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Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale.

ABSTRACT: Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ?20 ps (PSI and PSII), ?80 ps, ?440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future.

SUBMITTER: Krumova SB

PROVIDER: S-EPMC2941034 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale.

Krumova S B SB Laptenok S P SP Borst J W JW Ughy B B Gombos Z Z Ajlani G G van Amerongen H H

Biophysical journal 20100901 6

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I ( ...[more]

PMID: 20858447

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Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Project description:Cyanobacteria are promising industrial platforms owing to their ability to produce diverse natural secondary metabolites and nonnative value-added biochemicals from CO2 and light. To fully utilize their industrial potency, it is critical to understand their photosynthetic efficiency under various environmental conditions. In this study, we elucidated the inhibitory mechanisms of photosynthesis under high-light and low-temperature stress conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Under each stress condition, the transcript abundance and translation efficiency were measured using transcriptome sequencing (RNA-seq) and ribosome profiling, and the genome-wide transcription unit architecture was constructed by data integration of transcription start sites and transcript 3'-end positions obtained from differential RNA-seq and sequencing of 3'-ends (Term-seq), respectively. Our results suggested that the mode of photosynthesis inhibition differed between the two stress conditions; high light stress induced photodamage responses, while low temperature stress impaired the translation efficiency of photosynthesis-associated genes. In particular, poor translation of photosystem I resulted from ribosome stalling at the untranslated regions, affecting the overall photosynthetic yield under low temperature stress. Our comprehensive multiomics analysis with transcription unit architecture provides foundational information on photosynthesis for future industrial strain development. IMPORTANCE Cyanobacteria are a compelling biochemical production platform for their ability to propagate using light and atmospheric CO2 via photosynthesis. However, the engineering of strains is hampered by limited understanding of photosynthesis under diverse environmental conditions such as high-light and low-temperature stresses. Herein, we decipher the transcriptomic and translatomic responses of the photosynthetic efficiency to stress conditions using the integrative analysis of multiomic data generated by RNA-seq and ribosome profiling, respectively. Through the generated massive data, along with the guide of the genome-wide transcription unit architecture constructed by transcription start sites and transcript 3'-end positions, we identified the factors affecting photosynthesis at transcription, posttranscription, and translation levels. Importantly, the high-light stress induces photodamage responses, and the low-temperature stress cripples the translation efficiency of photosynthesis-associated genes. The resulting insights provide pivotal information for future cyanobacterial cell factories powered by the engineering toward robust photosynthesis ability.

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Dataset Information

Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale.

Publications

Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale.

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