12 research outputs found
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.</p
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.</p
Cytochrome cM 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 cM (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.</p
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.</p
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
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
Study of <i>O</i>‑Phosphorylation Sites in Proteins Involved in Photosynthesis-Related Processes in <i>Synechocystis</i> sp. Strain PCC 6803: Application of the SRM Approach
<i>O</i>-Phosphorylation
has been shown in photosynthesis-related
proteins in a cyanobacterium <i>Synechocystis</i> sp. strain
PCC 6803 (thereafter <i>Synechocystis</i> 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 <i>Synechocystis</i> 6803 using TiO<sub>2</sub> 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 CO<sub>2</sub> 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 <i>Synechocystis</i> 6803 cells challenged with various environmental
stresses
Quantitative analysis of the erythrocyte membrane proteins in polycythemia vera patients treated with hydroxycarbamide
More than 90% of polycythemia vera (PV) patients have a mutation in the protein JAK2, which is closely associated with the erythrocyte membrane. With the comparison of 1-D gels of erythrocyte membranes obtained from PV patients treated with hydroxycarbamide and those of untreated controls we observed significant differences in the region of 40–55 kDa. On the basis of the LC–MS/MS analysis of this region we report up-regulation of four protein disulfide isomerases, which was subsequently confirmed by targeted mass spectrometric analysis. In further studies it will be prudent to compare this in patients both treated and not treated with hydroxycarbamide
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
β-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 <i>in silico</i> 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