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.     

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophyenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.     

Photosystem II Cyclic Heterogeneity and Photoactivation in the Diazotrophic, Unicellular Cyanobacterium Cyanothece Species ATCC 51142

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N₂-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O₂-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (q_N) of PSII increased during N₂ fixation and persisted after treatments known to induce transitions to state 1. The q_N was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, ε, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N₂ fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O₂ evolution, altered dark stability of PSII centers, and substantial changes in q_N.     

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.     

Utilization of Light for Nitrogen Fixation by a New Synechocystis Strain Is Extended by Its Low Photosynthetic Efficiency

Performance of photosynthesis and nitrogenase activity in a novel cyanobacterium, Synechocystis sp. strain BO 8402, isolated from Lake Constance, located at the northern fringe of the Alps in central Europe, and of a stable derivative, strain BO 9201, were examined. Strain BO 8402 is characterized by an extraordinarily high level of autofluorescence originating from paracrystalline phycobiliprotein-linker complexes located in inclusion bodies (W. Reuter, M. Westermann, S. Brass, A. Ernst, P. Böger, and W. Wehrmeyer, J. Bacteriol. 176:896-904, 1994). Energy transfer between paracrystalline phycobiliproteins and the photosystems is inefficient, resulting in a high oxygen compensation point and a decreased growth rate. The derivative strain BO 9201 exhibits hemidiscoidal phycobilisomes that support a high growth rate, even under low light intensities. Because of the differences in photosynthetic performance, anaerobic light-stimulated nitrogenase activity is maintained at higher light intensity in the original strain BO 8402 than in the derivative strain BO 9201. The results indicate that the formation of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 represents a hitherto-unknown means for a unicellular cyanobacterium to extend its capacity to fix nitrogen in the light.     

Nitrogen Fixation Dynamics of Two Diazotrophic Communities in Mono Lake, California

Two types of diazotrophic microbial communities were found in the littoral zone of alkaline hypersaline Mono Lake, California. One consisted of anaerobic bacteria inhabiting the flocculent surface layers of sediments. Nitrogen fixation (acetylene reduction) by flocculent surface layers occurred under anaerobic conditions, was not stimulated by light or by additions of organic substrates, and was inhibited by O₂, nitrate, and ammonia. The second community consisted of a ball-shaped association of a filamentous chlorophyte (Ctenocladus circinnatus) with diazotrophic, nonheterocystous cyanobacteria, as well as anaerobic bacteria (Ctenocladus balls). Nitrogen fixation by Ctenocladus balls was usually, but not always, stimulated by light. Rates of anaerobic dark fixation equaled those in the light under air. Fixation in the light was stimulated by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and by propanil [N-(3,4-dichlorophenyl)propanamide]. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea-elicited nitrogenase activity was inhibited by ammonia (96%) and nitrate (65%). Fixation was greatest when Ctenocladus balls were incubated anaerobically in the light with sulfide. Dark anaerobic fixation was not stimulated by organic substrates in short-term (4-h) incubations, but was in long-term (67-h) ones. Areal estimates of benthic N₂ fixation were measured seasonally, using chambers. Highest rates (~29.3 μmol of C₂H₄ m⁻² h⁻¹) occurred under normal diel regimens of light and dark. These estimates indicate that benthic N₂ fixation has the potential to be a significant nitrogen source in Mono Lake.     

Comparative Amperometric Study of Uptake Hydrogenase and Hydrogen Photoproduction Activities between Heterocystous Cyanobacterium Anabaena cylindrica B629 and Nonheterocystous Cyanobacterium Oscillatoria sp. Strain Miami BG7

Heterocystous filamentous cyanobacterium Anabaena cylindrica B629 and nonheterocystous filamentous cyanobacterium Oscillatoria sp. strain Miami BG7 were cultured in media with N₂ as the sole nitrogen source; and activities of oxygen-dependent hydrogen uptake, photohydrogen production, photooxygen evolution, and respiration were compared amperometrically under the same or similar experimental conditions for both strains. Distinct differences in these activities were observed in both strains. The rates of hydrogen photoproduction and hydrogen accumulation were significantly higher in Oscillatoria sp. strain BG7 than in A. cylindrica B629 at every light intensity tested. The major reason for the difference was attributable to the fact that the heterocystous cyanobacterium had a high rate of oxygen-dependent hydrogen consumption activity and the nonheterocystous cyanobacterium did not. The activity of oxygen photoevolution and respiration also contributed to the difference. Oscillatoria sp. strain BG7 had lower O₂ evolution and higher respiration than did A. cylindrica B629. Thus, the effect of O₂ on hydrogen photoproduction was minimized in Oscillatoria sp. strain BG7.     

