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Metabolic Activities of Isolated Heterocysts of the Blue-green Alga Anabaena cylindrica
Abstract
KNOWLEDGE of the nature of the heterocysts of blue-green algae has been limited by the lack of suitable techniques for investigation of the physiology and biochemistry of these puzzling structures. The association of the presence of heterocysts with the ability to fix free nitrogen and the findings1,2 that ammonia suppresses both nitrogen fixation and heterocyst formation have led to the suspicion that these cells might be involved in the nitrogen-fixation process. In this communication we report a successful technique of `differential cell disruption' for the isolation of heterocysts of Anabaena cylindrica. We also present the first results concerning the metabolic activities of isolated heterocysts.
... The addition of sulfydryl agents increased N2 uptake by maintaining reducing conditions. Since 02 rapidly inactivates nitrogenase in isolated heterocysts (95,253,320), stringent anaerobic conditions must be applied during their isolation (90). ...
... The faint appearance of heterocysts under the light microscope suggests that pigment composition has been modified during differentiation and that both the organization of the photosynthetic apparatus and the photochemical activities of the heterocysts have been altered. Early studies have shown that heterocysts contain significantly reduced quantities of photosynthetic pigments, particularly phycobiliproteins (86,338,367), and that they have lost the ability to fix CO2 in the light (95,320,363) and to evolve 02 when illuminated (29,48,334). The changes were thought to indicate that heterocysts lack a functional PS II and the reductive pentose phosphate pathway. ...
The enigmatic coexistence of O2-sensitive nitrogenase and O2-evolving photosynthesis in diazotrophic cyanobacteria has fascinated researchers for over two decades. Research efforts in the past 10 years have revealed a range of O2 sensitivity of nitrogenase in different strains of cyanobacteria and a variety of adaptations for the protection of nitrogenase from damage by both atmospheric and photosynthetic sources of O2. The most complex and apparently most efficient mechanisms for the protection of nitrogenase are incorporated in the heterocysts, the N2-fixing cells of cyanobacteria. Genetic studies indicate that the controls of heterocyst development and nitrogenase synthesis are closely interrelated and that the expression of N2 fixation (nif) genes is regulated by pO2.
... One major aspect of the metabolic reprogramming of vegetative cells undergoing differentiation as heterocysts involves shutdown of oxygenic photosynthesis, which is not compatible with the activity of the O 2 -inactivated enzyme nitrogenase. Early studies showing movement of reduced carbon compounds from vegetative cells into heterocysts suggested that there was no CO 2 fixation in heterocysts (Fay and Walsby, 1966;Wolk, 1968), consistent with the absence in those cells of key enzymes of the Calvin cycle, such as ribulose-bisphosphate carboxylase/ oxygenase (Rubisco) or phosphoribulokinase (PRK; Codd and Stewart, 1977;Codd et al., 1980;Cossar et al., 1985;Elhai and Wolk, 1990). In agreement with those observations, a quantitative proteomic study in Nostoc punctiforme showed that proteins involved in the carbon concentrating mechanism and enzymes involved in CO 2 fixation were present in reduced amounts in the heterocysts versus the vegetative cells (Sandh et al., 2014). ...
In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen. This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO2 fixation in heterocysts, to provide a microaerobic environment suitable for N2 fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of NsiR4 (Nitrogen stress-inducible RNA 4) in post-transcriptionally regulating the expression of two genes involved in CO2 fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5’-UTR of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO2 assimilation.
... We note that genus Anabaena has been renamed to Dolichospermum but here we use the term Anabaena as it has been more commonly used. They form a chain of cells (trichome) (Fig. 5), within which there are heterocysts [64,171,172]. Specifically, heterocysts are visually distinct with thick glycolipid layers on the cell membrane, which protects the cytoplasm and thus nitrogenase from O 2 [65,73,173]. Some studies show that bacteria specifically associated with heterocysts can provide respiratory protection from O 2 [174]. ...
Nitrogen-fixing organisms are of importance to the environment, providing bioavailable nitrogen to the biosphere. Quantitative models have been used to complement the laboratory experiments and in situ measurements, where such evaluations are difficult or costly. Here, we review the current state of the quantitative modeling of nitrogen-fixing organisms and ways to enhance the bridge between theoretical and empirical studies.
... Conspicuous reorganization of the thylakoid membranes, concomitant with loss of photosystem II function (and thus oxygen production), as well as increased respiration in the so-called honeycomb membranes contributes to reduced O 2 levels [for a review see Magnuson and Cardona (2016)]. Early studies showing movement of reduced carbon compounds from the vegetative cells into heterocysts suggested that there was no CO 2 fixation in heterocysts (Fay andWalsby 1966, Wolk 1968). In addition, the CO 2 -fixing enzyme ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) is not detected in the heterocyst (Codd and Stewart 1977, Cossar et al. 1985, Elhai and Wolk 1990. ...
