The Role of Serine/Threonine-Specific Protein Kinases in Cyanobacteria - SpkB Is Involved in Acclimation to Fluctuating Conditions in Synechocystis sp. PCC 6803.

Protein phosphorylation via serine/threonine protein kinases (Spk) is a widespread mechanism to adjust cellular processes toward changing environmental conditions. To study their role(s) in cyanobacteria, we investigated a collection of 11 completely segregated spk mutants among the 12 annotated Spks in the model cyanobacterium Synechocystis sp. PCC 6803. Screening of the mutant collection revealed that especially the mutant defective in SpkB encoded by slr1697 showed clear deviations regarding carbon metabolism, that is, reduced growth rates at low CO2 or in the presence of glucose, and different glycogen accumulation patterns compared to WT. Alterations in the proteome of ΔspkB indicated changes of the cell surface but also metabolic functions. A phospho-proteome analysis revealed the absence of any phosphorylation in two proteins, while decreased phosphorylation of the carboxysome-associated protein CcmM and increased phosphorylation of the allophycocyanin alpha subunit ApcA was detected in ΔspkB. Furthermore, the regulatory PII protein appeared less phosphorylated in the mutant compared to WT, which was verified in Western blot experiments, indicating a clearly delayed PII phosphorylation in cells shifted from nitrate-containing to nitrate-free medium. Our results indicate that SpkB is an important regulator in Synechocystis that is involved in phosphorylation of the PII protein and additional proteins.

Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria.

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

Alterations in the CO2 availability induce alterations in the phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803.

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 phosphoproteomics 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 c. 70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the noncorrelating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phosphoproteins harboring over 200 site-specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phosphoproteins with reduced phosphorylation levels at lower CO2 , whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding ΔspkC 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 phosphoproteomics. Collectively, our study highlights the importance of posttranscriptional regulation of protein abundances as well as posttranslational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria.