Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.     

Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142.

It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.     

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.     

Relation between nitrogenase synthesis and activity in a marine unicellular diazotrophic strain, Gloeothece sp. 68DGA (Cyanophyte), grown under different light/dark regimens

The relationship between the abundance of nitrogenase and its activity was studied in the marine unicellular cyanobacterium Gloeothece sp. 68DGA cultured under different light/dark regimens. The Fe‐ and MoFe‐protein of nitrogenase and nitrogen (N₂)‐fixing (acetylene reduction) activity were detected only during the dark phase when the cells were grown under a 12 h light/12 h dark cycle (12L/12D). Nitrogenase activity appeared about 4 h after entering the dark phase. Maximum nitrogenase activity occurred at around the middle of the dark phase, and the activity rapidly decreased to zero before the start of the light phase. The rapid decrease of nitrogenase activity and the Fe‐protein of nitrogenase near the end of the dark phase in 12L/12D were partly recovered by the addition of l ‐methionine‐sulfoximine, an inhibitor of glutamine synthetase. Diurnal oscillation of the abundance of nitrogenase was maintained in the first subjective dark phase (i.e. the period corresponding to the dark phase) after the cells were transferred from 12L/12D to continuous illumination. However, enzyme activity was detected only when photosynthetic oxygen (O₂) evolution was completely suppressed by reducing the light intensity or by the addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Nitrogenase always appeared in the cells about 16 h after starting the light phase, even when the 12L/12D cycle was modified by the addition or subtraction of a single 6 h period of light or dark. These results suggest the following: (i) N₂‐fixation by Gloeothece sp. 68DGA is primarily regulated by an endogenous circadian oscillator at the level of nitrogenase synthesis. (ii) The endogenous circadian rhythm resets on a shift of the timing of the light phase. (iii) Nitrogenase activity is not always reflected in the presence of nitrogenase. (iv) The activity of nitrogenase is negatively regulated by fixed nitrogen and the concentration of ambient O₂.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

GROWTH,PHYSIOLOGY, AND ULTRASTRUCTURE OF A DIAZOTROPHIC CYANOBACTERIUM,CYANOTHECE SP. STRAIN ATCC 51142, IN MIXOTROPHIC AND CHEMOHETEROTROPHIC CULTURES1

The growth, physiology, and ultrastructure of the marine, unicellular, diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, was examined under mixotrophic and chemoheterotrophic conditions. Several organic substrates were tested for the capacity to support heterotrophic growth. Glycerol was the only substrate capable of enhancing mixotrophic growth in the light and supporting chemoheterotrophic growth in the dark. Dextrose enhanced mixotrophic growth but could not support chemoheterotrophic growth. Chemoheterotrophic cultures in continuous darkness grew faster and to higher densities than photoautotrophic cultures, thus demonstrating the great respiratory capacity of this cyanobacterial strain. Only small differences in the pigment content and ultrastructure of the heterotrophic strains were observed in comparison to photoautotrophic control strains. The chemoheterotrophic strain grown in continuous darkness and the mixotrophic strain grown in light/dark cycles exhibited daily metabolic oscillations in N₂ fixation and glycogen accumulation similar to those manifested in photoautotrophic cultures grown in light/dark cycles or continuous light. This “temporal separation” helps protect O₂-sensitive N₂ fixation from photosynthetic O₂ evolution. The rationale for cyclic glycogen accumulation in cultures with an ample source of organic carbon substrate is unclear, but the observation of similar daily rhythmicities in cultures grown in light/dark cycles, continuous light, and continuous dark suggests an underlying circadian mechanism.     

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.     

The effects of different light–dark cycles on the metabolism of the diazotrophic,unicellular cyanobacteria Cyanothece sp. ATCC 51142, and Cyanothecesp. PCC 7822

The diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 demonstrates circadian patterns in nitrogenase activity, H₂ production and glycogen storage when grown under nitrogen‐fixing, 12:12 light:dark (L:D) conditions. In this study, we grew Cyanothece sp. ATCC 51142, and another strain in this genus, Cyanothece sp. PCC 7822, under long‐day (16:8 L:D) and short‐day (8:16 L:D) nitrogen‐fixing conditions to determine if they continued to display circadian rhythms. Both strains demonstrated similar circadian patterns for all three metabolic parameters when grown under long‐day conditions. However, the strains responded differently to short‐day growth conditions. Cyanothece sp. ATCC 51142 retained reasonable circadian patterns under 8:16 L:D conditions, whereas Cyanothece sp. PCC 7822 had quite damped patterns without a clear circadian pattern. In particular, glycogen storage changed very little throughout the day and we ascribe this to the difference in the type of glycogen granules in Cyanothece sp. PCC 7822 which has small β‐granules, compared to the large, starch‐like granules in Cyanothece sp. ATCC 51142. The results suggested that both mechanistic and regulatory processes play a role in establishing the basis for these metabolic oscillations.     

Hydrogen metabolism of three unicellular nitrogen-fixing cyanobacteria

Abstract The enzyme activities responsible for the evolution and consumption of hydrogen in three unicellular cyanobacteria were investigated. Gloeothece sp. 6909 and Cyanothece sp. 7822 performed an oxygen-tolerant nitrogen fixation, whereas the nitrogenase activity of Synechococcus sp. 7425 was much more sensitive to oxygen. While in Gloeothece the net hydrogen production during nitrogen fixation was relatively low due to recycling by an uptake hydrogenase, little hydrogen consumption was detected in Cyanothece and Synechococcu . On the other hand a reversible hydrogenase was demonstrated in the latter strains. However, only Cyanothece shows hydrogenase-catalysed hydrogen production in vivo under anaerobic conditions in the dark. It is suggested that hydrogen is a fermentation product, and that the physiological function of this reversible hydrogenase is the removal of excess reduction equivalents under such conditions.     

Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N₂) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N₂ fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N₂ fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N₂ fixation and photosynthesis commonly observed. However, N₂ fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell.     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.     

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.     

Physiological conditions for nitrogen fixation in a unicellular marine cyanobacterium, Synechococcus sp. strain SF1.

A marine, unicellular, nitrogen-fixing cyanobacterium was isolated from the blades of a brown alga, Sargassum fluitans. This unicellular cyanobacterium, identified as Synechococcus sp. strain SF1, is capable of photoautotrophic growth with bicarbonate as the sole carbon source and dinitrogen as the sole nitrogen source. Among the organic carbon compounds tested, glucose and sucrose supported growth. Of the nitrogen compounds tested, with bicarbonate serving as the carbon source, both ammonia and nitrate produced the highest growth rates. Most amino acids failed to support growth when present as sole sources of nitrogen. Nitrogenase activity in Synechococcus sp. strain SF1 was induced after depletion of ammonia from the medium. This activity required the photosynthetic utilization of bicarbonate, but pyruvate and hydrogen gas were also effective sources of reductant for nitrogenase activity. Glucose, fructose, and sucrose also supported nitrogenase activity but to a lesser extent. Optimum light intensity for nitrogenase activity was found to be 70 microE/m2 per s, while the optimum oxygen concentration in the gas phase for nitrogenase activity was about 1%. A hydrogenase activity was coinduced with nitrogenase activity. It is proposed that this light- and oxygen-insensitive hydrogenase functions in recycling the hydrogen produced by nitrogenase under microaerobic conditions.     

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.