Upon nitrogen deficiency, some filamentous cyanobacteria differentiate specialized cells, called heterocysts, devoted to N2 fixation. Heterocysts appear regularly spaced along the filaments and exhibit structural and metabolic adaptations, such as loss of photosynthetic CO2 fixation or increased respiration, to provide a proper microaerobic environment for its specialized function. Heterocyst development is under transcriptional control of the global nitrogen regulator NtcA and the specific regulator HetR. Transcription of a large number of genes is induced or repressed upon nitrogen deficiency specifically in cells undergoing differentiation. In recent years, the HetR regulon has been described to include heterocyst-specific trans-acting small RNAs and antisense RNAs, suggesting that there is an additional layer of postranscriptional regulation involved in heterocyst development. Here we characterize in the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 an antisense RNA, that we call as_glpX, transcribed within the glpX gene encoding the Calvin cycle bifunctional enzyme sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase). Transcription of as_glpX is restricted to heterocysts and is induced very early during the process of differentiation. Expression of as_glpX RNA promotes the cleavage of the glpX mRNA by RNase III, resulting in a reduced amount of SBPase. Therefore, the early expression of this antisense RNA could contribute to the quick shut down of CO2 fixation in those cells in the filament that are undergoing differentiation into heterocysts. In summary, as_glpX is the first naturally occurring antisense RNA shown to rapidly and dynamically regulate metabolic transformation in Nostoc heterocysts. The use of antisense transcripts to manipulate gene expression specifically in heterocysts could became a useful tool for metabolic engineering in cyanobacteria.
... The need of ATP, which is provided by the photosystem I, and of NADPH to maintain nitrogen fixation, could explain why we found a slight increase of phosphorus in the heterocysts as well. Due to the loss of photosystem II, heterocyst have a much lower CO 2 fixing capability compared to vegetative cells 23 , as this process would compete with nitrogen fixation for reductant and ATP (reviewed by Haselkorn) 16 . Another reason for the lack of polyphosphate inclusions could be the alkalinisation of the heterocysts due to the forming of ammonium via nitrogen fixation 24,25 , which may cause the hydrolysis of polyphosphates. ...
The cyanobacterium Nodularia spumigena is a species that frequently forms blooms in the Baltic Sea. Accumulation of the vital nutrient phosphorus (P) apparently plays an important role in the ability of this and other cyanobacteria to grow even when dissolved inorganic phosphorus is depleted. However, until now, this has not been studied in N. spumigena at the cellular level. Therefore, in this study, phosphorus incorporation and distribution in cyanobacterial filaments over time was examined by scanning electron microscopy in combination with energy dispersive X-ray analysis (SEM/EDX) and nanoscale secondary ion mass spectrometry (NanoSIMS). Immediately after phosphate addition to a phosphorus-depleted population, the phosphate concentration decreased in the water while intracellular polyphosphate accumulated. Microscopically, phosphorus in form of polyphosphate granules was stored preferentially in vegetative cells, whereas heterocysts remained low in intracellular phosphorus. This information is an essential step towards understanding the phosphorus dynamics of this species and demonstrates that the division of tasks between vegetative cells and heterocysts is not restricted to nitrogen fixation.
... Kulasooriya et al. (1972) reported on the differentiation of heterocysts in Anabaena cylindrica and also demonstrated how it coincided with the restoration of nitrogenase activity. Isolated heterocysts of A. cylindrica showed higher rates of respiration than vegetative cells and their absorption spectrum coincided more closely with the action spectrum of nitrogenase activity by whole filaments than that of the vegetative cells (Fay & Walsby, 1966). The isolated heterocysts contained very little chlorophyll-a and were devoid of phycocyanin and phycoerythrin pigments, which are associated with the O 2 evolving photosystem-II of photosynthesis (Thomas, 1970 Janaki and Wolk (1982) showed the presence of nitrogenase in isolated heterocysts. ...
Cyanobacteria are unique in their ability to conduct the two incompatible processes of O2 evolving photosynthesis and O2sensitive N2 fixation within non-compartmentalised prokaryotic cells. Among the early life forms of the primitive Earth, cyanobacteria were the first to perform oxygenic photosynthesis. This process slowly changed the original reducing atmosphere of the Earth to an oxidising one, triggering off a dramatic evolution of global biodiversity. Under the original reducing atmosphere N2 fixation was widespread among the organisms that inhabited the Earth. As this atmosphere gradually became oxygenated, some including certain cyanobacteria developed mechanisms to protect their nitrogenase enzyme from damage by O2. Among the filamentous cyanobacteria a common adaptation is the formation of heterocysts, which are specialised for N2 fixation, but many unicellular species and some non-heterocystous filamentous forms have also developed mechanisms for aerobic N2 fixation. Recent findings have revealed the diversity and importance of novel diazotrophic unicellular cyanobacteria and their associations in the oligotrophic deep oceans. The discovery of the occurrence of unculturable marine diazotrophic cyanobacteria that possessed an unknown photo-fermentative metabolism, which was later found to be a symbiotic association is a case in point. Such findings indicate that there could be many new organisms and systems yet to be discovered and open up new vistas for future research. They also lend support to the novel hypothesis of symbiogenetic evolution. These reports have elevated the role of unicellular N2 fixing cyanobacteria and their symbiotic systems in the deep oceans. The pioneering habitation of the early Earth by the ancestors of present day cyanobacteria is reflected in their ubiquitous global distribution from barren tropical deserts to freezing environments of glaziers. Together with certain Proteobacteria and Archaea they are common inhabitants of extreme environments that are generally inhospitable to most organisms. The role of N2 fixing cyanobacteria are exemplified by the significant contribution they make to the nitrogen budget of the oceans, which occupy more than 80 % of the Earth’s surface. Cyanobacteria are also found in freshwater and terrestrial habitats spanning across all latitudes and longitudes of the world and contribute significantly to the carbon and nitrogen cycles of wetlands, freshwater and brackish ecosystems as free-living, symbiotic, epiphytic, benthic and periphyton organisms. Certain N2 fixing cyanobacteria form blooms in lentic water bodies and some of them produce cyanotoxins. N2 fixing cyanobacteria therefore play both positive and negative roles in nature. Cyanobacteria form symbiotic associations with all other major groups of organisms and contribute to the nutrition of their hosts. Certain cyanobacteria and their symbiotic systems like Azolla are used as biofertilizers, particularly in rice production. This paper is a review of literature on N2 fixing cyanobacteria, their diversity, their roles in nature and their utilisation.