Effects of Ammonium Ions, Oxygen, Carbon Monoxide, and Acetylene on Anaerobic and Aerobic Hydrogen Formation by Anabaena cylindrica B629

An investigation was made of various factors, both experimental and physiological, which influenced the formation of hydrogen gas by the heterocystous cyanobacterium Anabaena cylindrica B629 when incubated in both argon and air. A. cylindrica B629 produces hydrogen in air in the presence of carbon monoxide, acetylene, or both, with a short lag period. The rate of production in air at optimal concentrations of these compounds was found to be comparable with that in an argon atmosphere. Whereas under argon, ammonium ions (0.5 to 6 mM) were found to inhibit hydrogen formation in a manner which was dependent on light intensity and not relieved by oxygen (1 to 20% of gas phase), in air-carbon monoxide-acetylene, these ions (up to at least 0.5 mM) slightly stimulated hydrogen production for at least 24 h. Conclusions are drawn about short-term aerobic and anaerobic hydrogen formation by A. cylindrica B629 and the effects of ammonium ions, oxygen, carbon monoxide, and acetylene on these processes.     

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.     

Development of chlorophyll and hill activity

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding δ-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with δ-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.     

Oxygen evolution and the permeability of the outer envelope of isolated whole chloroplasts

A rapid oxygraph method of studying the permeability of the envelope of isolated chloroplasts was used. The outer envelope of aqueously isolated whole spinach (Spinacia oleracea L.) chloroplasts in buffer is readily permeable to 3-phosphoglyceric acid, which induces an immediate light dependent oxygen evolution. This light dependent oxygen evolution was completely eliminated by swelling these plastids in an osmotically dilute solution. Exogenous adenosine diphosphate, but not inorganic phosphate, strongly stimulated this oxygen evolution. This indicated that the chloroplast envelope is relatively permeable to adenosine diphosphate.

Oxygen evolution and swelling studies indicated that the chloroplast envelope is relatively impermeable to NADP and to ferredoxin.

A method is described whereby the percent of whole chloroplasts present in a chloroplast preparation may be rapidly estimated.

     

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.     

Aerobic Hydrogen Accumulation by a Nitrogen-Fixing Cyanobacterium, Anabaena sp

Hydrogen evolution by a nitrogen-fixing cyanobacterium, Anabaena sp. strain N-7363, was tested in order to develop a water biophotolysis system under aerobic conditions. A culture of the strain supplemented with carbon dioxide under an air atmosphere evolved hydrogen and oxygen gas, which reached final concentrations of 9.7 and 69.8%, respectively, after 12 days of incubation. Hydrogen uptake activity was not observed during incubation, and nitrogenase was thought to be the sole enzyme responsible for the hydrogen evolution.     

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth.

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.     

Weak red light plays an important role in awakening the photosynthetic machinery following desiccation in the subaerial cyanobacterium Nostoc flagelliforme

The subaerial cyanobacterium Nostoc flagelliforme can survive for years in the desiccated state and light exposure may stimulate photosynthetic recovery during rehydration. However, the influence of light quality on photosynthetic recovery and the underlying mechanism remain unresolved. Exposure of field collected N. flagelliforme to light intensity ≥2 μmol photons m⁻² s⁻¹ showed that the speed of photosystem II (PSII) recovery was in the following order: red > green > blue ≈ violet light. Decreasing the light intensity showed that weak red light stimulated PSII recovery during rehydration. The chlorophyll fluorescence transient and oxygen evolution activity indicated that the oxygen evolution complex (OEC) was the activated site triggered by weak red light. The damaged D1 protein accumulated in the thylakoid membrane during dehydration and is degraded and resynthesized during dark rehydration. PsbO interaction with the thylakoid membrane was induced by weak red light. Thus, weak red light plays an important role in triggering OEC photoactivation and the formation of functional PSII during rehydration. In its arid habitats, weak red light could stimulate the awakening of dormant N. flagelliforme after absorbing water from nighttime dew or rain to maximize growth during the early daylight hours of the dry season.     

