Effect of nitrogen starvation on the morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus.

The effects of nitrogen starvation on the morphology and ultrastructure of the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The internal ultrastructural characteristics of vegetative cells changed markedly during nitrogen starvation. Carboxysomes were degraded, while polyphosphate bodies and lipid bodies accumulated. The ultrastructure of mature heterocysts was also affected by nitrogen starvation; their intracytoplasmic membranes vesiculated to form vacuolelike structures and, eventually, large empty regions in the cytoplasm. Nitrogen starvation stimulated extensive heterocyst differentiation in M. laminosus, producing heterocyst frequencies of 17.5% in narrow filaments and 28.3% in wide filaments within 44 h after transfer to N-free conditions. Cells in wide filaments differentiated so extensively that only 16.8% of them failed to initiate the differentiation process within 44 h.     

A new method for identification of heterocysts and proheterocysts in morphologically complex cyanobacteria

A new light microscopic method for identifying heterocysts and proheterocysts in morphologically complex cyanobacteria was evaluated for reliability and usefulness. Mature heterocysts and proheterocysts could be distinguished readily from vegetative cells in 0.25 micron sections of fixed and embedded material after staining with toluidine blue. Examination by light and electron microscopy of the same specimens indicated that the staining reactions which served to differentiate these cell types were both reproducible and accurate. Light microscopic analysis of serial sections stained with toluidine blue greatly facilitated localization of heterocysts and proheterocysts in the complex, branching cyanobacterium, Mastigocladus laminosus, even when its filaments of cells were intertwined in thick mats.     

A New Method for Identification of Heterocysts and Proheterocysts in Morphologically Complex Cyanobacteria

A new light microscopic method for identifying heterocysts and proheterocysts in morphologically complex cyanobacteria was evaluated for reliability and usefulness. Mature heterocysts and proheterocysts could be distinguished readily from vegetative cells in 0.25 µm sections of fixed and embedded material after staining with toluidine blue. Examination by light and electron microscopy of the same specimens indicated that the staining reactions which served to differentiate these cell types were both reproducible and accurate. Light microscopic analysis of serial sections stained with toluidire blue greatly facilitated localization of heterocysts and proheterocysts in the complex, branching cyanobacterium, Mastigocla-dus laminosus, even when its filaments of cells were intertwined in thick mats.     

Functional dissection and evidence for intercellular transfer of the heterocyst‐differentiation PatS morphogen

The formation of a diazotrophic cyanobacterial filament represents a simple example of biological development. In Anabaena, a non‐random pattern of one nitrogen‐fixing heterocyst separated by about 10 photosynthetic vegetative cells results from lateral inhibition elicited by the cells differentiating into heterocysts. Key to this process is the patS gene, which has been shown to produce an inhibitor of heterocyst differentiation that involves the C‐terminal RGSGR pentapeptide. Complementation of a ΔpatS Anabaena mutant with different versions of PatS, including point mutations or tag fusions, showed that patS is translated into a 17‐amino acid polypeptide. Alterations in the N‐terminal part of PatS produced inhibition of heterocyst differentiation, thus this part of the peptide appears necessary for proper processing and self‐immunity in the producing cells. Alterations in the C‐terminal part of PatS led to over‐differentiation, thus supporting its role in inhibition of heterocyst differentiation. A polypeptide, produced in proheterocysts, consisting of a methionine followed by the eight, but not the five, terminal amino acids of PatS recreated the full activity of the native peptide. Immunofluorescence detection showed that an RGSGR‐containing peptide accumulated in the cells adjacent to the producing proheterocysts, illustrating intercellular transfer of a morphogen in the cyanobacterial filaments.     

Morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus growing under nitrogen-fixing conditions

The morphological and ultrastructural characteristics of the cyanobacterium Mastigocladus laminosus growing under N₂-fixing conditions were examined with light and electron microscopy. Vegetative cells in narrow filaments contained randomly arranged segments of thylakoid membrane, centrally located carboxysomes (polyhedral bodies), peripherally located lipid bodies, and large numbers of polysaccharide granules in addition to nuclear material and ribosomes. The ultrastructural characteristics of cells in wide filaments were similar, except for increased numbers of carboxysomes and lipid bodies. Heterocytes and proheterocysts developed at a variety of locations in narrow filaments, wide filaments, and the lateral branches off of wide filaments. Akinetes were not observed in any of the filaments. The morphological characteristics of heterocysts and proheterocysts were variable and depended on those of the vegative cells from which the heterocysts and proheterocysts developed. Mature M. laminosus heterocysts were somewhat similar to those formed in other cyanobacterial genera, but they possessed a number of distinct and unique ultrastructural characteristics, including (i) the absence of a fibrous and, possibly, a laminated wall layer, (ii) the presence many closely packed membranes throughout most of the cytoplasm, and (iii) the presence of unidentified, spherical inclusion bodies of variable electron density.     

Role for hetC in the transition to a nondividing state during heterocyst differentiation in Anabaena sp

Nitrogen-deprived filaments of wild-type or hetC Anabaena sp. produce respectively, at semiregular intervals, heterocysts and weakly fluorescent cells. Unlike heterocysts, the latter cells can divide and elongate, producing a pattern of spaced series of small cells. Because a hetR::gfp fusion is expressed most strongly in the small cells, we propose that these small cells represent a very early stage of heterocyst differentiation. hetC::gfp is expressed most strongly in proheterocysts and heterocysts.     