... The absence of oxygen evolution in heterocysts was first proposed by Fay et al. [150] in 1968. However, it should be noted that during most heterocyst purifications the samples are subjected to significant stress, to such an extent that the first preparations did not even show any nitrogen fixation at all [151]. Heterocyst preparations usually require prolonged incubation with lysozyme at elevated temperatures around 38°C, and are performed under continued illumination, usually in the absence of protease inhibitors, and in buffers that are not optimal to preserve PSII activity [8]. ...
Multicellular cyanobacteria form different cell types in response to environmental stimuli. Under nitrogen limiting conditions a fraction of the vegetative cells in the filament differentiate into heterocysts. Heterocysts are specialized in atmospheric nitrogen fixation and differentiation involves drastic morphological changes on the cellular level, such as reorganization of the thylakoid membranes and differential expression of thylakoid membrane proteins. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase by developing an extra polysaccharide layer that limits air diffusion into the heterocyst and by upregulating heterocyst-specific respiratory enzymes. In this review article, we summarize what is known about the thylakoid membrane in heterocysts and compare its function with that of the vegetative cells. We emphasize the role of photosynthetic electron transport in providing the required amounts of ATP and reductants to the nitrogenase enzyme. In the light of recent high-throughput proteomic and transcriptomic data, as well as recently discovered electron transfer pathways in cyanobacteria, our aim is to broaden current views of the bioenergetics of heterocysts. This article is part of a Special Issue entitled: Organization and dynamics of bioenergetic systems in bacteria. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.
... The development of this protocol was aimed to preserve the integrity of the heterocysts and for purities higher than 90%. The first heterocyst purifications were established by Fay and Walsby (1966) using a French press treatment, a comprehensive comparison of different methods for purification was published by Fay and Lang (1971). In these studies the extent of the damage was inspected by electron microscopy ultrastructural analysis. ...
... In addition, the absence of the Hill reaction in isolated heterocysts has been documented (Bradley and Carr 1971;Tel-Or and Stewart 1975). Mature heterocysts have been claimed to lack the ability of assimilating CO 2 (Fay and Walshby 1966;Brusca et al. 1989), and it was shown that carboxysomes disintegrate during heterocyst differentiation. ...
Changes of photosynthetic activity in vivo of individual heterocysts and vegetative cells in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 during the course of diazotrophic acclimation were determined using fluorescence kinetic microscopy (FKM). Distinct phases of stress and acclimation following nitrogen step-down were observed. The first was a period of perception, in which the cells used their internally stored nitrogen without detectable loss of PS II activity or pigments. In the second, the stress phase of nitrogen limitation, the cell differentiation occurred and an abrupt decline of fluorescence yield was observed. This decline in fluorescence was not paralleled by a corresponding decline in photosynthetic pigment content and PS II activity. Both maximal quantum yield and sustained electron flow were not altered in vegetative cells, only in the forming heterocysts. The third, acclimation phase started first in the differentiating heterocysts with a recovery of PS II photochemical yields \(F_{\text{v}} /F_{\text{m}} ,\;F^{\prime}_{\text{v}} /F^{\prime}_{\text{m}}.\) Afterwards, the onset of nitrogenase activity was observed, followed by the restoration of antenna pigments in the vegetative cells, but not in the heterocysts. Surprisingly, mature heterocysts were found to have an intact PS II as judged by photochemical yields, but a strongly reduced PS II-associated antenna as judged by decreased F
0. The possible importance of the functional PS II in heterocysts is discussed. Also, the FKM approach allowed to follow in vivo and evaluate the heterogeneity in photosynthetic performance among individual vegetative cells as well as heterocysts in the course of diazotrophic acclimation. Some cells along the filament (so-called “superbright cells”) were observed to display transiently increased fluorescence yield, which apparently proceeded by apoptosis.
... At that time there was no reason to suppose that nitrogenase activity and oxygenic photosynthesis needed to be separated and, indeed, I had argued for a close connection between the two processes (Fogg & Than-Tun 1958) [(21)].… Fay & Walsby (1966) devised a neat and effective way of separating [heterocysts] from normal cells and investigated the properties of the isolated heterocysts. They were metabolically active, as shown by the high rate of respiration, but incapable of either photosynthesis or nitrogen fixation. ...
Gordon Elliott (Tony) Fogg was a botanist who pioneered studies in cyanobacterial heterocysts, nitrogen fixation, extracellular products of algae and phytoplankton ecophysiology. He was a gifted lecturer and writer, and the author of books on algae, phytoplankton ecology and Antarctic science. After many years in Botany at University College and then Westfield College in the University of London, he completed his career in Marine Biology at the University College of North Wales. His natural diplomacy led to appointments on many scientific committees, where he was valued for his succinct observations and his ability to settle disputes behind the scenes. He made important contributions to many organizations, including the Institute of Biology, the Freshwater Biological Association, the British Antarctic Survey and the Royal Botanic Gardens, Kew. He was artistic, a fine water-colourist, and he enjoyed walking in Anglesey and Snowdonia.
... Members of the Nostocales differentiate specialized cells, called heterocysts , under nitrogen-fixing conditions. Several features of heterocyst biochemistry and structure, including the loss of 02-evolving photosystem I1 activity (Thomas 1970; Donze et al. 1972; Tel-Or and Stewart 1977), enhancement of respiratory capacity (Fay and Walsby 1966; Winkenbach and Wolk 1973) and synthesis of a thick cell wall, postulated to provide a barrier to the ingress of O2 (Larnbein and Wolk 1973), suggest that the heterocyst provides an anaerobic site for nitrogenase function. It is unlikely, however, that O2 is completely excluded from the heterocyst since dark nitrogenase activity is O2 dependent (Weare and Benemann 1973; Fay 1976). ...
