Ultradian Metabolic Rhythm in the Diazotrophic Cyanobacterium Cyanothece sp. ATCC 51142.

The unicellular cyanobacterium Cyanothece sp. American Type Culture Collection (ATCC) 51142 is capable of performing oxygenic photosynthesis during the day and microoxic nitrogen fixation at night. These mutually exclusive processes are possible only by temporal separation by circadian clock or another cellular program. We report identification of a temperature-dependent ultradian metabolic rhythm that controls the alternating oxygenic and microoxic processes of Cyanothece sp. ATCC 51142 under continuous high irradiance and in high CO2 concentration. During the oxygenic photosynthesis phase, nitrate deficiency limited protein synthesis and CO2 assimilation was directed toward glycogen synthesis. The carbohydrate accumulation reduced overexcitation of the photosynthetic reactions until a respiration burst initiated a transition to microoxic N2 fixation. In contrast to the circadian clock, this ultradian period is strongly temperature-dependent: 17 h at 27 °C, which continuously decreased to 10 h at 39 °C. The cycle was expressed by an oscillatory modulation of net O2 evolution, CO2 uptake, pH, fluorescence emission, glycogen content, cell division, and culture optical density. The corresponding ultradian modulation was also observed in the transcription of nitrogenase-related nifB and nifH genes and in nitrogenase activities. We propose that the control by the newly identified metabolic cycle adds another rhythmic component to the circadian clock that reflects the true metabolic state depending on the actual temperature, irradiance, and CO2 availability

Transcriptional Analysis of the Unicellular, Diazotrophic Cyanobacterium Cyanothece ATCC 51142 Grown Under Short Day/Night Cycles.

Cyanothece sp. strain ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N2-fixing conditions. We have performed a global transcription analysis of this organism using 6 h light:dark (L:D) cycles in order to determine the response of the cell to these conditions and to differentiate between diurnal and circadian-regulated genes. In addition, we used a context-likelihood of relatedness (CLR) analysis with these data and those from 2 d L:D and L:D plus continuous light experiments to better differentiate between diurnal and circadian-regulated genes. Cyanothece sp. acclimated in several ways to growth under short L:D conditions. Nitrogen was fixed in every second dark period and only once in each 24 h period. Nitrogen fixation was strongly correlated to the energy status of the cells and glycogen breakdown, and high respiration rates were necessary to provide appropriate energy and anoxic conditions for this process. We conclude that glycogen breakdown is a key regulatory step within these complex processes. Our results demonstrated that the main metabolic genes involved in photosynthesis, respiration, nitrogen fixation, and central carbohydrate metabolism have strong (or total) circadian-regulated components. The short L:D cycles enable us to identify transcriptional differences among the family of psbA genes, as well as the differing patterns of the hup genes, which follow the same pattern as nitrogenase genes, relative to the hoxgenes, which displayed a diurnal, dark-dependent gene expression

Modeling And Identification Of Differentially Regulated Genes Using Transcriptomics And Proteomics Data

Photosynthetic organisms are complex dynamical systems, showing a remarkable ability to adapt to different environmental conditions for their survival. Mechanisms underlying the coordination between different cellular processes in these organisms are still poorly understood. In this dissertation we utilize various computational and modeling techniques to analyze transcriptomics and proteomics data sets from several photosynthetic organisms. We try to use changes in expression levels of genes to study responses of these organisms to various environmental conditions such as availability of nutrients, concentrations of chemicals in growth media, and temperature. Three specific problems studied here are transcriptomics modifications in photosynthetic organisms under reduction-oxidation: redox) stress conditions, circadian and diurnal rhythms of cyanobacteria and the effect of incident light patterns on these rhythms, and the coordination between biological processes in cyanobacteria under various growth conditions. Under redox stresses caused by high light treatments, a strong transcriptomic level response, spread across many biological processes, is discovered in the cyanobacterium Synechocystis sp. PCC 6803. Based on statistical tests, expression levels of about 20% of genes in Synechocystis 6803 are identified as significantly affected due to influence of high light. Gene clustering methods reveal that these responses can mainly be classified as transient and consistent responses, depending on the duration of modified behaviors. Many genes related to energy production as well as energy utilization are shown to be strongly affected. Analysis of microarray data under two stress conditions, high light and DCMU treatment, combined with data mining and motif finding algorithms led to a discovery of novel transcription factor, RRTF1 that responds to redox stresses in Arabidopsis thaliana. Time course transcriptomics data from Cyanothece sp. ATCC 51142 have shown strong diurnal rhythms. By combining multiple experimental conditions and using gene classification algorithms based on Fourier scores and angular distances, it is shown that majority of the diurnal genes are in fact light responding. Only about 10% of genes in the genome are categorized as being circadian controlled. A transcription control model based on dynamical systems is employed to identify the interactions between diurnal genes. A phase oscillator network is proposed to model the behavior of different biological processes. Both these models are shown to carry biologically meaningful features. To study the coordination between different biological processes to various environment and genetic modifications, an interaction model is derived using Bayesian network approach, combining all publicly available microarray data sets for Synechocystis sp. PCC 6803. Several novel relationships between biological processes are discovered from the model. Model is used to simulate several experimental conditions, and the response of the model is shown to agree with the experimentally observed behaviors

