Jana Stöckel

Global transcriptomic analysis of Cyanothece 51142 reveals robust diurnal oscillation of central metabolic processes

Cyanobacteria are photosynthetic organisms and are the only prokaryotes known to have a circadian lifestyle. Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 produce oxygen and can also fix atmospheric nitrogen, a process exquisitely sensitive to oxygen. To accommodate such antagonistic processes, the intracellular environment of Cyanothece oscillates between aerobic and anaerobic conditions during a day–night cycle. This is accomplished by temporal separation of the two processes: photosynthesis during the day and nitrogen fixation at night. Although previous studies have examined periodic changes in transcript levels for a limited number of genes in Cyanothece and other unicellular diazotrophic cyanobacteria, a comprehensive study of transcriptional activity in a nitrogen-fixing cyanobacterium is necessary to understand the impact of the temporal separation of photosynthesis and nitrogen fixation on global gene regulation and cellular metabolism. We have examined the expression patterns of nearly 5,000 genes in Cyanothece 51142 during two consecutive diurnal periods. Our analysis showed that ≈30% of these genes exhibited robust oscillating expression profiles. Interestingly, this set included genes for almost all central metabolic processes in Cyanothece 51142. A transcriptional network of all genes with significantly oscillating transcript levels revealed that the majority of genes encoding enzymes in numerous individual biochemical pathways, such as glycolysis, oxidative pentose phosphate pathway, and glycogen metabolism, were coregulated and maximally expressed at distinct phases during the diurnal cycle. These studies provide a comprehensive picture of how a physiologically relevant diurnal light–dark cycle influences the metabolism in a photosynthetic bacterium.

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 N2-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.

Novel Metabolic Attributes of the Genus Cyanothece, Comprising a Group of Unicellular Nitrogen-Fixing Cyanobacteria

ABSTRACT 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 N2 fixation and H2 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. 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 N2 fixation and H2 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.

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

BackgroundLife 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.ResultsFourier 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.ConclusionWe 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.

Use of Degradation Tags To Control Protein Levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803

ABSTRACT We generated a collection of ssrA-based C-terminal protein degradation tags with different degradation strengths. The steady-state fluorescence levels of different enhanced yellow fluorescent protein (eYFP) tag variants in a Synechocystis sp. indicated a tunable range from 1% to 50% of untagged eYFP.

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.

A protein related to prokaryotic UMP kinases is involved in psaA / B transcript accumulation in Arabidopsis

Dpt1 (defect inpsaA/Btranscript accumulation 1) is a novel photosystem (PS) I mutant in Arabidopsis. dpt1 mutants fail to grow photoautotrophically, and are impaired in the accumulation of psaA/B transcripts while the transcript levels for the remaining PSI subunits, for subunits of the PSII, the cyt-b6/f-complex, and the ribulose-1,5-bisphosphate carboxylase are comparable to the wild type. In-organello run-on transcription assays demonstrate that the lower psaA/B transcript abundance in dpt1-1 is not caused by the inability to transcribe the psaA/psaB/rps14 operon. psaA/B transcripts in the mutant are associated with polyribosomes and translated. Thus, the mutation affects post-transcriptional processes specific for psaA/B. The dpt1 gene was isolated by map-based cloning. The protein is localized in the stroma of the chloroplast and exhibits striking similarities to UMP kinases of prokaryotic origin. Our results show that the nuclear encoded protein Dpt1 is essential for retaining photosynthetic activity in higher plant chloroplasts and involved in post-transcriptional steps of psaA/B transcript accumulation. We discuss that Dpt1 may be a bifunctional protein that couples the pyrimidine metabolism to the photosynthetic electron transport.

Proteomic profiles of five strains of oxygenic photosynthetic cyanobacteria of the genus Cyanothece.

Members of the cyanobacterial genus Cyanothece exhibit considerable variation in physiological and biochemical characteristics. The comparative assessment of the genomes and the proteomes has the potential to provide insights on differences among Cyanothece strains. By applying Sequedex, an annotation-independent method for ascribing gene functions, we confirmed significant species-specific differences of functional genes in different Cyanothece strains, particularly in Cyanothece PCC7425. Using a shotgun proteomics approach based on prefractionation and tandem mass spectrometry, we detected ∼28-48% of the theoretical Cyanothece proteome, depending on the strain. The expression of a total of 642 orthologous proteins was observed in all five Cyanothece strains. These shared orthologous proteins showed considerable correlations in their abundances across different Cyanothece strains. Functional classification indicated that the majority of proteins involved in central metabolic functions such as amino acid, carbohydrate, protein, and RNA metabolism, photosynthesis, respiration, and stress responses were observed to a greater extent in the core proteome, whereas proteins involved in membrane transport, iron acquisition, regulatory functions, flagellar motility, and chemotaxis were observed to a greater extent in the unique proteome. Considerable differences were evident across different Cyanothece strains. Notably, the analysis of Cyanothece PCC7425, which showed the highest number of unique proteins (682), provided direct evidence of evolutionary differences in this strain. We conclude that Cyanothece PCC7425 diverged significantly from the other Cyanothece strains or evolved from a different lineage.

Reply to Zhang et al.: Identification of origins of replication in the Cyanothece 51142 genome

We recently reported (1) our analysis of the Cyanothece 51142 genome, which included an attempt to identify the origins of replication in the circular and linear chromosomes in this genome. We used the web-based Ori-Finder software developed by Gao and Zhang (2). The program output at the time of submission of the revised version of our article is shown in Fig. 1.