Dataset Information

Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803.

ABSTRACT: BACKGROUND: The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model microbe for studying biochemistry, genetics and molecular biology of photobiological processes. Importance of this bacterium in basic and applied research calls for a systematic, genome-wide description of its transcriptional regulatory capacity. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. RESULTS: We designed, validated and used Synechocystis genome-wide oligonucleotide (70-mer) microarray (representing 96.7% of all chromosomal ORFs annotated at the time of the beginning of this project) to study transcriptional activity of the cyanobacterial genome in response to sulfur (S) starvation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate starvation were relatively moderate, but significant and consistent with growth kinetics; 2) S acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems I and II, cytochrome b6/f, and ATP synthase indicated reduced light-harvesting and photosynthetic activity; 3) S starvation elicited transcriptional responses associated with general growth arrest and stress; 4) A large number of genes regulated by S availability encode hypothetical proteins or proteins of unknown function; 5) Hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. CONCLUSION: The expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful S to S starvation, Synechocystis undergoes coordinated transcriptional changes, including changes in gene expression whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient starvation was dominated by a decrease in abundances of many transcripts. However, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. Down-regulation of transcripts encoding proteins whose function depends on a cellular S status indicated that the observed repression has a specific regulatory component. The repression of certain S-related genes was paralleled by activation of genes involved in internal and external S scavenging.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC2491639 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803.

Zhang Zhigang Z Pendse Ninad D ND Phillips Katherine N KN Cotner James B JB Khodursky Arkady A

BMC genomics 20080721

<h4>Background</h4>The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model microbe for studying biochemistry, genetics and molecular biology of photobiological processes. Importance of this bacterium in basic and applied research calls for a systematic, genome-wide description of its transcriptional regulatory capacity. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional reg ...[more]

PMID: 18644144

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Project description:The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model system for studying biochemistry, genetics and molecular biology of photobiological processes. Despite its importance in basic and applied research, the genome-wide picture of transcriptional regulation in this bacterium is limited. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. We designed, validated and used Synechocystis genome-wide oligonucleotide (70-mer) microarray (representing 96.7% of all chromosomal ORFs) to study transcriptional activity of the cyanobacterial genome in response to S deprivation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate withdrawal were relatively moderate, but significant and consistent with growth kinetics; 2) S acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems I and II, cytochrome b6/f, and ATP synthase indicated reduced light-harvesting and photosynthetic activity; 3) S deprivation elicited transcriptional responses associated with general growth arrest and stress; 4) A large number of genes regulated by S availability encode hypothetical proteins or proteins of unknown function; 5) Hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. The expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful sulfur to no sulfur, Synechocystis undergoes coordinated transcriptional changes, including genes whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient limitation was dominated by decrease in abundances of many transcripts. However, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. Down-regulation of transcripts encoding proteins whose function depends on a cellular sulfur status indicated that the observed repression has a specific regulatory component. The repression of certain sulfur-related genes was paralleled by activation of genes involved in internal and external S scavenging. Keywords: stress response, time course Synechocystis sp. PCC 6803 was grown photoautotrophically in BG-11 medium supplemented with 8mM NaHCO3 and buffered with 10mM HEPES (pH 7.4). The cells were grown in 250ml flasks at 32oC under a light intensity of 25µmol photons m-2 s-1. Cultures were bubbled with sterile air containing 1% (v/v) CO2. Log phase cells (OD730nm=0.6) were harvested by centrifugation (2000×g for 12 min) washed once and then re-suspended in sulfate-free media (MgSO4 replaced by the same molarity of MgCl2). In addition, all S-containing trace metals in BG-11 were replaced by non-S containing metals. Cells were harvested and fixed for microarray analysis by adding 10% (v/v) ice-cold 5% phenol in ethanol stop solution at the following time points: before S-depravation (time 0, control), 1, 3, 6, 12, 24, 48 and 72 hr after S-depravation. S-deprivation with HEPES buffering control experiment was performed as described above, except that HEPES buffer was used upon sulfate removal. Bacterial samples for a time course were taken at time 0, 1, 12 and 24 hrs after sulfate withdrawal. Growth stage control experiment was done in parallel with S deprivation experiments. Samples were taken at 0, 1, 2.5, 4, 7, 11 and 48 hr after OD730nm reached 0.60. All the experiments were done in biological replicates.

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:BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

Dataset Information

Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803.

Publications

Gene expression patterns of sulfur starvation in Synechocystis sp. PCC 6803.

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