Nitrogenase is known to be irreversibly inactivated by oxygen in vivo and in vitro. In time-course experiments using Anabaena cylindrica, cultures treated with antibiotics and incubated under various O2 tensions, in vivo acetylene reduction activity and immunologically determined Fe–Mo protein (component I) were lost at rates directly related to O2 tension. Activity was lost at a faster rate than cross-reactive material. The half-life of cross-reactive material was 25 h under microaerophilic conditions, 12 h under aerobic conditions, and 6.6 h under an O2 tension of 245% of air saturation. In vitro, cross-reactive material was lost in O2 exposed, but not in anaerobically prepared, crude cell extracts. Loss of cross-reactive material was prevented by freezing and by α-N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of histidine-residue proteases. These results indicate that nitrogenase is continuously inactivated by O2, hydrolyzed, and resynthesized during growth of this heterocystous cyanobacteria under aerobic conditions.
... We have recently been studying ways in which heterocysts are adapted to evolve 02, confirming an early report by Fay & Walsby (1966). This has been attributed to a lack of certain essential photosystem II accessory pigments (see Thomas, 1970 Heterocysts also photophosphorylate at rates which are several times higher than the rates of oxidative phosphorylation. ...
In this paper I wish to consider some aspects of the biology of blue-green algae which have caught my attention over the past few years. Some of the material is new, much of it is not, and a lot of it is a resumé of some work carried out in my laboratory over the last decade. It complements some excellent work being carried out in other laboratories both in Europe and in the United States (see Fogg et al., 1973; Carr & Whitton, 1973; Wolk, 1973). I have called these photosynthetic, 02-evolving, prokaryotes blue-green algae or cyanophytes, although Stanier et al. (1971) have recently rekindled the early view (Cohn, 1853) that they may be best classified as bacteria (cyanobacteria). I do not intend to enter such a controversy, but it would be inappropriate at this anniversary meeting of the British Phycological Society to call them anything other than algae or cyanophytes.
... We examined whether the Mehler reaction might serve as an important sink for O 2 in the heterocystic cyanobacterium A. flos-aquae and whether this activity is independent of PSII. Purified heterocysts have been shown to have increased O 2 -consumption rates in the dark relative to whole filaments (Fay and Walsby 1966, Smith et al. 1985 ), but light-dependent O 2 consumption has not been investigated in purified hetereocysts . We measured oxygen consumption rates in heterocysts purified from six independent cultures and observed that 16 O 2 -and 18 O 2 -consumption rates were equal and were enhanced 3-fold (dark rate = 0.17 ± 0.06 lmol O 2 AE mg chl a )1 AE min )1 ; light rate = 0.51 ± 0.11 lmol O 2 mg AE chl a )1 AE min )1 ; P < 0.001, Student's t-test) in the presence of light. ...
All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O2 through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O2, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O2. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer 18O2 and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O2 consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos-aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O2 concentrations are maintained at low levels by the light-dependent reduction of oxygen via the Mehler reaction. In N2-fixing Trichodesmium colonies, Mehler activity can consume ∼75% of gross O2 production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ∼10% of gross O2 production. Moreover, evidence for the coupling between N2 fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O2 consumption by 3-fold. Our results suggest that a major role for PSI in N2-fixing cyanobacteria is to effectively act as a photon-catalyzed oxidase, consuming O2 through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.
... High amounts of respiration are characteristic of heterocysts (Fay and Walsby, 1966). Respiration has two functions related to nitrogen fixation: removal of oxygen to maintain an anaerobic environment for nitrogenase and synthesis of ATP for N 2 reduction (Fay, 1992). ...
Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919TK and CBRH7919WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.
The name Cyanophyceae has recently been generally adopted for this class previously known as the Myxophyceae. Apart from the main feature that the cells lack true nuclei (see p. 1), they show other features which, with the bacteria, distinguish them from the Eucaryota (Echlin and Morris, 1965). The cell envelope in the Cyanophyceae consists of an outer sheath (capsule in the bacteria), an outer membrane and an inner one (present in gram + ve bacteria, but lacking in gram − ve) and a plasma membrane. The cell envelope is composed largely of mucopeptides built up from amino sugars (e.g. glucosamine) and amino acids (e.g. muramic, diaminopimelic), whereas the cell wall of other algae lack such amino constituents.
Chlorophyllous cells under atmospheric conditions photooxidize water, releasing O2 and simultaneously reducing CO2 to carbohydrate. This is a reaction unique to chlorophyll α-containing cells and is termed photosynthesis. The photosynthesis of green plants has long been considered a rigid process which can be enhanced or retarded by external conditions, but whose chemical metabolism is unalterable. This is not universally true. Indeed, Nakamura (1938) found that certain diatoms and blue-green algae could use H2S for the reduction of CO2. Recent reports (Stewart and Pearson, 1970; Cohen et al., 1975) showed that in contrast to water being the electron donor resulting in the evolution of O2, in blue-green algae H2S serves as an electron donor for CO2 photoassimilation.
