Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O₂-sensitive N₂ fixation from oxygenic photosynthesis. The energy and reducing power needed for N₂ fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture. Received: 30 August 1996 / Accepted: 24 October 1996     

Differential transcriptional analysis of the cyanobacterium Cyanothece sp. strain ATCC 51142 during light-dark and continuous-light growth

We analyzed the metabolic rhythms and differential gene expression in the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under N(2)-fixing conditions after a shift from normal 12-h light-12-h dark cycles to continuous light. We found that the mRNA levels of approximately 10% of the genes in the genome demonstrated circadian behavior during growth in free-running (continuous light) conditions. The genes for N(2) fixation displayed a strong circadian behavior, whereas photosynthesis and respiration genes were not as tightly regulated. One of our main objectives was to determine the strategies used by these cells to perform N(2) fixation under normal day-night conditions, as well as under the greater stress caused by continuous light. We determined that N(2) fixation cycled in continuous light but with a lower N(2) fixation activity. Glycogen degradation, respiration, and photosynthesis were also lower; nonetheless, O(2) evolution was about 50% of the normal peak. We also demonstrated that nifH (encoding the nitrogenase Fe protein), nifB, and nifX were strongly induced in continuous light; this is consistent with the role of these proteins during the assembly of the enzyme complex and suggested that the decreased N(2) fixation activity was due to protein-level regulation or inhibition. Many soluble electron carriers (e.g., ferredoxins), as well as redox carriers (e.g., thioredoxin and glutathione), were strongly induced during N(2) fixation in continuous light. We suggest that these carriers are required to enhance cyclic electron transport and phosphorylation for energy production and to maintain appropriate redox levels in the presence of elevated O(2), respectively.     

Analysis of chlorophyll-protein complexes from the cyanobacterium Cyanothece sp. ATCC 51142 by non-denaturing gel electrophoresis

The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 temporally separates N(2) fixation from photosynthesis. We are analyzing the mechanism by which photosynthesis is down-regulated so that O(2) evolution is minimized during N(2) fixation. Previous results suggested changes in photosynthesis that are mediated through the redox poise of the plastoquinone pool (a process involving state transitions, in which the redistribution of excitation energy between the two photosystems helps to optimize photosynthetic yield) and the oligomerization state of the photosystems. Our working hypothesis was that the regulation of photosynthesis involved changes in the oligomerization of the photosystems. To analyze this hypothesis, we utilized a low-ionic strength, non-denaturing gel electrophoresis system to study the Chl-protein complexes. We determined that PSI is mostly trimeric, whereas PSII appears mainly as monomers. We demonstrated that most of the Chl-protein complexes in Cyanothece sp. remained constant throughout the diurnal cycle, except for the transient accumulation of a Chl-protein complex (band C) which appeared only during the late light period. Based on the size of this complex, band C represents either an interaction of PSI and PSII or a PSII dimer. These results provide support for the dynamic nature of the photosystems with respect to the diurnal cycle.     

Genome-scale modeling of light-driven reductant partitioning and carbon fluxes in diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.     

Rhythmic oscillations in KaiC1 phosphorylation and ATP/ADP ratio in nitrogen-fixing cyanobacterium Cyanothece sp. ATCC 51142

Cyanobacterial circadian clock composed of the Kai oscillator has been unraveled in the model strain Synechococcus elongatus PCC 7942. Recent studies with nitrogen-fixing Cyanothece sp. ATCC 51142 show rhythmic oscillations in the cellular program even in continuous light albeit with a cycle time of ~11 h. In the present study, we investigate correlation between cellular rhythms, KaiC1 phosphorylation cycle, ATP/ADP ratio, and the redox state of plastoquinone pool in Cyanothece. KaiC1 phosphorylation cycle of Cyanothece was similar to that of Synechococcus under diurnal cycles. However, under continuous light, the cycle time was shorter (11 h), in agreement with physiological and gene expression studies. Interestingly, the ATP/ADP ratio also oscillates with an 11 h period, peaking concomitantly with the respiratory burst. We propose a mathematical model with C/N ratio as a probable signal regulating the clock in continuous light and emphasize the existence of a single timing mechanism regardless of the cycle time.     

Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions

Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several “proof of principle” studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by ¹³C metabolic flux analysis (¹³C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report ¹³C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of ¹²C and ¹³C from unlabeled CO₂ and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism’s preferred mode under nitrogen-fixing conditions. The ¹³C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism’s distinct metabolic features under nitrogen-fixing and -non-fixing conditions.     

Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a (13)C(15)N-l-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen-sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 414 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly, and degradation showed higher levels of isotope incorporation, suggesting that these biochemical pathways are important for growth under continuous light. Calculation of relative isotope abundances (RIA) values allowed the measurement of actual active protein synthesis over time for different biochemical pathways under high light exposure. Overall results demonstrated the utility of "non-steady state" pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.     

Analysis of the nifHDK operon and structure of the NifH protein from the unicellular, diazotrophic cyanobacterium, Cyanothece strain sp. ATCC 51142(1)

Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium that demonstrates diurnal rhythms for photosynthesis and N(2) fixation, with peaks of O(2) evolution and nitrogenase activity approximately 12 h out of phase. We cloned and sequenced the nifHDK operon, and determined that the amino acid sequences of all three proteins were highly conserved relative to those of other cyanobacteria and bacteria. However, the Fe-protein, encoded by the nifH gene, demonstrated two differences from the related protein in Azotobacter vinelandii, for which a 3-D structure has been determined. First, the Cyanothece Fe-protein contained a 37 amino acid extension at the N-terminus. This approximately 4 kDa addition to the protein appeared to fold as a separate domain, but remained a part of the active protein, as was verified by migration on acrylamide gels. In addition, the Cyanothece Fe-protein had amino acid differences at positions involved in formation of the Fe-protein dimer-dimer contacts in A. vinelandii nitrogenase. There were also changes in residues involved with interaction between the Fe-protein and the MoFe-protein when compared with A. vinelandii. Since the Cyanothece Fe-protein is quickly degraded after activity, it is suggested that the extension and the amino acid alterations were somehow involved in this degradative process.     

Temporal Changes in State Transitions and Photosystem Organization in the Unicellular,Diazotrophic Cyanobacterium Cyanothece sp. ATCC 51142

The unicellular Cyanobacterium Cyanothece sp. ATCC 51142, grown under alternating 12-h light/12-h dark conditions, temporally separated N2 fixation from photosynthesis. The regulation of photosynthesis was studied using fluorescence spectra and kinetics to determine changes in state transitions and photosystem organization. The redox poise of the plastoquinone (PQ) pool appeared to be central to this regulation. Respiration supported N2 fixation by oxidizing carbohydrate granules, but reduced the PQ pool. This induced state 2 photosystem II monomers and lowered the capacity for O2 evolution. State 2 favored photosystem I trimers and cyclic electron transport, which could stimulate N2 fixation; the stimulation suggested an ATP limitation to N2 and CO2 fixation. The exhaustion of carbohydrate granules at around 6 h in the dark resulted in reduced respiratory electron flow, which led to a more oxidized PQ pool and produced a sharp transition from state 2 to state 1. This transient state 1 returned to state 2 in the remaining hours of darkness. In the light phase, photosystem II dimerization correlated with increased phycobilisome coupling to photosystem II (state 1) and increased rates of O2 evolution. However, dark adaptation did not guarantee state 2 and left photosystem I centers in a mostly monomeric state at certain times.     

Analysis of carbohydrate storage granules in the diazotrophic cyanobacterium Cyanothece sp. PCC 7822

The unicellular diazotrophic cyanobacteria of the genus Cyanothece demonstrate oscillations in nitrogenase activity and H₂ production when grown under 12 h light–12 h dark cycles. We established that Cyanothece sp. PCC 7822 allows for the construction of knock-out mutants and our objective was to improve the growth characteristics of this strain and to identify the nature of the intracellular storage granules. We report the physiological and morphological effects of reduction in nitrate and phosphate concentrations in BG-11 media on this strain. We developed a series of BG-11-derived growth media and monitored batch culture growth, nitrogenase activity and nitrogenase-mediated hydrogen production, culture synchronicity, and intracellular storage content. Reduction in NaNO₃ and K₂HPO₄ concentrations from 17.6 and 0.23 to 4.41 and 0.06 mM, respectively, improved growth characteristics such as cell size and uniformity, and enhanced the rate of cell division. Cells grown in this low NP BG-11 were less complex, a parameter that related to the composition of the intracellular storage granules. Cells grown in low NP BG-11 had less polyphosphate, fewer polyhydroxybutyrate granules and many smaller granules became evident. Biochemical analysis and transmission electron microscopy using the histocytochemical PATO technique demonstrated that these small granules contained glycogen. The glycogen levels and the number of granules per cell correlated nicely with a 2.3 to 3.3-fold change from the minimum at L0 to the maximum at D0. The differences in granule morphology and enzymes between Cyanothece ATCC 51142 and Cyanothece PCC 7822 provide insights into the formation of large starch-like granules in some cyanobacteria.     

