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.
Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.
One of the earliest and largest transcriptional responses that occur during exposure of Synechocystis sp. PCC6803 to cold is the induction of the crhR RNA helicase transcript. We show that crhR deletion results in failure to cold acclimate: there is reduced growth at 24 degrees C and marked impairment of growth at 20 degrees C. 2D-DIGE, using five biological replicates, was used to analyze the proteomic differences between the wild-type and DeltacrhR strains grown at (1) 34 degrees C and (2) following transfer from 34 to 24 degrees C (cold-acclimation). Sixteen significantly differentially expressed proteins were identified between the two strains grown at 34 degrees C. Forty-three distinct proteins were identified that responded to cold-acclimation of the wild-type and 34 proteins for the mutant, with only 26 proteins common to both. A large proportion of the proteomic responses (76.5%) could not be predicted from published transcriptomic data. Only modest similarity is observed between proteomic and transcriptomic responses (r = 0.54-0.70). We propose functions for three previously hypothetical proteins. We suggest molecular targets for CrhR action and identify downstream regulated events in metabolism.
The unicellular cyanobacteria Synechocystis sp. (PCC6803) has become a model organism for a range of biochemical and molecular biology studies aimed at investigating environmental stress responses. In this study the soluble proteins of Synechocystis were analysed using narrow pH range (pH 4.5-5.5) zoom gels, automated matrix-assisted laser desorption/ionization mass spectrometry acquisition, spectral processing and database searching. The work sets the foundation for investigations of proteomic changes following stress treatment. One hundred and ninety-two protein spots were analysed and 105 proteins identified, of these 37 were novel proteins not previously seen on two-dimensional gels. Proteins involved in amino acid biosynthesis, energy metabolism and protein modification were identified using this fully automated procedure demonstrating that automated acquisition and processing will be a useful tool for proteomic analyses on this organism.
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