ABSTRACT: 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.
