmarks of marine diatom biology is their ability to cope with rapid changes in light availability due to mixing of the water column and the lens effect. We investigated how irradiance fluctuations influence the relative abundance of key photosynthetic proteins in the centric diatom Thalassiosira pseudonana by means of mass-spectrometry-based approaches for relative protein quantitation. Most notably, fluctuating-light conditions lead to a substantial overall up-regulation of light-harvesting complex proteins as well as several subunits of photosystems II and I. Despite an initial delay in growth under FL, there were no indications of FL-induced photosynthesis limitation, in contrast to other photosynthetic organisms. Our findings further strengthen the notion that diatoms use a qualitatively different mechanism of photosynthetic regulation in which chloroplast-mitochondria interaction has overtaken crucial regulatory processes of photosynthetic light reactions that are typical for the survival of land plants, green algae, and cyanobacteria.

Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) is a well-established model species in oxygenic photosynthesis research and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study, we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with a targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short- and long-term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short- and long-term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low-abundance proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.

The Flavodiiron Protein Flv3 Functions as a Homo-Oligomer During Stress Acclimation and is Distinct from the Flv1/Flv3 Hetero-Oligomer Specific to the O2 Photoreduction Pathway.

The flavodiiron proteins (FDPs) Flv1 and Flv3 in cyanobacteria function in photoreduction of O2 to H2O, without concomitant formation of reactive oxygen species, known as the Mehler-like reaction. Both Flv1 and Flv3 are essential for growth under fluctuating light (FL) intensities, providing protection for PSI. Here we compared the global transcript profiles of the wild type (WT), Δflv1 and Δflv1/Δflv3 grown under constant light (GL) and FL. In the WT, FL induced the largest down-regulation in transcripts involved in carbon-concentrating mechanisms (CCMs), while those of the nitrogen assimilation pathways increased as compared with GL. Already under GL the Δflv1/Δflv3 double mutant demonstrated a partial down-regulation of transcripts for CCM and nitrogen metabolism, while in FL conditions the transcripts for nitrogen assimilation were strongly down-regulated. Many alterations were specific only for Δflv1/Δflv3, and not detected in Δflv1, suggesting that certain transcripts are affected primarily because of the lack of flv3 By constructing the strains overproducing solely either Flv1 or Flv3, we demonstrate that the homo-oligomers of these proteins also function in acclimation of cells to FL, by catalyzing reactions with as yet unidentified components, while the presence of both Flv1 and Flv3 is a prerequisite for the Mehler-like reaction and thus the electron transfer to O2 Considering the low expression of flv1, it is unlikely that the Flv1 homo-oligomer is present in the WT.

Ovarian endometriosis signatures established through discovery and directed mass spectrometry analysis.

New molecular information on potential therapeutic targets or tools for noninvasive diagnosis for endometriosis are important for patient care and treatment. However, surprisingly few efforts have described endometriosis at the protein level. In this work we enumerate the proteins in patient endometrium and ovarian endometrioma by extensive and comprehensive analysis of minute amounts of cryosectioned tissues in a three-tiered mass spectrometric approach. Quantitative comparison of the tissues revealed 214 differentially expressed proteins in ovarian endometrioma and endometrium. These proteins are reported here as a resource of SRM (selected reaction monitoring) assays that are unique, standardized, and openly available. Pathway analysis of the proteome measurements revealed a potential role for Transforming growth factor ß-1 in ovarian endometriosis development. Subsequent mRNA microarray analysis further revealed clear ovarian endometrioma specificity for a subset of these proteins, which was also supported by further in silico studies. In this process two important proteins emerged, Calponin-1 and EMILIN-1, that were additionally confirmed in ovarian endometrioma tissues by immunohistochemistry and Western blotting. This study provides the most comprehensive molecular description of ovarian endometriosis to date and researchers with new molecular methods and tools for high throughput patient screening using the SRM assays.

Patterning of the Autotrophic, Mixotrophic, and Heterotrophic Proteomes of Oxygen-Evolving Cyanobacterium Synechocystis sp. PCC 6803.