In many filamentous cyanobacteria, oxygenic photosynthesis is restricted to vegetative cells, whereas N-2 fixation is confined to microoxic heterocysts. The heterocyst has an envelope that provides a barrier to gas exchange: N-2 and O-2 diffuse into heterocysts at similar rates, which ensures that concentrations of N-2 are high enough to saturate N-2 fixation while respiration maintains O-2 at concentrations low enough to prevent nitrogenase inactivation. I propose that the main gas-diffusion pathway is through the terminal pores that connect heterocysts with vegetative cells. Transmembrane proteins would make the narrow pores permeable enough and they might provide a means of regulating the rate of gas exchange, increasing it by day, when N-2 fixation is most active, and decreasing it at night, minimizing O-2 entry. Comparisons are made with stomata, which regulate gas exchange in plants.
Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO2 fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.
The chapter discusses a comparative review of the known mechanisms of inorganic nitrogen assimilation in free-living micro-organisms. The direct assimilation of nitrate to form first nitropropionic acid is known to occur in some fungi, but it is doubtful whether this or analogous systems, are of widespread significance in micro-organisms. The conversion of ammonia into glutamate is a key reaction in assimilation of inorganic nitrogen, whether the nitrogen source is molecular nitrogen, nitrate, or ammonia itself. The related enzyme glutamine synthetase has long been known to be subject to elaborate control mechanisms, and to be produced in greater quantities than would seem to be required, were its functions solely those known prior to the realization that this enzyme was involved in ammonia assimilation into cc-amino compounds. Blue-green algae are ubiquitous both in aquatic environments and soils, and are found in greatest abundance in tropical regions. Their ecological significance in relation to nitrogen fixation is especially established in aquatic habitats. Nitrogen fixation in extracts of blue-green algae requires for activity both ATP and a reducing system. Assimilatory nitrate reduction is catalyzed by at least two enzymes. Nitrate reductase reduces nitrate to nitrite, while nitrite reductase reduces nitrite to ammonia.
Heterocysts of autotrophically grown Anabaena cylindrica contain about 77% as much chlorophyll as do vegetative cells, have little phycocyanin, and appear to lack myxoxanthophyll. Heterocysts have a photosystem I. Lipids of the two cell types differ.
Heterocysts of Anabaena cylindrica, isolated rapidly in the cold, were found-in contrast to earlier reports-to contain all of the same lipids and lipophilic pigments, and in about the same proportions, as vegetative cells. In broken filaments and in heterocysts damaged during isolation, the membrane lipids and certain pigments (myxoxanthophyll and an unidentified red pigment) break down rapidly. The glycolipids specific to heterocyst-forming blue-green algae are localized in the laminated layer of the heterocyst envelope. A possible role of the laminated layer is discussed.
Enhancement of carbon fixation was demonstrated in the bluegreen alga, Anabaena cylindrica, grown in either aerobic or microaerobic conditions. Under identical conditions no enhancement of acetylene reduction was observed. Light absorbed by photosystem I supported relatively more acetylene reduction than carbon fixation. No competition between the two processes was observed under light-limiting conditions. The findings suggest that carbon fixation and acetylene reduction may depend on different pools of reductant and ATP. When aerobically grown cells were placed in the dark or at limiting light intensities, acetylene reduction was higher in air than under argon. In contrast, carbon fixation was lower in air than in argon.
Heterocysts of the cyanobacterium Anabaena flos-aquae retain gas vacuoles for several days after differentiation. It is demonstrated that the rate of gas diffusion into a heterocyst that is near an overlying gas phase can be determined approximately from observations on the rate of gas pressure rise required to collapse 50% of its gas vacuoles. The mean permeability coefficient (alpha ) of heterocysts to O2 and N2 was found to be 0.3 s-1. From this it was calculated that the average permeability (kappa ) of the heterocyst surface layer is about 0.4 mu m s-1 (within a factor of 2). This is probably within the range that could be provided by a few layers of the 26-C glycolipids in the heterocyst envelope. It is likely, but not proven, that the main route for gas diffusion is through the envelope rather than through the terminal pores of the heterocyst. From measurements of cell nitrogen content (2.7 pg), doubling time (3 days) and heterocyst: vegetative cell ratio (1:24) it was calculated that the average heterocyst fixed 5.9 × 10-18 mol N2 s-1; this must equal the diffusion rate of N2 into the heterocyst. This rate would be sustained by a concentration of N2 inside the average heterocyst that was 22% below the outside air-saturated concentration. The maximum N2 fixation rate allowed by the estimated permeability coefficient would be 2.7 × 10-17 mol s-1 per heterocyst, slightly greater than the maximum calculated N2 fixation rate. The observed permeability coefficient is low enough for the oxygen concentration in the heterocyst to be maintained close to zero by the probable rate of respiration, providing an anaerobic environment for nitrogenase. The rate of O2 diffusion will limit the N2-fixation rate in the dark by limiting the rate at which ATP is supplied by oxidative phosphorylation.
The present study gives evidence for the presence of cellulose in the heterocyst envelope of blue-green algae by means of electron microscopy, cellulase treatments and specific staining and demonstrates the role of this cellulose for the protection of the heterocyst nitrogenase during acetylene reduction. Experiments with lysozyme and cellulase suggest that nitrogen fixation in heterocystous blue-green algae under aerobic conditions is functionally effective only when an intimate relationship exists between vegetative cells and heterocysts and both cell types have intact wall structures.
Transient exposure to elevated temperature (37-40 °C) inhibited N2 fixation (acetylene reduction) in cultures of the heterocystous cyanobacterium Anabaena cylindrica (Lemmermann) ATCC 27899 and the unicellular, non-heterocystous cyanobacterium Gloeothece sp. (Nägeli) ATCC 27152. In neither organism was inhibition of N2 fixation due to thermal inactivation of nitrogenase. Rather, inactivation of N2 fixation at elevated temperature was a consequence of increased sensitivity to inhibition by O2 In A. cylindrica, thermally induced inactivation of N2 fixation by O2 could be correlated with inhibition of uptake hydrogenase. However, in Gloeothece, inhibition of carbon-supported respiratory O2 consumption by elevated temperature was the probable cause of the increased sensitivity of N2 fixation to O2.
