Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria.

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

Carbon availability affects diurnally controlled processes and cell morphology of Cyanothece 51142.

Cyanobacteria are oxygenic photoautotrophs notable for their ability to utilize atmospheric CO2 as the major source of carbon. The prospect of using cyanobacteria to convert solar energy and high concentrations of CO2 efficiently into biomass and renewable energy sources has sparked substantial interest in using flue gas from coal-burning power plants as a source of inorganic carbon. However, in order to guide further advances in this area, a better understanding of the metabolic changes that occur under conditions of high CO2 is needed. To determine the effect of high CO2 on cell physiology and growth, we analyzed the global transcriptional changes in the unicellular diazotrophic cyanobacterium Cyanothece 51142 grown in 8% CO2-enriched air. We found a concerted response of genes related to photosynthesis, carbon metabolism, respiration, nitrogen fixation, ribosome biosynthesis, and the synthesis of nucleotides and structural cell wall polysaccharides. The overall response to 8% CO2 in Cyanothece 51142 involves different strategies, to compensate for the high C/N ratio during both phases of the diurnal cycle. Our analyses show that high CO2 conditions trigger the production of carbon-rich compounds and stimulate processes such as respiration and nitrogen fixation. In addition, we observed that high levels of CO2 affect fundamental cellular processes such as cell growth and dramatically alter the intracellular morphology. This study provides novel insights on how diurnal and developmental rhythms are integrated to facilitate adaptation to high CO2 in Cyanothece 51142.

Variations in the rhythms of respiration and nitrogen fixation in members of the unicellular diazotrophic cyanobacterial genus Cyanothece.

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.

Novel metabolic attributes of the genus cyanothece, comprising a group of unicellular nitrogen-fixing Cyanothece.

UNLABELLED: The genus Cyanothece comprises unicellular cyanobacteria that are morphologically diverse and ecologically versatile. Studies over the last decade have established members of this genus to be important components of the marine ecosystem, contributing significantly to the nitrogen and carbon cycle. System-level studies of Cyanothece sp. ATCC 51142, a prototypic member of this group, revealed many interesting metabolic attributes. To identify the metabolic traits that define this class of cyanobacteria, five additional Cyanothece strains were sequenced to completion. The presence of a large, contiguous nitrogenase gene cluster and the ability to carry out aerobic nitrogen fixation distinguish Cyanothece as a genus of unicellular, aerobic nitrogen-fixing cyanobacteria. Cyanothece cells can create an anoxic intracellular environment at night, allowing oxygen-sensitive processes to take place in these oxygenic organisms. Large carbohydrate reserves accumulate in the cells during the day, ensuring sufficient energy for the processes that require the anoxic phase of the cells. Our study indicates that this genus maintains a plastic genome, incorporating new metabolic capabilities while simultaneously retaining archaic metabolic traits, a unique combination which provides the flexibility to adapt to various ecological and environmental conditions. Rearrangement of the nitrogenase cluster in Cyanothece sp. strain 7425 and the concomitant loss of its aerobic nitrogen-fixing ability suggest that a similar mechanism might have been at play in cyanobacterial strains that eventually lost their nitrogen-fixing ability. IMPORTANCE: The unicellular cyanobacterial genus Cyanothece has significant roles in the nitrogen cycle in aquatic and terrestrial environments. Cyanothece sp. ATCC 51142 was extensively studied over the last decade and has emerged as an important model photosynthetic microbe for bioenergy production. To expand our understanding of the distinctive metabolic capabilities of this cyanobacterial group, we analyzed the genome sequences of five additional Cyanothece strains from different geographical habitats, exhibiting diverse morphological and physiological attributes. These strains exhibit high rates of N(2) fixation and H(2) production under aerobic conditions. They can generate copious amounts of carbohydrates that are stored in large starch-like granules and facilitate energy-intensive processes during the dark, anoxic phase of the cells. The genomes of some Cyanothece strains are quite unique in that there are linear elements in addition to a large circular chromosome. Our study provides novel insights into the metabolism of this class of unicellular nitrogen-fixing cyanobacteria.

