Genome-wide changes in the circadian transcriptional program in Synechococcus elongatus PCC 7942
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ABSTRACT:
ORGANISM(S): Synechococcus elongatus PCC 7942 = FACHB-805
PROVIDER: GSE59112 | GEO | 2014/07/08
SECONDARY ACCESSION(S): PRJNA254452
REPOSITORIES: GEO
Similar Datasets
Project description:Circadian input kinas A (cikA) null cells severly distorts the genome-wide output of the circadian clock Synechococcus cultures (both wildtype and M-NM-^TcikA null cells) were synchronized with two light-dark (12h:12h) cycle before release into continuous light. Samples were collected at 4 h intervals for the next 24 h, total RNA was isolated from samples collected at each time points and genome-wide expression was measured using custom-designed microarrays
2014-07-08 | E-GEOD-59112 | biostudies-arrayexpress
Project description:The circadian clock of cyanobacteria is composed of KaiA, KaiB, and KaiC proteins, and the SasA-RpaA two-component system has been implicated in the regulation of one of the output pathways of the clock. In this study, we show that another response regulator that is essential for viability, the RpaA paralog, RpaB, plays a central role in the transcriptional oscillation of clock-regulated genes. In vivo and in vitro analyses revealed that RpaB and not RpaA could specifically bind to the kaiBC promoter, possibly repressing transcription during subjective night. This suggested that binding may be terminated by RpaA to activate gene transcription during subjective day. Moreover, we found that rpoD6 and sigF2, which encode group-2 and group-3 ? factors for RNA polymerase, respectively, were also targets of the RpaAB system, suggesting that a specific group of ? factors can propagate genome-wide transcriptional oscillation. Our findings thus reveal a novel mechanism for a circadian output pathway that is mediated by two paralogous response regulators.
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Project description:In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.
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Project description:Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.
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2023-10-24 | PXD030282 | Pride
Project description:Phosphoglycerate-mutase (PGM) is an ubiquitous glycolytic enzyme, which in eukaryotic cells can be found in different compartments. In prokaryotic cells, several PGMs are annotated/localized in one compartment. The identification and functional characterization of PGMs in prokaryotes is therefore important for better understanding of metabolic regulation. Here we introduce a method, based on a multi-level kinetic model of the primary carbon metabolism in cyanobacterium Synechococcus elongatus PCC 7942, that allows the identification of a specific function for a particular PGM. The strategy employs multiple parameter estimation runs in high CO2, combined with simulations testing a broad range of kinetic parameters against the changes in transcript levels of annotated PGMs. Simulations are evaluated for a match in metabolic level in low CO2, to reveal trends that can be linked to the function of a particular PGM. A one-isoenzyme scenario shows that PGM2 is a major regulator of glycolysis, while PGM1 and PGM4 make the system robust against environmental changes. Strikingly, combining two PGMs with reverse transcriptional regulation allows both features. A conclusion arising from our analysis is that a two-enzyme PGM system is required to regulate the flux between glycolysis and the Calvin-Benson cycle, while an additional PGM increases the robustness of the system.
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Project description:Ethylene is a volatile alkene which is used in large commercial scale as a precursor in plastic industry, and is currently derived from petroleum refinement. As an alternative production strategy, photoautotrophic cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been previously evaluated as potential biotechnological hosts for producing ethylene directly from CO2, by the over-expression of ethylene forming enzyme (efe) from Pseudomonas syringae. This work addresses various open questions related to the use of Synechococcus as the engineering target, and demonstrates long-term ethylene production at rates reaching 140 µL L-1 h-1 OD750-1 without loss of host vitality or capacity to produce ethylene. The results imply that the genetic instability observed earlier may be associated with the expression strategies, rather than efe over-expression, ethylene toxicity or the depletion of 2-oxoglutarate-derived cellular precursors in Synechococcus. In context with literature, this study underlines the critical differences in expression system design in the alternative hosts, and confirms Synechococcus as a suitable parallel host for further engineering.
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Project description:The psbAI gene of the cyanobacterium Synechococcus elongatus PCC 7942 is one of three psbA genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of psbAI is downregulated under high-intensity light (high light) in contrast to induction of the other two family members. We show that, in addition to a known accelerated degradation of the psbAI message, promoter activity decreases upon exposure to high light. Unlike the other psbA genes, additional sequences upstream of the psbAI -35 element are required for expression. Mutagenizing the atypical psbAI -10 element from TCTCCT to TATAAT increased the magnitude of expression from both psbAI::lacZ and psbAI::luxAB fusions but did not affect downregulation under high light. Inactivation of group 2 sigma factor genes rpoD2 and sigC, in both wild-type and -10-element mutagenized backgrounds, resulted in elevated psbAI::luxAB expression but did not alter the response to high light. The results are consistent with redundancy of promoter recognition among cyanobacterial group 2 sigma factors. Electrophoretic mobility shift assays showed that the DNA sequence corresponding to the untranslated leader of the psbAI message binds one or more proteins from an S. elongatus extract. The corresponding region of psbAII efficiently competed for this binding activity, suggesting a shared regulatory factor among these disparately regulated genes.
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Project description:Gene expression timecourse in clock rescue strains
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Project description:We analyzed the stress responses of three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the cyanobacterium Synechococcus elongatus PCC 7942. A reporter assay showed that under stress conditions the expression of only the dnaK2 gene was induced, suggesting a functional assignment of these homologues. RNA blot hybridization indicated a typical stress response of dnaK2 to heat and high-light stress. Primer extension mapping showed that dnaK2 was transcribed from similar sites under various stress conditions. Although no known sequence motif was detected in the upstream region, a 20-bp sequence element was highly conserved in dnaK2; it was essential not only for the stress induction but also for the basal expression of dnaK2. The ubiquitous upstream localization of this element in each heat shock gene suggests its important role in the cyanobacterial stress response.
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