Nitrogen fixation and heterocysts in the blue-green alga Anabaena cylindrica

The site of nitrogen fixation in the blue-green alga Anabaenacylindrica Lemra (Fogg strain) was investigated. Less than 4%of the total nitrogen fixed during a relatively short period(5-15 min) was recovered in heterocysts. When estimated on thecellular nitrogen basis, vegetative cells can fix molecularnitrogen at the same rate as do heterocysts. There was no positivecorrelation between nitrogen fixation and heterocyst formation.Results do not support the hypothesis that the heterocyst isthe main site for nitrogen fixation in blue-green algae. ¹ This work was supported by grant (No. 38814) from the Ministryof Education. (Received July 23, 1971; )     

Acetylene reduction by nitrogen-fixing blue-green algae

Summary Known nitrogen-fixing species of blue-green algae are capable of reducing acetylene to ethylene, but acetylene is not reduced by Anacystis nidulans, which does not fix nitrogen. Cycad root nodules which contain blue-green algae as endophytes reduce acetylene. Acetylene reduction is inhibited by carbon monoxide. Nitrate or ammonium-nitrogen has no immediate effect on algae reducing acetylene, but algae grown on nitrate-nitrogen gradually lose their capacity to reduce acetylene. Nitrate-nitrogen also inhibits heterocyst formation in these algae and there is a fairly direct correlation between the abundance of heterocysts in a particular sample and its capacity to reduce acetylene. Aphanizomenon flosaquae reduces acetylene and fixes nitrogen in unialgal culture and there is strong presumptive evidence that these reductions are carried out by the alga rather than by associated bacteria. The molar ratios of ethylene: ammonia produced vary within the range 1.4–1.8.     

THE HETEROCYSTS OF BLUE-GREEN ALGAE (MYXOPHYCEAE)

1. Heterocysts are found in many species of filamentous blue-green algae. They are cells of slightly larger size and with a more thickened wall than the vegetative cells. 2. Structural details of the heterocyst are: the presence of three additional wall layers, the absence of granules, sparse thylakoid network throughout, except at the poles where a dense coiling of membranes occurs. Other characters include the two pores at opposite poles ‘plugged’ with refractive material called the polar granule. 3. Peculiarities in the pigment composition of the heterocyst include an abundance of carotenoids and absence of phycobilins, and a short-wave form of chlorophyll a. 4. Unique glycolipids and an acyl lipid, not found in the vegetative cells of the algae or in other plant cells, are associated with the heterocyst. The glycolipids constitute the laminated layer of the wall and probably regulate diffusion of substances through it, whereas the acyl lipids are supposed to function as carriers and intermediates in the biosynthesis of the wall. 5. The heterocysts develop from vegetative cells, and the visible changes during differentiation include cell enlargement, synthesis of additional wall layers, disappearance of granules and reorientation and synthesis of the thylakoids. 6. Heterocysts are formed sequentially with characteristic cellular spacing during the growth of cultures in medium free from combined nitrogen. 7. Various sources of combined nitrogen inhibit heterocyst formation when supplied in the culture medium. Ammonium salts are among the most powerful inhibitors. Heterocysts are formed simultaneously and within a short period after transference of ammonia-grown non-heterocystous filaments to ammonia-free medium. 8. Incompletely differentiated heterocysts or proheterocysts are found in cultures grown in the presence of combined nitrogen. If two or more proheterocysts are close together generally a single one develops to maturity after a competitive interaction in medium free from combined nitrogen. This indicates that heterocyst formation is completed in two phases: phase I, synthesis and conservation of macromolecules, which takes place during growth in ammonia-containing medium: and phase 11, morphological differentiation of the heterocyst which is unaccompanied by growth in cell number. In the ammonia-free medium phase 11 quickly succeeds phase 1 and the whole process appears as a continuum. 9. Heterocyst formation shows a definite requirement for light. Red light favours heterocyst formation, whereas green and blue light do not. The effects of light seem to be mainly due to photosynthesis, although some effects may be morphogenetic. 10. Studies with metabolic inhibitors have revealed the involvement of photosynthesis, respiration and protein synthesis in heterocyst formation. Photosynthesis provides carbon skeletons, whereas ATP is most probably supplied by oxidative metabolism. 11. Various functions have been assigned to the heterocyst from time to time. Their role in akinete formation is suggested by (i) the formation of akinetes adjacent to the heterocysts and (ii) prevention of sporulation by detachment of the heterocysts from the vegetative cells (potential akinetes). Despite substantial evidence for such a role, it is not applicable to all akinete-forming genera. 12. Heterocysts are now widely believed to be the site of nitrogen fixation in blue-green algae. The main facts in favour of such a role are: (i) fixation of nitrogen by all heterocystous algae, (ii) inhibition of heterocyst formation by combined nitrogen and (iii) direct observations on acetylene reduction by isolated heterocysts. 13. Some non-heterocystous and unicellular algae, and vegetative cells of heterocystous algae fix nitrogen under microaerophilic conditions suggesting that absence of oxygen favours nitrogenase activity. Heterocysts lack the oxygen-evolving photo-system 11, possess oxidative enzymes, and reduce externally supplied tetrazolium salts - all indicating that they are the most suitable sites for harbouring nitrogenase in aerobic conditions. 14. Heterocysts probably originated in the Precambrian in response to the earth's changing environment and seem to be the first example of morphological differentiation in the plant kingdom.     