Levels of Daily Light Doses Under Changed Day-Night Cycles Regulate Temporal Segregation of Photosynthesis and N2 Fixation in the Cyanobacterium Trichodesmium erythraeum IMS101

While the diazotrophic cyanobacterium Trichodesmium is known to display inverse diurnal performances of photosynthesis and N₂ fixation, such a phenomenon has not been well documented under different day-night (L-D) cycles and different levels of light dose exposed to the cells. Here, we show differences in growth, N₂ fixation and photosynthetic carbon fixation as well as photochemical performances of Trichodesmium IMS101 grown under 12L:12D, 8L:16D and 16L:8D L-D cycles at 70 μmol photons m^-2 s^-1 PAR (LL) and 350 μmol photons m^-2 s^-1 PAR (HL). The specific growth rate was the highest under LL and the lowest under HL under 16L:8D, and it increased under LL and decreased under HL with increased levels of daytime light doses exposed under the different light regimes, respectively. N₂ fixation and photosynthetic carbon fixation were affected differentially by changes in the day-night regimes, with the former increasing directly under LL with increased daytime light doses and decreased under HL over growth-saturating light levels. Temporal segregation of N₂ fixation from photosynthetic carbon fixation was evidenced under all day-night regimes, showing a time lag between the peak in N₂ fixation and dip in carbon fixation. Elongation of light period led to higher N₂ fixation rate under LL than under HL, while shortening the light exposure to 8 h delayed the N₂ fixation peaking time (at the end of light period) and extended it to night period. Photosynthetic carbon fixation rates and transfer of light photons were always higher under HL than LL, regardless of the day-night cycles. Conclusively, diel performance of N₂ fixation possesses functional plasticity, which was regulated by levels of light energy supplies either via changing light levels or length of light exposure.     

Role of light in the response of PSII photochemistry to salt stress in the cyanobacterium Spirulina platensis

The role of light in the effect of salt stress on PSII photochemistry in the cyanobacterium Spirulina platensis grown at 50 micromol m(-2) s(-1) was investigated. The time-course of changes in PSII photochemistry in response to high salinity (0.8 M NaCl) incubated in the dark and at 30, 50 and 100 micromol m(-2) s(-1) was composed of two phases. The first phase, which was independent of light, was characterized by a rapid decrease (20-50%) in the maximal efficiency of PSII photochemistry (F:(v)/F:(m)), the efficiency of excitation energy capture by open PSII reaction centres (F(1)(v)/F(1)(m)), photochemical quenching (q(P)), and the quantum yield of PSII electron transport (Phi(PSII)) in the first 15 min, followed by a recovery of up to about 86-92% of their initial levels after 4 h of incubation. The second phase took place after 4 h, in which a further decline in the above parameters occurred only in the light but not in the dark, reaching levels as low as 32-56% of their initial levels after 12 h. Moreover, the higher incubation light intensity, the greater the decrease in the above parameters. At the same time, Q(B)-non-reducing PSII reaction centres increased significantly in the first 15 min and then recovered to the initial level during the first phase, but increased again in the light in the second phase. Photosynthetic oxygen evolution activity decreased sharply by 70% in the first 5 min, and then kept largely constant until 12 h. The changes in oxygen evolution activity were independent of light intensity during both phases.     

Intracellular spheroid bodies of Rhopalodia gibba have nitrogen-fixing apparatus of cyanobacterial origin

Nitrogen fixation is not regarded as a eukaryotic invention. The process has only been reported as being carried out by bacteria. These prokaryotes typically interact with their eukaryotic hosts as extracellular and temporary nonobligate nitrogen-fixing symbionts. However, intracellular permanent "spheroid bodies" have been reported within the fresh-water diatom Rhopalodia gibba, and these, too, have been speculated as being able to provide nitrogen to their host diatom. These spheroid bodies have gram-negative characteristics with thylakoids. We demonstrate that they fix nitrogen under light conditions. We also show that phylogenetic analyses of their 16rRNA and nif D genes predict that their genome is closely related to that of Cyanothece sp. ATCC 51.142, a free-living diazotrophic cyanobacterium. We suggest that the intracellular spheroid bodies of Rhopalodia gibba may represent a vertically transmitted, permanent endosymbiotic stage in the transition from a free-living diazotrophic cyanobacterium to a nitrogen-fixing eukaryotic organelle.