Diurnal rhythms result in significant changes in the cellular protein complement in the cyanobacterium Cyanothece 51142.

Cyanothece sp. ATCC 51142 is a diazotrophic cyanobacterium notable for its ability to perform oxygenic photosynthesis and dinitrogen fixation in the same single cell. Previous transcriptional analysis revealed that the existence of these incompatible cellular processes largely depends on tightly synchronized expression programs involving ∼30% of genes in the genome. To expand upon current knowledge, we have utilized sensitive proteomic approaches to examine the impact of diurnal rhythms on the protein complement in Cyanothece 51142. We found that 250 proteins accounting for ∼5% of the predicted ORFs from the Cyanothece 51142 genome and 20% of proteins detected under alternating light/dark conditions exhibited periodic oscillations in their abundances. Our results suggest that altered enzyme activities at different phases during the diurnal cycle can be attributed to changes in the abundance of related proteins and key compounds. The integration of global proteomics and transcriptomic data further revealed that post-transcriptional events are important for temporal regulation of processes such as photosynthesis in Cyanothece 51142. This analysis is the first comprehensive report on global quantitative proteomics in a unicellular diazotrophic cyanobacterium and uncovers novel findings about diurnal rhythms.

Global proteomics reveal an atypical strategy for carbon/nitrogen assimilation by a cyanobacterium under diverse environmental perturbations.

Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen.

Integrative analysis of large scale expression profiles reveals core transcriptional response and coordination between multiple cellular processes in a cyanobacterium.

BACKGROUND: Cyanobacteria are the only known prokaryotes capable of oxygenic photosynthesis. They play significant roles in global biogeochemical cycles and carbon sequestration, and have recently been recognized as potential vehicles for production of renewable biofuels. Synechocystis sp. PCC 6803 has been extensively used as a model organism for cyanobacterial studies. DNA microarray studies in Synechocystis have shown varying degrees of transcriptome reprogramming under altered environmental conditions. However, it is not clear from published work how transcriptome reprogramming affects pre-existing networks of fine-tuned cellular processes. RESULTS: We have integrated 163 transcriptome data sets generated in response to numerous environmental and genetic perturbations in Synechocystis. Our analyses show that a large number of genes, defined as the core transcriptional response (CTR), are commonly regulated under most perturbations. The CTR contains nearly 12% of Synechocystis genes found on its chromosome. The majority of genes in the CTR are involved in photosynthesis, translation, energy metabolism and stress protection. Our results indicate that a large number of differentially regulated genes identified in most reported studies in Synechocystis under different perturbations are associated with the general stress response. We also find that a majority of genes in the CTR are coregulated with 25 regulatory genes. Some of these regulatory genes have been implicated in cellular responses to oxidative stress, suggesting that reactive oxygen species are involved in the regulation of the CTR. A Bayesian network, based on the regulation of various KEGG pathways determined from the expression patterns of their associated genes, has revealed new insights into the coordination between different cellular processes. CONCLUSION: We provide here the first integrative analysis of transcriptome data sets generated in a cyanobacterium. This compilation of data sets is a valuable resource to researchers for all cyanobacterial gene expression related queries. Importantly, our analysis provides a global description of transcriptional reprogramming under different perturbations and a basic framework to understand the strategies of cellular adaptations in Synechocystis.

A systems-level analysis of the effects of light quality on the metabolism of a cyanobacterium.