Components and activity of the photosynthetic electron transport system of intact heterocysts isolated from the blue-green alga Nostoc muscorum

Heterocysts of the blue-green alga Nostoc muscorum have been isolated by prolonged treatment with lysozyme. Quantitative data are presented which show the occurrence of cytochromes c-553, f-557 and b-563 in heterocysts in amounts comparable to vegetative cells. Particularly the content of the water-soluble cytochrome c-553 can be used to evaluate the intactness of a heterocyst preparation. Cytochrome f-557 has been partially purified and found to be a c-type cytochrome corresponding to cytochrome f of higher plants and other algae. Cytochrome b-559 is present in vegetative cells but not in heterocysts. The content of plastoquinone in heterocysts is reduced to 42% of the amount present in vegetative cells. These data suggest a degradation of Photosystem II during heterocyst differentiation. Measurements of photosynthetic electron transport in heterocysts proved the inability of the photosynthetic apparatus to carry out electron transport with electrons donated by water or diphenylcarbazide. In Tris-washed thylakoids from vegetative cells, however, diphenylcarbazide can act as an electron donor to Photosystem II.     

Action of 2,4-dichlorophenoxyacetic acid and rifampicin on heterocyst differentiation in the blue-green alga,Nostoc linckia

2,4-Dichlorophenoxyacetic acid, a commonly used herbicide, increased the growth of the filamentous blue-green alga,Nostoc linckia at doses upto 100 μg /ml. The herbicidetreated N₂-cultures showed enhanced heterocyst frequency and N₂-growth. Thus, the herbicide stimulated algal growth at the expense of molecular nitrogen under aerobic growth conditions. Rifampicin caused chain formation of heterocysts. This was effectively counteracted by 2,4-dichlorophenoxyacetic acid, suggesting a biological interaction between them at the level of the heterocyst spacing control mechanism.     

Effect of ammonia and sulfide on rifampicin-induced heterocyst formation inNostoc linckia

The effect of ammonia and sulfide on rifampicin-induced heterocyst differentiation was studied in the nitrogen-fixing cyanobacteriumNostoc linckia. Aerobic growth with nitrogen gas of the cyanobacterium was greatly affected by rifampicin with formation of multiple heterocysts in chains in the filaments whereas ammonia in the medium reversed the rifampicin inhibition of growth and prevented the induction of heterocysts. In a sulfide medium the suppression exerted by rifampicin on aerobic growth with nitrogen gas and heterocyst induction was found to be considerably reduced. The results suggest two interesting points,viz. that (i) rifampicin interferes with the nitrogen-fixing function of heterocysts, and (ii) it checks the synthesis of an unknown heterocyst, inhibitor and thus permits the adjacent vegetative cells to differentiate into heterocysts in chains.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Ascorbic Acid and Heterocyst Development in the Blue–Green Alga Amabaema ambigua

The nitrogen–fixing blue–green alga Anabaena ambigua was grown in a medium which contained either ammonium chloride as nitrogen source or molecular nitrogen. In the latter case the alga produced heterocysts. The material was analysed for ascorbic acid, dehydro-ascorbic acid and diketogulonic acid. The amount of a,scorbic acid was found always to he higher in the alga grown with molecular nitrogen. When the alga grown with combined nitrogen was transferred to the medium lacking it, there was an increase in the ascorbic acid content. Conversely, when material cultured on the nitrogen–free medium was suspended in the medium containing ammonium chloride, there was a decrease in the cellular ascorbic acid. Esogenously added ascorbic acid, up to 0.5 mg per ml, increased the heterocyst frequency to nearly three times that of the control. D–isoascorbic acid, an analogue of ascorbic acid, also showed an enhancement of heterocyst production. Algal extracts were fractionated by poiyacrylamide electro–phoresis, and the presence of ascorbic acid oxidase was detected on the gels. Two bands, with Rf values 0.34 and 1.0, were found to give positive test: for the enzyme. The total enzyme activity was 16.7 % higher in cells grown with molecular nitrogen than in those grown with combined nitrogen. The exact location of the enzyme in the alga ist not known although the heterocysts were earlier shown to contain ascorbic acid. Cytochemical tests, however, indicated strong per–oxidase activity in the heterocysts.     