The relationship between ATP and nitrogenase activity has been investigated using the obligate photoautotroph Anabaena cylindrica and Anabaenopsis circularis strain 6720, a photoautotroph which can also use exogenously supplied carbon. In the light photophosphorylation supports maximum observed rates of nitrogenase activity (0.60 and 1.2 μmoles C2H4 mg chl a−1 min−1 under aerobic and microaerobic conditions respectively). The ATP pools sustained under both conditions are similar at 160–200 μmoles mg chl a−1. Oxidative phosphorylation and substrate level phosphorylation are unimportant in supporting nitrogenase activity in the light. Cyclic photophosphorylation can supply all the necessary ATP provided fixed carbon compounds from photosynthetic CO2 fixation (Anabaena) or supplied exogenously (0.2%, w/v, fructose) (Anabaenopsis) are available. When the algae are placed in the dark nitrogenase activity declines. In Anabaenopsis, after an initial rapid drop, nitrogenase activity can be sustained in the presence of exogenous carbohydrate in the dark at up to 70% of the light rate under air, but not under anaerobic conditions. This decreased nitrogenase activity in the dark is not due to a lack of nitrogenase synthesis or of penetration of fixed carbon into the cells, but appears to be due to an inherent inability of dark metabolism to generate sufficient ATP and/or reductant for maximum nitrogenase activity. The ATP pool can be sustained at maximum levels when photophosphorylation, cyclic photophosphorylation, and oxidative phosphorylation operate individually, and to a lesser extent when substrate-level phosphorylation operates. No consistent correlations were found between the ATP pool and nitrogenase activity, except under dark anaerobic conditions where there was a positive correlation. The data suggest either that the ATP pool which supports nitrogenase activity is distinct from that which supplies ATP for general filament metabolism, and/or that the supply of ATP is not the most critical short-term factor in controlling nitrogenase activity in Anabaena and Anabaenopsis.
The enigmatic coexistence of O2-sensitive nitrogenase and O2-evolving photosynthesis in diazotrophic cyanobacteria has fascinated researchers for over two decades. Research efforts in the past 10 years have revealed a range of O2 sensitivity of nitrogenase in different strains of cyanobacteria and a variety of adaptations for the protection of nitrogenase from damage by both atmospheric and photosynthetic sources of O2. The most complex and apparently most efficient mechanisms for the protection of nitrogenase are incorporated in the heterocysts, the N2-fixing cells of cyanobacteria. Genetic studies indicate that the controls of heterocyst development and nitrogenase synthesis are closely interrelated and that the expression of N2 fixation (nif) genes is regulated by pO2.
The purpose of this article is not to review exhaustively all that is known about nitrogen-fixing microbes but to provide a reasonable coverage of more recent discoveries in the field. It is primarily concerned with the microbiology of the free-living nitrogen-fixing organisms, although casual reference to symbiotic systems has been made. With this in mind, I shall attempt not to burden the reader with microbiological minutiae but will restrict coverage to those biological aspects which help us understand how such a diverse assortment of microbes can grow with N2 gas as their sole nitrogen source. The discovery of the nitrogen-fixing character in a certain microorganism sometimes necessitates a reevaluation of its taxonomic position. Where necessary, this has been done.
There are several reports on the isolation of heterocysts However, there is no report on the factors reesponsible for maintaining the metabolic activity of isolated heterocysts. So the purpose of this work was to isolate metabolically active heterocysts from marine heterocystous cyanobacteria and study the factors that modify or maintain the activity of the isolated heterocysts
Isolated heterocysts of the N2-fixing Anabaena cylindrica, prepared by a combination of lysozyme and Yeda press treatments, are metabolically active with over 90% of the measurable nitrogenase activity being located in the heterocyst preparations after disruption of the intact filaments. The photosynthetic activities of such isolated heterocysts are characterized by an inability to carry out the photolysis of water or to fix CO2. The lack of O2 evolution appears to be due in part to the depletion during heterocyst differentiation of Mn, a central component of the photosystem II reaction centre in O2-evolving algae. There is evidence that components of the photosynthetic electron transport chain on the reducing side of the photosystem II reaction centre are present and functional in heterocysts. These include cytochrome c554, plastocyanin, plastoquinone, cytochrome b559, P700, cytochrome b563, and iron-sulphur proteins which appear to correspond to centre A and centre B of higher plant chloroplasts. Soluble, or loosely bound ferredoxin is also present and involved in electron transport from ferredoxin to NADP. Isolated heterocysts photoreduce methylviologen when reduced 2,6-dichlorophenolindophenol and diphenylcarbazide serve as electron donors. They show P700 photo-oxidation and photoreduction, photosynthetic electron transport which is inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone an antagonist of plastoquinone, photophosphorylation, oxidative phosphorylation and ferredoxin-NADP oxidoreductase mediated reactions. The photosynthetic modifications of the heterocyst are such that electron transport and the generation of ATP for nitrogenase can occur without concomitant O2 evolution and without nitrogenase having to compete with CO2 fixation for ATP and reductant.