Photosynthetic organisms experience changes in light quantity and light quality in their natural habitat. In response to changes in light quality, these organisms redistribute excitation energy and adjust photosystem stoichiometry to maximize the utilization of available light energy. However, the response of other cellular processes to changes in light quality is mostly unknown. Here, we report a systematic investigation into the adaptation of cellular processes in Synechocystis species PCC 6803 to light that preferentially excites either photosystem II or photosystem I. We find that preferential excitation of photosystem II and photosystem I induces massive reprogramming of the Synechocystis transcriptome. The rewiring of cellular processes begins as soon as Synechocystis senses the imbalance in the excitation of reaction centers. We find that Synechocystis utilizes the cyclic photosynthetic electron transport chain for ATP generation and a major part of the respiratory pathway to generate reducing equivalents and carbon skeletons during preferential excitation of photosystem I. In contrast, cytochrome c oxidase and photosystem I act as terminal components of the photosynthetic electron transport chain to produce sufficient ATP and limited amounts of NADPH and reduced ferredoxin during preferential excitation of photosystem II. To overcome the shortage of NADPH and reduced ferredoxin, Synechocystis preferentially activates transporters and acquisition pathways to assimilate ammonia, urea, and arginine over nitrate as a nitrogen source. This study provides a systematic analysis of cellular processes in cyanobacteria in response to preferential excitation and shows that the cyanobacterial cell undergoes significant adjustment of cellular processes, many of which were previously unknown.

Effect of continuous light on diurnal rhythms in Cyanothece sp. ATCC 51142.

BACKGROUND: Life on earth is strongly affected by alternating day and night cycles. Accordingly, many organisms have evolved an internal timekeeping system with a period of approximately 24 hours. Cyanobacteria are the only known prokaryotes with robust rhythms under control of a central clock. Numerous studies have been conducted to elucidate components of the circadian clock and to identify circadian-controlled genes. However, the complex interactions between endogenous circadian rhythms and external cues are currently not well understood, and a direct and mathematical based comparison between light-mediated and circadian-controlled gene expression is still outstanding. Therefore, we combined and analyzed data from two independent microarray experiments, previously performed under alternating light-dark and continuous light conditions in Cyanothece sp. ATCC 51142, and sought to classify light responsive and circadian controlled genes. RESULTS: Fourier Score-based methods together with random permutations and False Discovery Rates were used to identify genes with oscillatory expression patterns, and an angular distance based criterion was applied to recognize transient behaviors in gene expression under constant light conditions. Compared to previously reported mathematical approaches, the combination of these methods also facilitated the detection of modified amplitudes and phase-shifts of gene expression. Our analysis showed that the majority of diurnally regulated genes, essentially those genes that are maximally expressed during the middle of the light and dark period, are in fact light responsive. In contrast, most of the circadian controlled genes are up-regulated during the beginning of the dark or subjective dark, and are greatly enriched for genes associated with energy metabolism. Many of the circadian controlled and light responsive genes are found in gene clusters within the Cyanothece sp. ATCC 51142 genome. Interestingly, in addition to cyclic expression patterns with a period of 24 hours, we also found several genes that oscillate with an ultradian period of 12 hours, a novel finding among cyanobacteria. CONCLUSION: We demonstrate that a combination of different analytical methods significantly improved the identification of cyclic and transient gene expression in Cyanothece sp. ATCC 51142. Our analyses provide an adaptable and novel analytical tool to study gene expression in a variety of organisms with diurnal, circadian and ultradian behavior.

Arabidopsis transcriptome reveals control circuits regulating redox homeostasis and the role of an AP2 transcription factor.