Some characteristics of two morphological mutants of Nostoc linckia induced by nitrosoguanidine.

The blue-green alga Nostoc linckia was treated with nitrosoguanidine and two classes of morphological mutant clones were isolated. One class shows certain abnormal phenotypic features of vegetative cells, spores, and heterocysts. It has increased heterocyst frequency and impaired growth rate. The other class exhibits an altered heterocyst spacing pattern. Both classes of mutants have reduced nitrogenase activity.     

The Presence of Cellulose in Heterocyst Envelopes of Blue-Green Algae and its Role in Relation to Nitrogen Fixation

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.     

Mutagenesis of the blue-green alga,Anabaena doliolum Bharadwaja

Mutagenesis in the blue-green alga, Anabaena doliolum Bharadwaja has been investigated with particular reference to N₂ fixation. Several types of mutant have been isolated after induction with UV, NG, acridine orange and acriflavine. From a comparative characterization it is concluded that the heterocyst is not the sole site of N₂ fixation. There does not appear to be a linkage between N₂ fixation and heterocyst or spore differentiation: they seem to be independent processes probably regulated either by different genes or by a single regulatory gene with independent operons. A common genetic determinant has also been suggested for nitrogenase and nitrate and nitrite reductases.     

Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena

Filamentous, N₂‐fixing, heterocyst‐forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ–GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild‐type Anabaena, were notably enlarged in the SepJ‐overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ‐overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ‐related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ‐overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation.     

Some Observations on the Pattern of Sporulation in a Blue-green Alga, Anabaena doliolum

Sporulation in Anabaena doliolum begins in the middle of thetwo heterocysts and proceeds towards the heterocystous ends.Two inorganic nitrogen sources—potassium nitrate and ammoniumchloride inhibit sporulation, whereas glucose promotes it. Duringsporulation, the reductive ability of the heterocyst graduallydiminishes. It is concluded that spore differentiation in this alga is controlledby critical levels of nitrogen and of sugar in the cell. Thecritical levels are probably regulated by the heterocyst.     

Action of 2,4-Dichlorophenoxyacetic Acid onNostoc linckia: Impact of Glucose and Tryptophan

2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated growth and heterocyst differen- tiation ofNostoc linckia in nitrogen-free medium at lower concentrations (100 μ.g/mL) while its higher concentrations inhibited both processes and 1500 μg/mL proved to be lethal. Dry mass and specific growth rate of the alga declined with increasing concentration of 2,4-D in the range of 100–1500 μg/mL. Glucose slightly increased the heterocyst frequency without any lag in their differentiation. Tryptophan promoted growth of the alga, and formation of heterocysts (nearly three-fold). Tryptophan (50 μg/mL) complex medium with 1 mg 2,4-D per mL did not produce mature heterocysts. The filaments were fragmented at the point of hererocyst development and detached heterocysts germinatedin situ. Glucose and tryptophan protected the alga, its growth and heterocyst differentiation even at the lethal concentration of the herbicide. We are grateful to the Head, Department of Botany,Banaras Hindu University, Varanasi, for providing the necessary facilities. The first author is also grateful to the Principal,K.D. College, Kutir-Chakkey, Jaunpur, for his interest in this study.     

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.     

Chromatic adaptation and photoreversal in blue-green algaCalothrix clavata West

Complementary chromatic adaptation, a well-established phenomenon in some blue-green algae, has been observed inCalothrix clavata, a heterocystous blue-green alga of the family Rivulariaceae. The chromatic adaptation has been observed for fluorescent and incandescent light by measuring the absorption spectra. The material grown in fluorescent light forms more of phycoerythrin whereas more of phycocyanin tends to be formed in incandescent light. Besides this, photoreversal was observed by transferring the incandescent light grown alga to fluorescent light conditions and vice-versa. Effect of photoreversal and chromatic adaptation has also been discussed for this alga under different monochromatic light conditions. The influence of different light conditions on morphological changes, heterocysts and hormogonia formation has also been investigated. Both chromatic adaptation and photomorphogentic phenolmena in this alga show the involvement of some photoreversible (red:green) pigment.     

The hetZ gene indirectly regulates heterocyst development at the level of pattern formation in Anabaena sp. strain PCC 7120

Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen‐fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein–protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.     

The Ultrastructure of a Cyanophage Attack on Anabaena variabilis

Cyanophages multiplying on the nitrogen fixing blue-green alga Anabaena variabilis Kütz. were revealed by electron microscopy. Severe ultrastructural changes have been observed in the vegetative cells, whereas the heterocysts appeared resistant to the cyanophage. A lytic cycle was observed from adsorption to lysis.