Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum

Evident differentiation of vegetative cells into hetero-cysts in Anabaena sp. strain PCC 7120 is prevented by Insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild-type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored, in contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Observation of microplasmodesmata in both heterocyst-forming and non-heterocyst forming filamentous cyanobacteria by freeze-fracture electron microscopy

Structures which may establish cytoplasmic continuity between adjacent cells of filamentous cyanobacteria have been observed by freeze-fracture electron microscopy. They are visible in the septum region of the plasma membrane as pits on the E-face (EF) and corresponding protrusions on the P-face (PF). Between 100 and 250 of these structures, termed microplasmodesmata, were present between adjacent vegetative cells in all four strains of heterocyst-forming filamentous cyanobacteria, Anabaena cylindrica Lemm, A. variabilis (IUCC B377), A. variabilis Kütz. (ATCC 29413) and Nostoc muscorum, examined. Only 30–40 microplasmodesmata were observed between adjacent cells in two species, Phormidium luridum and Plectonema boryanum, that do not form heterocysts. The results suggest that in species that form heterocysts a greater degree of cytoplasmic continuity is established, presumably to facilitate the exchange of metabolites. In species capable of forming heterocysts, the number of microplasmodesmata per septum between two adjacent vegetative cells remained constant whether the filaments were grown in the presence of NH₄ and lacked heteroxysts or under N₂-fixing conditions and contained heterocysts. When a vegetative cell differentiates into a heterocyst, about 80% of the existing microplasmodesmata are destroyed as the poles of the cell become constricted into narrow necks leaving smaller areas of contact with the adjacent vegetative cells.     

Distribution of nitrogen-fixing cyanobacteria (Nostocaceae) during rice cultivation in fertilized and unfertilized paddy fields

This study describes the development of nitrogen-fixing cyanobacteria (Nostocaceae) during different stages of rice seedlings plantation in fertilized and unfertilized paddy fields in north Bihar, India. A heterogeneous population of cyanobacteria was randomly harvested around day 20, 40 and 60 of rice seedlings plantation, and the diversity was analyzed. Thirtytwo species (7 genera) were identified from unfertilized fields, of which Anabaena was represented by 12 species, Anabaenopsis and Aulosira by 3 each, Cylindrospermum by 4, Nostoc by 8 and Aphanizomenon and Nodularia by 1 species each. However, fertilized fields contained only 25 species (7 genera), of which 8 each belonged to Nostoc and Anabaena , 3 to Cylindrospermum , 2 to each Anabaenopsis and Aulosira and 1 to each Aphanizomenon and Nodularia . Although Nostoc and Anabaena were dominant in both fertilized and unfertilized paddy fields, a marked decrease in nitrogen-fixing cyanobacteria was recorded from fertilized fields. In both treatments, the diversity of nitrogen-fixing cyanobacteria was at a maximum around day 60 after seedling plantation. The current study concludes that there is a negative effect of nitrogenous fertilizers on the development of heterocystous cyanobacteria in rice fields. It is proposed that early appearing efficient nitrogen-fixers should be used as nitrogen fertilizers in the management for better establishment and exploitation of heterocystous cyanobacteria for sustainable agricultural practices.     

A NUMERICAL TAXONOMIC STUDY OF NOSTOC AND ANABAENA1

Thirty characteristics of 14 Nostoc and 10 Anabaena species were analyzed from previously published data. Using standard numerical taxonomic methods, simple matching coefficients were calculated and a phenogram drawn. The analysis revealed that some of the central characteristics of Nostoc are: a punctiforme stage; motile reproductive stage; plant mass with a dull to shiny luster, non-veined surface, and nonfimbriate margin; some spherical vegetative cells; no cylindrical heterocysts; and some spherical, but no cylindrical akinetes. Some of the central characteristics of Anabaena that were revealed are: no punctiforme stage; a motile vegetative stage; plant mass with a shiny luster, veined surface, and fimbriate margin; no spherical vegetative cells; some cylindrical heterocysts; and some cylindrical, but no spherical, akinetes. In general, Anabaena has larger akinetes and vegetative cells than Nostoc. Based on 30 morphological characteristics and the clustering data of the phenogram, keys were constructed for the Nostoc and Anabaena species studied. The data clearly support two separate and distinct, though similar genera and, less sharply, the separation of the 24 species. The more useful characteristics for separation of the species are size and shape of akinetes, vegetative cells, and heterocysts; color and luster of plant mass; veined plant mass surface; margin fimbriate; and shape of plant mass in nature.     