Sensors and regulatory circuits that maintain redox homeostasis play a central role in adjusting plant metabolism and development to changing environmental conditions. We report here control networks in Arabidopsis (Arabidopsis thaliana) that respond to photosynthetic stress. We independently subjected Arabidopsis leaves to two commonly used photosystem II inhibitors: high light (HL) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Microarray analysis of expression patterns during the period of redox adjustment to these inhibitors reveals that 20% and 8% of the transcriptome are under HL and DCMU regulation, respectively. Approximately 6% comprise a subset of genes common to both perturbations, the redox responsive genes (RRGs). A redox network was generated in an attempt to identify genes whose expression is tightly coordinated during adjustment to homeostasis, using expression of these RRGs under HL conditions. Ten subnetworks were identified from the network. Hierarchal subclustering of subnetworks responding to the DCMU stress identified novel groups of genes that were tightly controlled while adjusting to homeostasis. Upstream analysis of the promoters of the genes in these clusters revealed different motifs for each subnetwork, including motifs that were previously identified with responses to other stresses, such as light, dehydration, or abscisic acid. Functional categorization of RRGs demonstrated involvement of genes in many metabolic pathways, including several families of transcription factors, especially those in the AP2 family. Using a T-DNA insertion in one AP2 transcription factor (redox-responsive transcription factor 1 [RRTF1]) from the RRGs, we showed that the genes predicted to be within the subnetwork containing RRTF1 were changed in this insertion line (Deltarrtf1). Furthermore, Deltarrtf1 showed greater sensitivity to photosynthetic stress compared to the wild type.

The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle.

Unicellular cyanobacteria have recently been recognized for their contributions to nitrogen fixation in marine environments, a function previously thought to be filled mainly by filamentous cyanobacteria such as Trichodesmium. To begin a systems level analysis of the physiology of the unicellular N(2)-fixing microbes, we have sequenced to completion the genome of Cyanothece sp. ATCC 51142, the first such organism. Cyanothece 51142 performs oxygenic photosynthesis and nitrogen fixation, separating these two incompatible processes temporally within the same cell, while concomitantly accumulating metabolic products in inclusion bodies that are later mobilized as part of a robust diurnal cycle. The 5,460,377-bp Cyanothece 51142 genome has a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of a linear element in the genome of a photosynthetic bacterium. On the 429,701-bp linear chromosome is a cluster of genes for enzymes involved in pyruvate metabolism, suggesting an important role for the linear chromosome in fermentative processes. The annotation of the genome was significantly aided by simultaneous global proteomic studies of this organism. Compared with other nitrogen-fixing cyanobacteria, Cyanothece 51142 contains the largest intact contiguous cluster of nitrogen fixation-related genes. We discuss the implications of such an organization on the regulation of nitrogen fixation. The genome sequence provides important information regarding the ability of Cyanothece 51142 to accomplish metabolic compartmentalization and energy storage, as well as how a unicellular bacterium balances multiple, often incompatible, processes in a single cell.

Integration of carbon and nitrogen metabolism with energy production is crucial to light acclimation in the cyanobacterium Synechocystis.

Light drives the production of chemical energy and reducing equivalents in photosynthetic organisms required for the assimilation of essential nutrients. This process also generates strong oxidants and reductants that can be damaging to the cellular processes, especially during absorption of excess excitation energy. Cyanobacteria, like other oxygenic photosynthetic organisms, respond to increases in the excitation energy, such as during exposure of cells to high light (HL) by the reduction of antenna size and photosystem content. However, the mechanism of how Synechocystis sp. PCC 6803, a cyanobacterium, maintains redox homeostasis and coordinates various metabolic processes under HL stress remains poorly understood. In this study, we have utilized time series transcriptome data to elucidate the global responses of Synechocystis to HL. Identification of differentially regulated genes involved in the regulation, protection, and maintenance of redox homeostasis has offered important insights into the optimized response of Synechocystis to HL. Our results indicate a comprehensive integrated homeostatic interaction between energy production (photosynthesis) and energy consumption (assimilation of carbon and nitrogen). In addition, measurements of physiological parameters under different growth conditions showed that integration between the two processes is not a consequence of limitations in the external carbon and nitrogen levels available to the cells. We have also discovered the existence of a novel glycosylation pathway, to date known as an important nutrient sensor only in eukaryotes. Up-regulation of a gene encoding the rate-limiting enzyme in the hexosamine pathway suggests a regulatory role for protein glycosylation in Synechocystis under HL.