Proteomes of an oxygenic photosynthetic cyanobacterium, Synechocystis sp. PCC 6803, were analyzed under photoautotrophic (low and high CO2, assigned as ATLC and ATHC), photomixotrophic (MT), and light-activated heterotrophic (LAH) conditions. Allocation of proteome mass fraction to seven sub-proteomes and differential expression of individual proteins were analyzed, paying particular attention to photosynthesis and carbon metabolism-centered sub-proteomes affected by the quality and quantity of the carbon source and light regime upon growth. A distinct common feature of the ATHC, MT, and LAH cultures was low abundance of inducible carbon-concentrating mechanisms and photorespiration-related enzymes, independent of the inorganic or organic carbon source. On the other hand, these cells accumulated a respiratory NAD(P)H dehydrogenase I (NDH-11) complex in the thylakoid membrane (TM). Additionally, in glucose-supplemented cultures, a distinct NDH-2 protein, NdbA, accumulated in the TM, while the plasma membrane-localized NdbC and terminal oxidase decreased in abundance in comparison to both AT conditions. Photosynthetic complexes were uniquely depleted under the LAH condition but accumulated under the ATHC condition. The MT proteome displayed several heterotrophic features typical of the LAH proteome, particularly including the high abundance of ribosome as well as amino acid and protein biosynthesis machinery-related components. It is also noteworthy that the two equally light-exposed ATHC and MT cultures allocated similar mass fractions of the total proteome to the seven distinct sub-proteomes. Unique trophic condition-specific expression patterns were likewise observed among individual proteins, including the accumulation of phosphate transporters and polyphosphate polymers storing energy surplus in highly energetic bonds under the MT condition and accumulation under the LAH condition of an enzyme catalyzing cyanophycin biosynthesis. It is concluded that the rigor of cell growth in the MT condition results, to a great extent, by combining photosynthetic activity with high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, besides the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MT cultures with excellent conditions for growth that often exceeds that of mere ATHC.

Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120.

Nostoc (Anabaena) sp. PCC 7120 is a filamentous cyanobacterial species that fixes N2 to nitrogenous compounds using specialised heterocyst cells. Changes in the intracellular ratio of carbon to nitrogen (C/N balance) is known to trigger major transcriptional reprogramming of the cell, including initiating the differentiation of vegetative cells to heterocysts. Substantial transcriptional analysis has been performed on Nostoc sp. PCC 7120 during N stepdown (low to high C/N), but not during C stepdown (high to low C/N). In the current study, we shifted the metabolic balance of Nostoc sp. PCC 7120 cultures grown at 3% CO2 by introducing them to atmospheric conditions containing 0.04% CO2 for 1 h, after which the changes in gene expression were measured using RNAseq transcriptomics. This analysis revealed strong upregulation of carbon uptake, while nitrogen uptake and metabolism and early stages of heterocyst development were downregulated in response to the shift to low CO2. Furthermore, gene expression changes revealed a decrease in photosynthetic electron transport and increased photoprotection and reactive oxygen metabolism, as well a decrease in iron uptake and metabolism. Differential gene expression was largely attributed to change in the abundances of the metabolites 2-phosphoglycolate and 2-oxoglutarate, which signal a rapid shift from fluent photoassimilation to glycolytic metabolism of carbon after transition to low CO2. This work shows that the C/N balance in Nostoc sp. PCC 7120 rapidly adjusts the metabolic strategy through transcriptional reprogramming, enabling survival in the fluctuating environment.

Proteomics of cyanobacteria: current horizons.

Application of proteomics has made a profound impact on the cyanobacterial research. It has not only provided a global identification of expressed proteins in cyanobacterial cells, but has also brought valuable insights into dynamics of cell responses to environmental challenges, regulation mechanisms, structure of protein complexes, compartmentalization, and other important biological questions. In this review, we highlight current trends in proteomics of cyanobacteria and bring to focus rising techniques which have a huge potential in expanding our knowledge about cyanobacterial proteins and in developing cyanobacteria-based biotechnological applications.

Selenoprotein biosynthesis defect causes progressive encephalopathy with elevated lactate.

OBJECTIVE: We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate. METHODS: We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency. RESULTS: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain. CONCLUSIONS: These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation.