GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways.

BACKGROUND: Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. RESULTS: Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. CONCLUSIONS: The developed approach, based on parallel 13C tracer studies with GC-MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

The Entner-Doudoroff Pathway Contributes to Glycogen Breakdown During High to Low CO2 Shifts in the Cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria perform plant-like oxygenic photosynthesis to convert inorganic carbon into organic compounds and can also use internal carbohydrate reserves under specific conditions. A mutant collection with defects in different routes for sugar catabolism was studied to analyze which of them is preferentially used to degrade glycogen reserves in light-exposed cells of Synechocystis sp. PCC 6803 shifted from high to low CO2 conditions. Mutants defective in the glycolytic Embden-Meyerhof-Parnas pathway or in the oxidative pentose-phosphate (OPP) pathway showed glycogen levels similar to wild type under high CO2 (HC) conditions and were able to degrade it similarly after shifts to low CO2 (LC) conditions. In contrast, the mutant Δeda, which is defective in the glycolytic Entner-Doudoroff (ED) pathway, accumulated elevated glycogen levels under HC that were more slowly consumed during the LC shift. In consequence, the mutant Δeda showed a lowered ability to respond to the inorganic carbon shifts, displayed a pronounced lack in the reactivation of growth when brought back to HC, and differed significantly in its metabolite composition. Particularly, Δeda accumulated enhanced levels of proline, which is a well-known metabolite to maintain redox balances via NADPH levels in many organisms under stress conditions. We suggest that deletion of eda might promote the utilization of the OPP shunt that dramatically enhance NADPH levels. Collectively, the results point at a major regulatory contribution of the ED pathway for the mobilization of glycogen reserves during rapid acclimation to fluctuating CO2 conditions.

Glycolytic Shunts Replenish the Calvin-Benson-Bassham Cycle as Anaplerotic Reactions in Cyanobacteria.

The recent discovery of the Entner-Doudoroff (ED) pathway as a third glycolytic route beside Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (OPP) pathway in oxygenic photoautotrophs requires a revision of their central carbohydrate metabolism. In this study, unexpectedly, we observed that deletion of the ED pathway alone, and even more pronounced in combination with other glycolytic routes, diminished photoautotrophic growth in continuous light in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, we found that the ED pathway is required for optimal glycogen catabolism in parallel to an operating Calvin-Benson-Bassham (CBB) cycle. It is counter-intuitive that glycolytic routes, which are a reverse to the CBB cycle and do not provide any additional biosynthetic intermediates, are important under photoautotrophic conditions. However, observations on the ability to reactivate an arrested CBB cycle revealed that they form glycolytic shunts that tap the cellular carbohydrate reservoir to replenish the cycle. Taken together, our results suggest that the classical view of the CBB cycle as an autocatalytic, completely autonomous cycle that exclusively relies on its own enzymes and CO2 fixation to regenerate ribulose-1,5-bisphosphate for Rubisco is an oversimplification. We propose that in common with other known autocatalytic cycles, the CBB cycle likewise relies on anaplerotic reactions to compensate for the depletion of intermediates, particularly in transition states and under fluctuating light conditions that are common in nature.

Prevalence, Risk Factors and Assessment of Depressive Symptoms in Patients With Systemic Sclerosis.

Objectives: This study aims to evaluate the prevalence of depressive symptoms among systemic sclerosis (SSc) patients using the Major Depression Inventory (MDI), identify possible risk factors, and analyze the current standard of care to raise awareness and improve clinical care for SSc patients. Patients and methods: The study included 94 SSc patients (12 males, 82 females; mean age 58.3±13.6 years; range, 28 to 83 years) who completed the MDI, Short Form 36 Health Survey, Scleroderma Health Assessment Questionnaire, Brief Fatigue Inventory and Physical Activity Questionnaire. Clinical parameters were assessed according to standardized procedures. Discharge letters were analyzed for evaluation of depressive symptoms. Results: The prevalence of depressive symptoms was 22.3%. It correlated with female sex (p=0.047), underweight (p=0.002), fatigue (p<0.001), decreased quality of life (p<0.001) and less physical activity (p=0.048). The latter three were confirmed as independent risk factors in a multivariable regression analysis. The analysis of the current standard of care revealed no assessment of depressive symptoms in the majority of patients (89.4%), including 19 with depressive symptoms according to the MDI score. Conclusion: This study confirms the high prevalence of depressive symptoms in SSc patients. There is an unmet need of regular assessment of mental health during SSc consultations. Fatigue, decreased quality of life and reduced physical activity were ascertained as independent risk factors, while special attention should also be paid to weight loss and underweight.

Prevalence of sarcopenia in systemic sclerosis: assessing body composition and functional disability in patients with systemic sclerosis.

OBJECTIVE: We analyzed the prevalence of sarcopenia among systemic sclerosis (SSc) patients with respect to quality of life, disability, organ involvement, and muscle function. METHODS: A total of 129 patients who met the ACR/EULAR 2013 classification criteria were included. Body composition was measured using bioelectric impedance analysis. Sarcopenia was defined according to the criteria of the European Working Group on Sarcopenia in Older People. Handgrip and knee extension strength and pulmonary peak flow were measured. Physical function was assessed with the Short Form-36 Health Survey and Scleroderma Health Assessment Questionnaire. RESULTS: Sarcopenia was prevalent in 22.5% of patients. There were significant differences between patients with and without sarcopenia regarding handgrip strength (11.5 [2.0-30.0] versus 18.0 [1.0-41.0] kilogram force [kgf]; P <0.001) and knee extension strength (11.0 [3.5-32.5] versus 17.5 [3.5-88.0] kgf; P = 0.006), physical function (38.8 [9.9-85.0] versus 48.8 [0-88.0]; P = 0.032) and number of immunosuppressants (2 [0-4] versus 1 [0-5]; P = 0.009). There were no differences regarding age (57.0 [32.0-83.0] versus 60.5 [28.0-82.0] years; P = 0.350) and disease duration (8 [1-27] versus 7 [0-34] years; P = 0.350). CONCLUSIONS: Sarcopenia is common in patients with SSc and is associated with physical impairment that affects everyday life and participation in work. Interestingly, although age is the main risk factor for sarcopenia in the general population, it did not differ between sarcopenic and non-sarcopenic SSc patients in our study. Instead, the number of immunosuppressive drugs was significantly higher among sarcopenic patients.

A Specific Glycogen Mobilization Strategy Enables Rapid Awakening of Dormant Cyanobacteria from Chlorosis.

Many organisms survive stressful conditions via entry into a dormant state that can be rapidly exited when the stressor disappears; this ability provides a strong selective advantage. In the cyanobacterium Synechocystis sp. PCC 6803, the exit from nitrogen chlorosis takes less than 48 h and is enabled by the impressive metabolic flexibility of these cyanobacteria, which pass through heterotrophic and mixotrophic phases before reentering photoautotrophic growth. Switching between these states requires delicate coordination of carbohydrate oxidation, CO2 fixation, and photosynthesis. Here, we investigated the contribution of the different carbon catabolic routes by assessing mutants of these pathways during nitrogen chlorosis and resuscitation. The addition of nitrate to nitrogen-starved cells rapidly starts the awakening program. Metabolism switches from maintenance metabolism, characterized by residual photosynthesis and low cellular ATP levels, to an initial heterotrophic phase, characterized by respiration and an immediate increase in ATP levels. This respiration relies on glycogen breakdown catalyzed by the glycogen phosphorylase GlgP2. In the following transient mixotrophic phase, photosynthesis and CO2 fixation restart and glycogen is consumed. During the mixotrophic phase, parallel operation of the oxidative pentose phosphate cycle and the Entner-Doudoroff pathway is required for resuscitation to proceed; the glycolytic route via the Embden-Meyerhof-Parnas pathway has minor importance. Our data suggest that, during resuscitation, only the Entner-Doudoroff and oxidative pentose phosphate pathways supply the metabolic intermediates necessary for the anabolic reactions required to reconstitute a vegetative cell. Intriguingly, the key enzymes for glycogen catabolism are already expressed during the preceding chlorotic phase, in apparent preparation for rapid resuscitation.

The impact of malnutrition on quality of life in patients with systemic sclerosis.

BACKGROUND/OBJECTIVES: Our objective was to investigate the prevalence of malnutrition in patients with systemic sclerosis (SSc) and its impact on their quality of life (QoL). SUBJECTS/METHODS: One hundred and twenty-nine patients with SSc underwent clinical evaluation and were screened with the Malnutrition Universal Screening Tool (MUST). Malnutrition was defined as MUST score ≥2.To investigate QoL, all patients completed the Short Form 36 Questionnaire and the Scleroderma Health Assessment Questionnaire. The data were analyzed with IBM SPSS® Statistics by using χ2/Fisher's test, Mann-Whitney U test, correlation, and linear regression analysis. RESULTS: One hundred and twenty-nine patients were included in this study (mean age was 59.1 ± 13.8 years, 90.7% women). The prevalence of malnutrition was 10.9%. Age and disease duration were not significantly different between malnourished and well-nourished patients. All QoL scores (except bodily pain and self-reported health) were significantly impaired in malnourished patients. Furthermore, the Scleroderma Health Assessment Score score which assesses disease-specific QoL was significantly higher in the malnourished patients (1.6 ± 0.73 compared to 0.91 ± 0.61 in well-nourished patients; p = 0.001), reflecting a lower QoL due to disease-specific impairments. CONCLUSIONS: Severe malnutrition in SSc patients is associated with reduced QoL. Standardized nutritional screening should routinely be conducted to identify the risk of malnutrition in order to enable an intervention with multimodal treatment and avoid the serious consequences associated with severe malnutrition.

The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.

Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden-Meyerhof-Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner-Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO2 via the Calvin-Benson cycle.