NEW YORK (GenomeWeb News) – In a paper scheduled to appear online this week in the Proceedings of the National Academy of Science, a team of Japanese researchers used gene expression profiling to improve their understanding of circadian clock function in Synechococcus elongatus, a single-celled cyanobacterial species living in freshwater.
The team employed custom oligonucleotide arrays to investigate gene expression for S. elongatus under continuous light or continuous dark conditions and in the presence or absence of three circadian genes — kaiA, kaiB, and kaiC. Their results suggest that almost a third of the cyanobacteria's transcripts fluctuate with circadian cycles. Even so, they cautioned, more work is needed to tease apart how this impacts S. elongatus' protein repertoire and physiological processes.
"[T]he Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator," senior author Hideo Iwasaki, a researcher affiliated with Waseda University in Tokyo and the Japan Science and Technology Agency, and co-authors wrote.
Previous research indicates that light/dark cycles in general often involve transcription and translation feedback mechanisms acting on circadian clock genes. In S. elongatus, though, recent studies suggest circadian cycles involve enzymatic changes affecting the phosphorylation of the circadian protein KaiC, which interacts with the products of other circadian genes, kaiA, and kaiB to govern the expression of many other genes.
To more fully understand the circadian cycles in S. elongatus PCC 7942, Iwasaki and his team developed custom Affymetrix high-density oligonucleotide microarrays representing 2,515 potential protein-coding genes based on chromosomal gene sequences from another S. elongatus strain, PCC 6301. They then grew the PCC 792 cells under a range of different conditions, including continuous light and continuous dark.
Based on experiments done under continuous light conditions, the team found that Synechococcus' circadian clock regulates at least 800 of the 2,516 genes tested. Among them: kaiB and kaiC, which showed peak expression at a time coinciding with what would have been dawn under non-lab conditions. Another group of genes were maximally expressed at dusk. For example, the researchers noted that at least 87 high amplitude genes peaked at dawn while ten peaked at dusk.
Their subsequent analyses suggest around 1,322 of the S. elongatus genes tested fall under the control of 488 operons. On the other hand, the remaining 1,194 genes appear to be transcribed on their own.
The team also began characterizing the sorts of genes that are temporally controlled in S. elongatus, identifying genes for metabolic and photosynthesis-related enzymes whose expression varied with time.
But when the team used a similar approach to explore gene expression in cells missing the kaiABC genes, they found that these gene expression patterns all but disappeared. Nevertheless, they noted, 17 genes continued fluctuating, even when the kaiABC genes were gone, suggesting there may another circadian process acting in S. elongatus.
In contrast, when the team artificially ramped up the levels of KaiC, they found that the cells remained locked in a dawn-like gene expression pattern over long periods of time.
And, they found, under continuous dark conditions, gene expression was dramatically decreased — both for circadian-regulated and other genes. "We suggest that Synechococcus cells become nearly mRNA-deficient during the night phase, whereas transcription restarts from dawn so that they exhibit a clock-modified expression profile during the daytime," the researchers wrote.
But while the current work offers a window into the transcripts that are regulated by the S. elongatus circadian clock, the authors cautioned that these expression profiles do not necessarily correlate with protein levels in the cells. Rather, they noted, there seems to be "marked and interesting discrepancies between transcriptomic and proteomic profiles" in Synechococcus.
Consequently, the researchers called for more detailed analyses of the cyanobacteria to understand the role of circadian-related genes, concluding, "[T]he physiological relevance of the dynamic diurnal/nocturnal control of Synechococcus needs to be clarified by further proteomic analysis."