The heterocysts of Anabaena cylindrica were freed from filaments by differential disruption of vegetative cells using four techniques: mechanical disruption by French press, sonication, osmotic shock and lysozyme. The ultrastructure of isolated heterocysts was compared with that of heterocysts in intact filaments. The first three methods produced heterocysts whose internal structure showed different degrees of damage, involving in particular disruption of the heterocyst cell wall and plasmalemma. Isolation by the lysozyme method yielded heterocysts which appeared in the electron microscope to be intact and comparable with those of the untreated controls. These results suggest that earlier reports on the physiological properties of heterocysts isolated by means of the French press or sonication may require re-examination.
The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O2 for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O2 tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.
Two mutants of Anabaena sp. strain CA were used to demonstrate that oxygen-dependent hydrogen uptake was not the primary means to protect the nitrogenase enzyme complex from the deleterious effects of hyperbaric oxygen in vivo. Exposure to air caused the immediate and irreversible inactivation of nitrogenase activity in an oxygen-sensitive mutant, designated strain 22Y. Inactivation was concomitant with the destruction of the molybdo-iron (MoFe) protein of the nitrogenase complex. The mutant 22Y expressed an O2-stable, Ni2+-stimulated hydrogen uptake of up to 2.7 μM H2 per mg dry wt per h. Conversely, after exposure to 1% CO2-99% O2 for 3 h, both wild-type strain CA and a hydrogen uptake deficient (Hup−) mutant, strain N9AR, recovered 70–80% of their original acetylene reduction capacity with no apparent perturbations in the MoFe protein.
Nitrogenase activity and CO2 fixation differed in two subarctic lichens, Peltigera aphthosa (L.) Willd. and Stereocaulon paschale (L.) Fr., after exposure to 10-8-10-4 M concentrations of atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-s-triazine), diuron (3-[3,4-dichlorophenyl]-1,1-dimethylurea), or ioxynil (4-hydroxy-3, 5-diiodobenzonitrile). Nitrogenase activity in P. aphthosa was stimulated at 4.7 x 10-7 M atrazine and 2.3 x 10-8 M ioxynil but not in S. paschale. Inhibitions of nitrogenase activity in P. aphthosa at the highest concentrations of diuron, atrazine, and ioxynil were 90%, 80%, and 30%, respectively. In S. paschale, high concentrations of ioxynil completely inhibited nitrogenase activity, while diuron caused 50% and atrazine 35% deactivation. Positive net carbon assimilation in P. aphthosa was found after exposure to 10-6-10-8 M atrazine or diuron, but in S. paschale, only at 10-8 M diuron. Increased respiration in light was observed in S paschale after exposure to diuron, ioxynil, and a...
Occurrence andlocalization ofanuptake hydrogenase wereexamind inthree strains ofthebluelgeen alga, ADbDeMCD InmvoH2uptake was detected (0.60-1.44 pnoles/jmg ofchlrophyll aperhouri) Inalthree strains when grown with N2aSthesole source ofnitroge H2uptake (in vivo andinvitro) wasseverely suppressed incultures grown onNH4'and lacking heterocysts. H2uptake Incell-free extracts could bereadfly measured withamethyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation ofaerobically grownAnabhae 7120Indicates thattheuptake hydrogenase islocazed solely inthe heterocyst. Whenthesameorganm isgrown onNs/CO%, vegetatve cels mayaccount forupto21%ofthetotal hydrogease activity Inthefilaments. Theresults arediscussed intermsofaproposed functional relatiship between nitrogenase andhydrogenase.
The study focuses on the research involved in generating hydrogen using algae as a renewable energy resource. Due to the decline in fossil fuel resource, the energy derived from biomass seems to be the only major source of world's renewable energy. The hydrogen derived from algae is promising due to its sustainability, no green house gases emission during the combustion of hydrogen and security of its supply even at remote places. The novel approach of generating hydrogen at commercial scale from algae has been a curiosity among many researchers till today. This review study updates the research involved in hydrogen generation from algae based on light intensity and its photoperiod, nitrogen and sulfur content, fermentative metabolism and symbiosis. The following algal species had been widely investigated for hydrogen production namely: Chlamydomonas, Anabaena, Chorella, Oscillatoria, Scenedesmus and their mutant. The generation of hydrogen from algae is still at research level. Hence, this review would be an eye opener for researchers who are interested in generating hydrogen from algae.
This chapter discusses the methods for heterocyst isolation. Heterocysts display characteristic structural and functional properties. They appear under the light microscope as round yellowish-green cells, with a relatively homogenous content, surrounded by a thick secondary envelope. Heterocysts lack a functional photosystem II, fix no CO2, and evolve no O2 when illuminated, though retain an active photosystem I capable of photophosphorylation. They also possess an elevated respiratory metabolism and can carry out oxidative phosphorylation. The biochemical transformation of heterocysts is apparently essential in the performance of their principal function of atmospheric N2 fixation. A method devised for the isolation of heterocysts was based on the selective mechanical disruption of vegetative cells in a French pressure cell. It was observed during the preparation of cell-free extracts from blue-green algae that heterocysts resisted breakage at a pressure which caused the disruption of all vegetative cells. It is generally concluded that methods involving powerful mechanical forces used to disrupt the vegetative cells inevitably injure heterocysts. Only heterocysts isolated after selective enzymic destruction of the vegetative cells preserve their integrity compared to those in untreated filaments. Though the lysozyme treatment generally preferable, other methods may be favored in cases where loss of soluble or fine granular heterocyst content is unimportant.
A description is given of a large glass vessel suitable for growing algae and other microorganisms in shaken culture. The main feature of the flask is the coneshaped base around which water circultes when the flask is shaken on a reciprocal mechanism.