A Day in the Life of Microcystis aeruginosa Strain PCC 7806 as Revealed by a Transcriptomic Analysis

The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism

Circadian input kinases and their homologs in cyanobacteria: Evolutionary constraints versus architectural diversification

The circadian input kinase A (cikA) gene encodes a protein relaying environmental signal to the central circadian oscillator in cyanobacteria. The CikA protein has a variable architecture and usually consists of four tandemly arrayed domains: GAF, histidine kinase (HisKA), histidine kinase-like ATPase (HATPase-c), and a pseudo-receiver (REC). Among them, HisKA and HATPase-c are the least polymorphic, and REC is not present in heterocystic filamentous cyanobacteria. CikA contains several conserved motifs that are likely important for circadian function. There are at least three types of circadian systems, each of which possesses a different set of circadian genes. The originally described circadian system (kaiABC system) possesses both cikA and kaiA, while the others lack either only cikA (kaiABC Δ) or both (kaiBC). The results we obtained allowed us to approximate the time of the cikA origin to be about 2600-2200 MYA and the time of its loss in the species with the kaiABC Δ or kaiBC system between 1100 and 600 MYA. Circadian specialization of CikA, as opposed to its non-circadian homologs, is a result of several factors, including the unique conserved domain architecture and high evolutionary constraints of some domains and regions, which were previously identified as critical for the circadian function of the gene. © 2010 Springer Science+Business Media, LLC.postprin

Metabolic Engineering of Cyanobacteria for Photosynthetic Production of Drop-In Liquid Fuels

Cyanobacteria are oxygenic phototrophs with great potential as hosts for renewable fuel and chemical production. They grow very quickly (compared with plants) and can use sunlight for energy and CO2 as a carbon source (unlike yeast or E. coli). While cyanobacteria have been engineered to make many chemicals that are native and non-native parts of their metabolism, this work is concerned with the production of heptadecane in Synechocystis sp. PCC 6803. Heptadecane is in a class of natural products produced by all cyanobacteria, but in quantities insufficient for industrialization. Towards this future goal, we have built enabling systems for the overproduction of fuels and chemicals in Synechocystis 6803 and cyanoabacteria generally. These tools include plasmid vectors for the overproduction of heterologous proteins and genome- scale metabolic models that can predict strategies for metabolite overproduction. We have shown that the vectors we developed are helpful in controlling the level and timing of heterologous protein expression using a fluorescent reporter, and have made progress towards heptadecane overproduction. During this process, we have also found that heptadecane is crucial for cold tolerance and modulates cyclic electron flow in photosynthesis. In addition to measuring this phenotype in vivo, we have analyzed it in silico using our genome-scale metabolic model and have gained insight into the role of cyclic electron flow in photosynthesis generally

Dinitrogen fixation in the unicellular diazotroph Crocosphaera watsonii

Crocosphaera watsonii is an abundant organism in the tropical and subtropical ocean and is considered to be an important contributor to the marine nitrogen cycle due to its ability to fix dinitrogen (N2). Light-dark cycles were used to determine the diel variation in N2 fixation and photosynthesis in the unicellular diazotrophic cyanobacterium C. watsonii WH8501. A first set of experiments showed that N2 fixation and photosynthesis were separated temporally with N2 fixation during the dark and photosynthesis during the light periods. Due to the storage of carbon reserves during the day and subsequent respiration at night, C. watsonii links its cellular nitrogen and carbon metabolism. N2 fixation and photosynthesis appear to be regulated and optimized by a circadian rhythm. Gene expression analysis demonstrated cyclic patterns of the major genes involved in nitrogen and carbon metabolism. However, the patterns of expression did not coincide with patterns of activity with the gene expression peaking several hours prior to the activity. In a second set of experiments, N2 fixation and photosynthesis were simultaneously measurable at population level in the respective dark period, i.e. an artificial light period instead of a dark period. Single-cell analysis using nanoSIMS revealed that the co-occurrence of N2 fixation and photosynthesis at population level was probably due to each individual cell’s capability of coping with the O2-sensitivity of the nitrogenase enzyme. In addition, single-cell rates showed large variability with the highest rate about six times higher than the mean rate during the normal dark N2 fixation. The experimental work with a pure culture of a diazotroph provided the opportunity for a re-assessment of the two commonly used methods for the measurement of N2 fixation in both field and laboratory studies, i.e. the 15N2-tracer addition method and the acetylene reduction assay