Factors affecting the photoproduction of ammonia from dinitrogen and water by the cyanobacterium Anabaena sp. strain ATCC 33047

Synthesis of ammonia from dinitrogen and water by suspensions of Anabaena sp. Strain ATCC 33047 treated with the glutamine synthetase inhibitor L-methionine-D,L-sulfoximine is strictly dependent on light. Under otherwise optimal conditions, the yield of ammonia production is influenced by irradiance, as well as by the density, depth, and turbulence of the cell suspension. The interaction among these factors seems to determine the actual amount of light available to each single cell or filament in the suspension for the photoproduction process. Under convenient illumination, the limiting factor in the synthesis of ammonia seems to be the cellular nitrogenase activity level, but under limiting light conditions the limiting factor could, however, be the assimilatory power required for nitrogen fixation. Photosynthetic ammonia production from atmospheric nitrogen and water can operate with an efficiency of ca. 10% of its theoretical maximum, representing a remarkable process for the conversion of light energy into chemical energy.     

Coupling of Cellular Processes and Their Coordinated Oscillations under Continuous Light in Cyanothece sp. ATCC 51142, a Diazotrophic Unicellular Cyanobacterium

Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 (henceforth Cyanothece), temporally separate the oxygen sensitive nitrogen fixation from oxygen evolving photosynthesis not only under diurnal cycles (LD) but also in continuous light (LL). However, recent reports demonstrate that the oscillations in LL occur with a shorter cycle time of ~11 h. We find that indeed, majority of the genes oscillate in LL with this cycle time. Genes that are upregulated at a particular time of day under diurnal cycle also get upregulated at an equivalent metabolic phase under LL suggesting tight coupling of various cellular events with each other and with the cell’s metabolic status. A number of metabolic processes get upregulated in a coordinated fashion during the respiratory phase under LL including glycogen degradation, glycolysis, oxidative pentose phosphate pathway, and tricarboxylic acid cycle. These precede nitrogen fixation apparently to ensure sufficient energy and anoxic environment needed for the nitrogenase enzyme. Photosynthetic phase sees upregulation of photosystem II, carbonate transport, carbon concentrating mechanism, RuBisCO, glycogen synthesis and light harvesting antenna pigment biosynthesis. In Synechococcus elongates PCC 7942, a non-nitrogen fixing cyanobacteria, expression of a relatively smaller fraction of genes oscillates under LL condition with the major periodicity being 24 h. In contrast, the entire cellular machinery of Cyanothece orchestrates coordinated oscillation in anticipation of the ensuing metabolic phase in both LD and LL. These results may have important implications in understanding the timing of various cellular events and in engineering cyanobacteria for biofuel production.     

Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. under Culture Conditions Resulting in Enhanced H2 Production

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N₂-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N₂-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H₂-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H₂ production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H₂ production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H₂ production.     

Characterization of the Extracellular Polysaccharide Produced by a Marine Cyanobacterium, Cyanothece sp. ATCC 51142, and Its Exploitation Toward Metal Removal from Solutions

Cyanobacterium, Cyanothece sp. ATCC 51142 produces an exopolysaccharide at a high level. Physical analysis of the exopolysaccharide (EPS), such as nuclear magnetic resonance, infrared spectrum, were done to determine its possible structure. Thermal gravimetric analysis, differential scanning calorimeter, and differential thermal analysis of the polymer were done to find out the thermal behavior. Calcium content within the sample was found out. Some of the physicochemical properties, such as relative viscosity, specific viscosity, and intrinsic viscosity of the EPS were studied under different conditions. The phenomenon of gel formation by the EPS was investigated for its potential application in metal removal from solutions. Received: 23 August 1999 / Accepted: 12 November 1999