The functional aspects of specific associations between bluegreen algae and bacteria were investigated using both naturally occurring and cultured species of Anabaena. In take waters where bacteria were associated with Anabaena heterocysls, the bacteria exhibited a chemotactic response to a variety of amino acids and glucose. Earlier autoradiographic evidence that bacteria associated with heterocysts incorporate identical substrates indicates that associated bacteria probably benefit by utilizing algal excretion products. In return, the bacteria stimulate algal N2fixation. The most likely mechanism explaining such stimulation appeared to be bacterial oxygen removal in microzones (< 3 μm diam) bordering heterocysts during periods of high ambient oxygen concentrations. In the presence of bacteria, Anabaena rapidly overcame nitrogenase- inhibiting concentrations of oxygen. Axenic cullures had more extensive nitrogenase inhibition, and took longer to recover in response to oxygenation. Algal-bacterial mutualism aids Anabaena in maintaining concurrent optimal N2 fixation and high photosynthetic rates in highly oxygenated surface waters.
Spore (akinete) differentiation in Anabaena fertilissima in accompanied by changes in the composition of allophycocyanin, phycocyanin and phycoerythrin. Phycoerythrin in transformed from C-type in vegetative cells to R-type in spores. The apparent disappearance/reduction in the amount of allophycocyanin/phycocyanin in spores is mainly due to the loss of chromophores (bilins) rather than due to the loss of the apoproteins.
Heterocyst-forming cyanobacteria simultaneously photosynthesize, producing oxygen (O2), and fix dini-trogen (N2), initially into ammonia, using nitrogenase enzymes that are rapidly inactivated by O2. These cyanobacteria enable nitrogenases to function in an oxic environment by segregating them within specialized cells,
called heterocysts, in which O2 is not produced, respiration is highly active, and an envelope barrier of glycolipids greatly slows the rate of entry of
O2. We will describe the chemical structure of the heterocyst-specific glycolipids (Hgls), their physiological role, and what
is known of their deposition. We will then discuss the clustered genes that encode the proteins required for their biosynthesis,
how the glycolipids are believed to be synthesized, and what is known of the regulation of their biosynthesis. Finally, we
will examine the relationship between their biosynthetic enzymes and other polyketide synthases, with an emphasis on those
from cyanobacteria.
Culture studies with 6 genera of Cyanophyceae were undertaken in order to throw some light on the morphological and physiological properties of ecologically differing types.
Pseudanabaena turned out to be a good genus distinguishable from Anabaena in various ways.
Borzia, described 85 years ago but almost forgotten was discovered again and found like Pseudanabaena not to fit into the current taxonomical system of Cyanophyceae.
In the highly differentiated Cylindrospermum the morphogenetic influence of nitrogen supply proved to be particularly strong and distinct.
A Synechococcus of the size and appearance of a small bacterium exhibited strict mixotrophy, a mode of nutrition only recently discovered for the first time among Cyanophyceae in Lauterbornia (Anacystis) nidulans.
In a Merismopedia a behaviour of dividing cells was observed which caused a deviation from the development of the tabular colonies in a flat plain.
Founded on observations in nitrogenbinding Cyanophyceae the hypothesis was put forward that heterocysts are indispensable for the molecular nitrogen to be transformed into compounds for the metabolism of the organism.
The mode of locomotion in Cyanophyceae was analysed and discussed anew with respect to the special kind of motility found in filaments of Pseudanabaena where it recalls that of Chroococcaceae.
Simultaneous measurements of acetylene reduction by Anabaena variabilis and the concentration of dissolved oxygen in the suspension were made using a specially designed vessel which allowed measurements under steady-state conditions. The rate of acetylene reduction in the dark increased with increasing oxygen concentrations until a maximum value was reached at 300 M O2 (corresponding to 30% O2 in the gas phase at 35C). This presumably results from a requirement for energy provided by respiration. Measurements of the dependence of respiration rate on dissolved oxygen concentration were made under comparable conditions using an open system to allow conditions close to steady-state to be obtained. The respiration rate of diazotrophically grown Anabaena variabilis had a dependence on oxygen concentration corresponding to the sum of two activities. These had K
m values of 1.0 M and 69 M and values of V
max of similar magnitude. Only the high affinity activity was observed in nitrate-grown cyanobacteria lacking heterocysts, and this presumably represent activity in the vegetative cells. The oxygen concentration dependence of the low affinity activity resembled that for the stimulation of acetylene reduction. We interpret this as the result of oxygen uptake by the heterocysts. The results are consistent with the idea that in intact filaments of cyanobacteria O2 enters heterocysts much more slowly than it enters the vegetative cells.
A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (max 615 nm) and allophycocyanin (max 645 nm, shoulder 620 nm). Isolated heterocysts under H2 showed acetylene reduction rates of 57 nmol C2H4/mg dry wtmin (342 mol C2H4/mg chl ah), whereas intact filaments reduced at the rate of 18 nmol C2H4/mg dry wtmin (108 mol C2H4/mg chl ah). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H2 for more than 3 h. Addition of as little as 5% H2 under argon stimulated the C2H2 reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O2 either under H2 or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C2H2 reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.
The ultrastructure of the heterocyst and its development from the vegetative cell is described. The ultrastructure of the akinete is also described. The “mature” heterocyst (still attached to the filament) has an elaborate structure which is distinct from both that of the normal vegetative cell and the akinete (the normal reproductive cell).
Despite the extra structural detail seen in the electron micrographs, the observations do not indicate a likely physiological role for the heterocyst. However, since the developing-mature heterocyst has an organised structure, it is probable that it has an active metabolism. In contrast, the detached heterocyst has a highly disorganised structure and, for this reason, it is likely to be metabolically inactive and incapable of germination.
- GE Fogg