The fatty acid composition of akinetes, heterocysts and vegetative cells in Anabaena cylindrica

The fatty acid composition of akinetes, heterocysts and vegetativecells in Anabaena cylindrica was examined. Akinetes and heterocystscontained much less -linolenic acid than did vegetative cells.Furthermore, akinetes and heterocysts contained fatty acidswith less unsaturation as compared with vegetative cells. (Received February 19, 1972; )     

2-Methylhopanoids are maximally produced in akinetes of Nostoc punctiforme: geobiological implications

2-Methylhopanes, molecular fossils of 2-methylbacteriohopanepolyol (2-MeBHP) lipids, have been proposed as biomarkers for cyanobacteria, and by extension, oxygenic photosynthesis. However, the robustness of this interpretation is unclear, as 2-methylhopanoids occur in organisms besides cyanobacteria and their physiological functions are unknown. As a first step toward understanding the role of 2-MeBHP in cyanobacteria, we examined the expression and intercellular localization of hopanoids in the three cell types of Nostoc punctiforme : vegetative cells, akinetes, and heterocysts. Cultures in which N. punctiforme had differentiated into akinetes contained approximately 10-fold higher concentrations of 2-methylhopanoids than did cultures that contained only vegetative cells. In contrast, 2-methylhopanoids were only present at very low concentrations in heterocysts. Hopanoid production initially increased threefold in cells starved of nitrogen but returned to levels consistent with vegetative cells within 2 weeks. Vegetative and akinete cell types were separated into cytoplasmic, thylakoid, and outer membrane fractions; the increase in hopanoid expression observed in akinetes was due to a 34-fold enrichment of hopanoid content in their outer membrane relative to vegetative cells. Akinetes formed in response either to low light or phosphorus limitation, exhibited the same 2-methylhopanoid localization and concentration, demonstrating that 2-methylhopanoids are associated with the akinete cell type per se . Because akinetes are resting cells that are not photosynthetically active, 2-methylhopanoids cannot be functionally linked to oxygenic photosynthesis in N.   punctiforme .     

Antimicrobial effects of Cellvibrio on blue-green algae

Summary The culture fluids from two Cellvibrio strains, in the stationary phase of growth, are shown to contain heat-resistant, low molecular weight substances with antibiotic-like effects on blue-green algae. Morphological changes and lysis of cells were observed in various species of blue-green algae; ultrastructural changes were noted in the cell walls of growing vegetative cells of Anabaena inaequalis. The viability of resting cells, including heterocysts and akinetes was not affected.     

Surface lectin binding toAnabaena variabilis and to cultured and freshly isolatedAnabaena azollae

Five fluorescein isothiocyanate (FITC)-labeled lectins and Calcofluor white ST were tested for their binding abilities to vegetative cells and heterocysts of culturedAnabaena variabilis (AVA) and toA. azollae from four species ofAzolla; toAnabaena azollae freshly isolated fromAzolla pinnata (AP),A. caroliniana (AC),A. mexicana (AX), andA. filiculoides (AF); and to cultured akinetes ofAnabaena variabilis and four isolates ofA. azollae. Heterocysts of cultured cells of threeAnabaena isolates (APC, ACC, AXC) were most intensively surface-stained with soybean agglutinin fromGlycine max (SBA)-FITC; those of AX and AC were dimly stained with wheat germ agglutinin fromTriticum vulgaris (WGA); and only heterocysts of AX were dimly stained withDolichos biflorus agglutinin (DBA). Akinetes of cultured cells stained only with ConA. Vegetative cells and heterocysts of all four fresh isolates stained with Jack Beam aglutinin fromCanavalia ensiformis (ConA). None of the cell types were stained with either peanut agglutinin fromArachis hypogea (PNA) or Calcofluor.     

