Dataset Information

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803.

ABSTRACT: There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ?64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.

SUBMITTER: Mitschke J

PROVIDER: S-EPMC3033270 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803.

Mitschke Jan J Georg Jens J Scholz Ingeborg I Sharma Cynthia M CM Dienst Dennis D Bantscheff Jens J Voss Björn B Steglich Claudia C Wilde Annegret A Vogel Jörg J Hess Wolfgang R WR

Proceedings of the National Academy of Sciences of the United States of America 20110118 5

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS wer ...[more]

PMID: 21245330

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Project description:BACKGROUND:The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. RESULTS:The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. CONCLUSIONS:Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.

Project description:The production of biofuels in photosynthetic microalgae and cyanobacteria is considered a promising alternative to the generation of fuels from fossil resources. However, to be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria to forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Here we report the results of a monitoring experiment, in which the transcriptome-wide response to continuous ethanol production in the unicellular model cyanobacterium Synechocystis sp. PCC6803 was examined using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) EtOH d-1 M-BM-1 0.002 and 0.0303% (v/v) d-1 M-BM-1 0.002 were obtained over 18 consecutive days, measuring biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation in the producer strains and the development of a bleaching phenotype. Absorption spectroscopy indicated in particular a down-regulation of light harvesting capacity. Microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an about 4 fold reduction in cpcB (sll1577) and an about 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional operon discoordination in the cpcBA operon leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, Western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. We conclude that the changes in gene expression upon induction of long term ethanol production in Synechocystis sp. PCC6803 are highly specific. in particular we did not observe a comprehensive stress response contributing to a complex phenotype as might have been expected. Gene expression of Synechocystis sp. PCC 6803 WT (#621) and the isogenic ethanol producing strain #309 was monitored at 4, 7, 11 and 18 days after induction of ethanol production by copper depletion. Each condition was sampled in biological duplicates

Project description:The production of biofuels in photosynthetic microalgae and cyanobacteria is considered a promising alternative to the generation of fuels from fossil resources. However, to be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria to forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Here we report the results of a monitoring experiment, in which the transcriptome-wide response to continuous ethanol production in the unicellular model cyanobacterium Synechocystis sp. PCC6803 was examined using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) EtOH d-1 ± 0.002 and 0.0303% (v/v) d-1 ± 0.002 were obtained over 18 consecutive days, measuring biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation in the producer strains and the development of a bleaching phenotype. Absorption spectroscopy indicated in particular a down-regulation of light harvesting capacity. Microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an about 4 fold reduction in cpcB (sll1577) and an about 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional operon discoordination in the cpcBA operon leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, Western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. We conclude that the changes in gene expression upon induction of long term ethanol production in Synechocystis sp. PCC6803 are highly specific. in particular we did not observe a comprehensive stress response contributing to a complex phenotype as might have been expected.

Dataset Information

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803.

Publications

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803.

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