The daily light-dark cycle is a recurrent and predictable environmental phenomenon to which many organisms, including cyanobacteria, have evolved to adapt. Understanding how cyanobacteria alter their metabolic attributes in response to subjective light or dark growth may provide key features for developing strains with improved photosynthetic efficiency and applications in enhanced carbon sequestration and renewable energy. Here, we undertook a label-free proteomic approach to investigate the effect of extended light (LL) or extended dark (DD) conditions on the unicellular cyanobacterium Crocosphaera subtropica ATCC 51142. We quantified 2287 proteins, of which 603 proteins were significantly different between the two growth conditions. These proteins represent several biological processes, including photosynthetic electron transport, carbon fixation, stress responses, translation, and protein degradation. One significant observation is the regulation of over two dozen proteases, including ATP dependent Clp-proteases (endopeptidases) and metalloproteases, the majority of which were upregulated in LL compared to DD. This suggests that proteases play a crucial role in the regulation and maintenance of photosynthesis, especially the PSI and PSII components. The higher protease activity in LL indicates a need for more frequent degradation and repair of certain photosynthetic components, highlighting the dynamic nature of protein turnover and quality control mechanisms in response to prolonged light exposure. The results enhance our understanding of how Crocosphaera subtropica ATCC51142 adjusts its molecular machinery in response to extended light or dark growth conditions.

Proteomic analysis of unicellular cyanobacterium Crocosphaera subtropica ATCC 51142 under extended light or dark growth

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges. 

Interactome Analysis of ClpX Reveals Its Regulatory Role in Metabolism and Photosynthesis in Cyanobacteria.

Cyanobacteria (oxygenic photoautrophs) comprise a diverse group holding significance both environmentally and for biotechnological applications. The utilization of proteomic techniques has significantly influenced investigations concerning cyanobacteria. Application of proteomics allows for large-scale analysis of protein expression and function within cyanobacterial systems. The cyanobacterial proteome exhibits tremendous functional, spatial, and temporal diversity regulated by multiple factors that continuously modify protein abundance, post-translational modifications, interactions, localization, and activity to meet the dynamic needs of these tiny blue greens. Modern mass spectrometry-based proteomics techniques enable system-wide examination of proteome complexity through global identification and high-throughput quantification of proteins. These powerful approaches have revolutionized our understanding of proteome dynamics and promise to provide novel insights into integrated cellular behavior at an unprecedented scale. In this Review, we present modern methods and cutting-edge technologies employed for unraveling the spatiotemporal diversity and dynamics of cyanobacterial proteomics with a specific focus on the methods used to analyze post-translational modifications (PTMs) and examples of dynamic changes in the cyanobacterial proteome investigated by proteomic approaches.

Cyanobacterial Proteomics: Diversity and Dynamics.

Bacterial lifespan ranges from a few hours to geological timescales. The prolonged survival trait under extreme energy starvation is essential for the perpetuation of their existence. The theme for long-term survival [long-term stationary phase (LTSP)] in the non-growing state may be dependent on the diversity in the environmental niche and the lifestyle of the bacteria, exemplified by longevity studies, albeit few, with model organisms. In the present study, we characterized the LTSP of mycelial cells of Streptomyces Minutiscleroticus , which remain metabolically active, demonstrate ongoing protein synthesis—killed by protein synthesis inhibitors—and remarkably by the cell-wall synthesis inhibitors, vancomycin, and ampicillin, suggesting “growth.” Their rapid turnover is also evident in ~10-fold loss of colony-forming unit (CFU) over a year, suggesting that for the death of one “old” cell, slightly less than one “new” cell is born. This longevity is consequent to (i) induction of the gene expression program effected by non-metabolizable, non-ionic osmolyte, sucrose, thus conditional, and (ii) possibly rendering this carbon utilizable by the production of a slow hydrolytic activity generating glucose, reinforcing the relevance of low-level energy resource for long term survival in the starvation phase. The viability parameters of LTSP cells measured through up to 90 days suggest that the stationary phase transitioning into LTSP following nutrient exhaustion is nearly quantitative. Expectedly, the viability in LTSP is (p)ppGpp/RelA dependent. Whereas mutation in chaperone clpX , negatively affects survival in stationary phase, overexpression of signal sensor-transducer histidine kinase, SasA8, enhances cell survivability. The relevance of longevity functions identified here requires further deduction of the genetic program. 

Genetics of conditional extended mycelial cell viability of Streptomyces minutiscleroticus in deep starvation phase implicates the involvement of (p)ppGpp, clpX, and a histidine kinase sasA

Protein homeostasis is tightly regulated by protein quality control systems such as chaperones and proteases. In cyanobacteria, the ClpXP proteolytic complex is regarded as a representative proteolytic system and consists of a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. However, the functions and molecular mechanisms of ClpX in cyanobacteria remain unclear. This study aimed to decipher the unique contributions and regulatory networks of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We showed that the interruption of clpX led to slower growth, decreased high light tolerance, and impaired photosynthetic cyclic electron transfer. A quantitative proteomic strategy was employed to globally identify ClpX-regulated proteins in Synechocystis cells. In total, we identified 172 differentially expressed proteins (DEPs) upon the interruption of clpX. Functional analysis revealed that these DEPs are involved in diverse biological processes, including glycolysis, nitrogen assimilation, photosynthetic electron transport, ATP-binding cassette (ABC) transporters, and two-component signal transduction. The expression of 24 DEPs was confirmed by parallel reaction monitoring (PRM) analysis. In particular, many hypothetical or unknown proteins were found to be regulated by ClpX, providing new candidates for future functional studies on ClpX. Together, our study provides a comprehensive ClpX-regulated protein network, and the results serve as an important resource for understanding protein quality control systems in cyanobacteria.

/pdf/proteomic-analysis-of-the-regulatory-networks-of-clpx-in-a-1v3dkkij.pdf

Proteomic analysis of the regulatory networks of ClpX in a model cyanobacterium Synechocystis sp. PCC 6803

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Proteomic analysis of the regulatory networks of ClpX in a model cyanobacterium Synechocystis sp. PCC 6803