Endophyte transmission and activity in the Anabaena-Azolla association

Summary The survival of Azolla was studied in an artificial system which simulated the soil/water interface and the desiccation of soil during a fallow period in lowland rice culture. Tests with non-sporulating and sporulating Azolla fronds showed that Azolla only survives with sporulated fronds. At their reappearance the Azolla fronds already harboured the Anabaena endophyte. A detailed light microscopic and transmission electron microscopic study of macro- and micros-porocarp formation and development revealed that the endophyte is transmitted by the macrosporocarps and not by the microsporocarps. The Anabaena cells within the macrosporocarps are found just below the indusium cap. These cells are not nitrogen-fixing akinetes. The free-living Anabaena cells at the stem apex and below the overarching developing leaves do not bear heterocysts and accordingly are non nitrogen-fixing. During the development of the leaf the Anabaena enters the leaf cavity, but later the pore of this, cavity closes and the imprisoned cyanobacteria are lysed before the leaf decays. As the Azolla leaves age a nitrogen-fixing capability is successively built up concomittantly with the production of heterocysts. Heterocyst frequencies of 40–50% can be found inAnabaena azollae. Usually a gradient of nitrogen-fixing capacity occurs along the Azolla rhizome with two distinct peaks at leaf number 7/8 and at leaf number 13/14 from the apex.     

Immuno-gold localization of glutamine synthetase in a nitrogen-fixing cyanobacterium (Anabaena cylindrica)

Localization of glutamine synthetase in thin sections of nitrogen-fixing Anabaena cylindrica was performed using immuno-gold/transmission electronmicroscopy. The enzyme was present in all of the three cell types possible; vegetative cells, heterocysts and akinetes. The specific gold label was always more pronounced in heterocysts compared with vegetative cells, and showed a uniform distribution in all three types. No specific label was associated with subcellular inclusions such as carboxysomes, cyanophycin granules and polyphosphate granules. When anti-glutamine synthetase antiserum was omitted, no label was observed.Abbreviation GS glutamine synthetase     

APPLICATION OF GAS-LIQUID CHROMATOGRAPHY TO STUDY OF THE ENVELOPE LIPIDS OF HETEROCYSTS12

Gas-liquid chromatography of algal extracts provides a sensitive assay for their content of 1-(O-α-d -glycopyranosyl)-3, 25-hexacosanediol, the principal glycolipid from the envelope of heterocysts of Anabaena cylindrica. Different patterns of lipids were found in the heterocysts of 5 species of Anabaena and Nostoc. Lipids with comparable chromatographic properties were detected in Gloeocapsa.     

POLYSACCHARIDES FROM THE ENVELOPES OF HETEROCYSTS AND SPORES OF THE BLUE-GREEN ALGAE ANABAENA VARIABILIS AND CYLINDROSPERMUM LICHENIFORME1

The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.     

Morphological and habitat evolution in the Cyanobacteria using a compartmentalization approach

A phylogenomic approach was used to study the evolution of traits in the Cyanobacteria. A cyanobacterial backbone tree was constructed using multiple concatenated sequences from whole genome sequences. Additional taxa were added using a separate alignment that contained morphological characters, SSU (small subunit) and LSU (large subunit) rDNA, rpoC, rpoD, tufA, and gyrB genes. A compartmentalization approach was then used to construct a robust phylogeny with resolved deep branches. Additional morphological characters (e.g. unicellular or filamentous growth, presence or absence of heterocysts) were coded, mapped onto the backbone cyanobacterial tree, and the ancestral character states inferred. Our analyses show that the earliest cyanobacterial lineages were likely unicellular coccoid/ellipsoidal/short rods that lived in terrestrial/freshwater environments. Later cyanobacterial lineages independently gained the ability to colonize brackish, marine, and hypersaline environments while acquiring a large number of more complex traits: sheath, filamentous growth, nitrogen fixation, thermophily, motility, and use of sulphide as an electron donor. Many of these adaptations would have been important in the appearance of dense microbial mats early in Earth's history. Complex traits such as hormogonia, heterocysts, and akinetes had a single ancestor. Within the Nostocales, hormogonia and heterocysts arose before akinetes.     

NanoSIMS 50 elucidation of the natural element composition in structures of cyanobacteria and their exposure to halogen compounds

Aims: NanoSIMS (secondary ion mass spectrometry) is a powerful technique for mapping the elemental composition of a variety of small-scale samples (e.g. in Material Research, Cosmochemistry and Geology). However, its analytical features are making it also valuable to address biological questions. We demonstrate the ability of the NanoSIMS 50 to map elements at subcellular lateral resolution (approx. 50 nm) within cyanobacteria (Anabaena sp. and Cylindrospermum alatosporum) and its feasibility to investigate the uptake of bromine-containing substances (NaBr and deltamethrin). Methods and Results: Elemental maps of O, N, P and S were obtained from semi-thin sections of different cell types (chemically fixed and resin-embedded heterocysts, akinetes and vegetative cells). NanoSIMS enabled the detection of various characteristic cell sub-structures and inclusions. A homogenous bromine distribution was detected following NaBr and deltamethrin exposure, at Br-concentrations of 0·05, 0·5 (NaBr) and 0·0025 mmol l⁻¹ (deltamethrin). Conclusions: NanoSIMS allowed study of the mapping of common elements in cyanobacterial cells and the uptake of NaBr and deltamethrin. Significance and Impact of the Study: These results highlight the potential usefulness of NanoSIMS analysis for tracking elements within cell structures at the nanoscale and the ability to detect marker elements of xenobiotic compounds within exposed organisms.     

Superoxide dismutase in vegetative cells, heterocysts and akinetes of Anabaena cylindrica Lemm

Abstract: Superoxide dismutase (SOD) activity was assayed in vegetative cells, heterocysts and akinetes of Anabaena cylindrica Lemm. The iron-containing isoenzyme (Fe-SOD) was in all cases predominant over the manganese-containing isoenzyme (Mn-SOD). Differentiated cells maintained the same relative content of the two enzymes as in vegetative cells. However, heterocysts and akinetes contained only 20 and 35%, respectively, of the total SOD activity present in vegetative cells.
Both Mn-SOD and Fe-SOD activities increased in all types of cells isolated from A. cylindrica grown at high light intensity. The increase of SOD in heterocysts paralleled that of nitrogenase, suggesting a role of SOD in the protection mechanism of nitrogenase.     

The Anabaena sp. strain PCC 7120 asr1734 gene encodes a negative regulator of heterocyst development

The novel asr1734 gene of Anabaena (Nostoc) sp. strain PCC 7120 inhibited heterocyst development when present in extra copies. Overexpression of asr1734 inhibited heterocyst development in several strains including the wild type and two strains that form multiple contiguous heterocysts (Mch phenotype): a PatS null mutant and a hetR(R223W) mutant. Overexpression of asr1734 also caused increased nblA messenger RNA levels, and increased loss of autofluorescence in vegetative cells throughout filaments after nitrogen or sulphur depletion. Unlike the wild type, an asr1734 knockout mutant formed 5% heterocysts after a nitrogen shift from ammonium to nitrate, and formed 15% heterocysts and a weak Mch phenotype after step-down to medium lacking combined nitrogen. After nitrogen step-down, the asr1734 mutant had elevated levels of ntcA messenger RNA. A green fluorescent protein reporter driven by the asr1734 promoter, P(asr1734)-gfp, was expressed specifically in differentiating proheterocysts and heterocysts after nitrogen step-down. Strains overexpressing asr1734 and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show normal patterned upregulation 24 h after nitrogen step-down even though hetR expression was upregulated at 6 h. Apparent orthologues of asr1734 are found only in two other filamentous nitrogen-fixing cyanobacteria, Anabaena variabilis and Nostoc punctiforme.     

Nitrogenase activity of blue-green algae on seasonally flooded soils in Bangladesh

Blue-green algal communities formed an extensive cover on soils at five deepwater rice-growing locations in Bangladesh during the month before the arrival of floodwater. The principal taxa wereAnabaena, Cylindrospermum, Lyngbya, Microcoleus, Nostoc, Prophyrosiphon notarisii, Scytonema mirabile andTolypothrix byssoidea. One of two locations studied after the floodwaters had receded also had an extensive cover, mainlyScytonema mirabile. Nitrogenase activity assayed at mid-day was from one to three orders of magnitude higher per unit area of community than bare soil. Nostoc showed higher activity than Tolypothrix, whether expressed per unit area or biomass. Whole field estimates of N₂ fixed by blue-green algal communities during the pre-flood period ranged from 1.0 to 10.2 kg N ha^–1. Much of this is probably not recycled until floodwaters cover the fields. However N₂ fixed after floodwaters have receded is probably recycled rapidly due to ploughing.     

A study of several blue-green algae in the electron microscope

Summary All of the three blue-green algae, Anabaena cylindrica, Mastigocladus laminosus and Nostoc muscorum are characterized by the presence of multi-layered envelopes (sheath, wall and plasma membrane), photosynthetic lamellae and a variety of intracellular granules. Sections of heterocysts of Anabaena cylindrica showed the presence of an internal membrane system as well as lamellae. An unusual feature of the structure of Nostoc muscorum was the presence of densely stained intracellular membranes or lamellae. The results emphasize the variability in appearance of the internal structure of the blue-green algae and point to the need for detailed investigations of the influence of change in physiological environment on the anatomy of these organisms.