The aim of this work was to characterize in detail the response of Arthrospira to ionizing radiation, to better understand its radiation resistance capacity. Live cells of Arthrospira sp. PCC 8005 were irradiated with 60Co gamma rays. This study is the first, showing that Arthrospira is highly tolerant to gamma rays, and can survive at least 6400 Gy (dose rate of 527 Gy h−1), which identified Arthrospira sp. PCC 8005 as a radiation resistant bacterium. Biochemical, including proteomic and transcriptomic, analysis after irradiation with 3200 or 5000 Gy showed a decline in photosystem II quantum yield, reduced carbon fixation, and reduced pigment, lipid, and secondary metabolite synthesis. Transcription of photo-sensing and signaling pathways, and thiol-based antioxidant systems was induced. Transcriptomics did show significant activation of ssDNA repair systems and mobile genetic elements (MGEs) at the RNA level. Surprisingly, the cells did not induce the classical antioxidant or DNA repair systems, such superoxide dismutase (SOD) enzyme and the RecA protein. Arthrospira cells lack the catalase gene and the LexA repressor. Irradiated Arthrospira cells did induce strongly a group of conserved proteins, of which the function in radiation resistance remains to be elucidated, but which are a promising novel routes to be explored. This study revealed the radiation resistance of Arthrospira, and the molecular systems involved, paving the way for its further and better exploitation.
Cyanobacteria are photosynthetic prokaryotes that play a crucial role in the Earth's nitrogen and carbon cycles. Nitrogen availability is one of the most important factors in cyanobacterial growth. Interestingly, filamentous non-diazotrophic cyanobacteria, such as Arthrospira sp. PCC 8005, have developed survival strategies that enable them to adapt to nitrogen deprivation. Metabolic studies recently demonstrated a substantial synthesis and accumulation of glycogen derived from amino acids during nitrogen starvation. Nevertheless, the regulatory mechanism of this adaptation is poorly understood. To the best of our knowledge, this study is the first proteomic and cellular analysis of Arthrospira sp. PCC 8005 under nitrogen depletion. Label-free differential proteomic analysis indicated the global carbon and nitrogen reprogramming of the cells during nitrogen depletion as characterized by an upregulation of glycogen synthesis and the use of endogenous nitrogen sources. The degradation of proteins and cyanophycin provided endogenous nitrogen when exogenous nitrogen was limited. Moreover, formamides, cyanates and urea were also potential endogenous nitrogen sources. The transporters of some amino acids and alternative nitrogen sources such as ammonium permease 1 were induced under nitrogen depletion. Intriguingly, although Arthrospira is a non-diazotrophic cyanobacterium, we observed the upregulation of HetR and HglK proteins, which are involved in heterocyst differentiation. Moreover, after a long period without nitrate, only a few highly fluorescent cells in each trichome were observed, and they might be involved in the long-term survival mechanism of this non-diazotrophic cyanobacterium under nitrogen deprivation.
The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.
The edible cyanobacterium Arthrospira is resistant to ionising radiation. The cellular mechanisms underlying this radiation resistance are, however, still largely unknown. Therefore, additional molecular analysis was performed to investigate how these cells can escape from, protect against, or repair the radiation damage. Arthrospira cells were shortly exposed to different doses of 60Co gamma rays and the dynamic response was investigated by monitoring its gene expression and cell physiology at different time points after irradiation. The results revealed a fast switch from an active growth state to a kind of 'survival modus' during which the cells put photosynthesis, carbon and nitrogen assimilation on hold and activate pathways for cellular protection, detoxification, and repair. The higher the radiation dose, the more pronounced this global emergency response is expressed. Genes repressed during early response, suggested a reduction of photosystem II and I activity and reduced tricarboxylic acid (TCA) and Calvin-Benson-Bassham (CBB) cycles, combined with an activation of the pentose phosphate pathway (PPP). For reactive oxygen species detoxification and restoration of the redox balance in Arthrospira cells, the results suggested a powerful contribution of the antioxidant molecule glutathione. The repair mechanisms of Arthrospira cells that were immediately switched on, involve mainly proteases for damaged protein removal, single strand DNA repair and restriction modification systems, while recA was not induced. Additionally, the exposed cells showed significant increased expression of arh genes, coding for a novel group of protein of unknown function, also seen in our previous irradiation studies. This observation confirms our hypothesis that arh genes are key elements in radiation resistance of Arthrospira, requiring further investigation. This study provides new insights into phasic response and the cellular pathways involved in the radiation resistance of microbial cells, in particularly for photosynthetic organisms as the cyanobacterium Arthrospira.
The filamentous cyanobacterium Arthrospira platensis is an attractive feedstock for carbohydrate-based biofuels because it accumulated up to 74% of carbohydrates when nitrogen stressed. Nitrogen stressed A. platensis also settled spontaneously, and this occurred simultaneously with carbohydrates accumulation, suggesting a link between both phenomena. The increased settling velocity was neither due to production of extracellular carbohydrates, nor due to degradation of gas vacuoles, but was caused by an increase in the specific density of the filaments as a result of accumulation of carbohydrates under the form of glycogen. Settling velocities of carbohydrate-rich A. platensis reached 0.64mh(-1), which allowed the biomass to be harvested using a lamella separator. The biomass could be concentrated at least 15 times, allowing removal of 94% of the water using gravity settling, thus offering a potential application as a low-cost and high-throughput method for primary dewatering of carbohydrate-rich A. platensis.
Cyanobacteria have a strong potential for biofuel production due to their ability to accumulate large amounts of carbohydrates. Nitrogen (N) stress can be used to increase the content of carbohydrates in the biomass, but it is expected to reduce biomass productivity. To study this trade-off between carbohydrate accumulation and biomass productivity, we characterized the biomass productivity, biomass composition as well as the transcriptome and proteome of the cyanobacterium Arthrospira sp. PCC 8005 cultured under N-limiting and N-replete conditions. N limitation resulted in a large increase in the carbohydrate content of the biomass (from 14 to 74%) and a decrease in the protein content (from 37 to 10%). Analyses of fatty acids indicated that no lipids were accumulated under N-limited conditions. Nevertheless, it did not affect the biomass productivity of the culture up to five days after N was depleted from the culture medium. Transcriptomic and proteomic analysis indicated that de novo protein synthesis was down-regulated in the N-limited culture. Proteins were degraded and partly converted into carbohydrates through gluconeogenesis. Cellular N derived from protein degradation was recycled through the TCA and GS-GOGAT cycles. In addition, photosynthetic energy production and carbon fixation were both down-regulated, while glycogen synthesis was up-regulated. Our results suggested that N limitation resulted in a redirection of photosynthetic energy from protein synthesis to glycogen synthesis. The fact that glycogen synthesis has a lower energy demand than protein synthesis might explain why Arthrospira is able to achieve a similar biomass productivity under N-limited as under N-replete conditions despite the fact that photosynthetic energy production was impaired by N limitation.
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