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.     

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.     

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.     

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.     

A Light and Electron Microscopic Study of Anabaena volzii Lemm

Anabaena volzii Lemm. is a rare species of Cyanophyta. It possesses characteristics of prokary0tes. Young filaments of A. volzii consist of only vegetative cells. The filament leng- thens by the increase of its cell number owing to amitosis. A mature filament contains vegetative cells, heterocysts and akinetes; the latter two differentiate from the vegetative cells. Vegetative cells and heterocysts are short-cylindric shaped. An akinete in longitudinal sections of appear to be elliptical. Viewed with a transmission electron microscope, an electron-dense cell wall, plasmolemma, thylakoids (photosynthetic lamellae), nucleo-plasmic region and polyhedral bodies can be seen in the vegetative cell. The nucleo-plasmic region, which lacks a nuclear envelope, is surrounded or dissected, but often connected with the thylakoids. There are also some extremely electron-dense (if samples were post-fixed in osmic acid) cyanophycin granules in its cytoplasm. Heterocyst is larger than vegetative cells. Its remarkable features are a thick envelope, an electron-transparent cell wall and a distinctive plug-like body at both ends of the cell respectively. In the plug-like body is seen an irregular narrow channel. Somewhat dilated thylakoids in the heterocyst appear to be more winding and contorted (than those in vegetative cells), making a dedicate pattern. A long ellipticring-shaped membrane structure is formed in a heterocyst ,composed, of an electron-dense rod core surrounded by 14 concentric layers of lamellae. Akinete forms thick cell wall. A nucleo-plasmic region, fine and contorted thylakoids, many cyanophycin granules, and abundant ribosomes are found in akinetes.     

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.     

The mystique of irreversibility in cyanobacterial heterocyst formation: parallels to differentiation and senescence in eukaryotic cells

The formation of cyanobacterial heterocysts is unique in the prokaryotic world: it is the only irreversible collective process. This terminal differentiation resembles senescence and differentiation in the eukaryotic urkingdom. During their cell cycle eukaryotic cells at the restriction point may reversibly proceed from a vegetative phase (G1) into a quiescent state (G0), and then may irreversibly enter the way towards differentiated or senescent cells. In parallel, at commitment point 1 vegetative cells from filamentous cyanobacteria may reversibly form proheterocysts, and then may proceed irreversibly towards mature heterocysts at commitment point 2. While the signals paving the path for differentiation or senescence in eukaryotes are largely unknown, heterocyst development is clearly triggered by nitrogen starvation. The reasons for the irreversibility in both systems are poorly understood. We discuss these questions, especially in the light of recent advances in the molecular biology of cyanobacteria, with emphasis on self-stabilizing autocatalytic cycles.     

The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.     

Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae

Summary Heterocysts reduce triphenyl tetrazolium chloride (TTC) faster than vegetative cells apparently because the absence of the O₂-evolving photosystem II and the high electron transport activity in these cells. Although the rate of TTC reduction in vegetative cells is increased by the continuous removal of O₂ evolved in photosynthesis, it has not been possible to obtain rates of TTC reduction comparable with those in heterocysts probably because of the continued competition for electrons between TTC and O₂. The use of nitro-blue tetrazolium chloride (NBT) as a redox indicator has revealed the presence in filaments under aerobic conditions of a gradient of electron transport activity with strongest reducing power in the heterocysts, proheterocysts and vegetative cells next to heterocysts, and with gradually diminishing activity midway between two heterocysts. This pattern is indistinct in filaments grown under micro-aerophilic conditions. The strong electron transport activity in vegetative cells adjacent to heterocysts appears to promote reducing conditions in the heterocysts. Both, red-formazan formation in the heterocysts and blue-formazan deposition in vegetative cells greatly inhibit nitrogenase activity, and this was adversely affected also by the detachment of heterocysts from vegetative cells. The findings are consistent with the idea that the association of heterocysts with vegetative cells in essential for nitrogen fixation to occur in heterocystous blue-green algae.     

FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.     

Relationships between the ABC-Exporter HetC and Peptides that Regulate the Spatiotemporal Pattern of Heterocyst Distribution in Anabaena

In the model cyanobacterium Anabaena sp. PCC 7120, cells called heterocysts that are specialized in the fixation of atmospheric nitrogen differentiate from vegetative cells of the filament in the absence of combined nitrogen. Heterocysts follow a specific distribution pattern along the filament, and a number of regulators have been identified that influence the heterocyst pattern. PatS and HetN, expressed in the differentiating cells, inhibit the differentiation of neighboring cells. At least PatS appears to be processed and transferred from cell to cell. HetC is similar to ABC exporters and is required for differentiation. We present an epistasis analysis of these regulatory genes and of genes, hetP and asr2819, successively downstream from hetC, and we have studied the localization of HetC and HetP by use of GFP fusions. Inactivation of patS, but not of hetN, allowed differentiation to proceed in a hetC background, whereas inactivation of hetC in patS or patS hetN backgrounds decreased the frequency of contiguous proheterocysts. A HetC-GFP protein is localized to the heterocysts and especially near their cell poles, and a putative HetC peptidase domain was required for heterocyst differentiation but not for HetC-GFP localization. hetP is also required for heterocyst differentiation. A HetP-GFP protein localized mostly near the heterocyst poles. ORF asr2819, which we denote patC, encodes an 84-residue peptide and is induced upon nitrogen step-down. Inactivation of patC led to a late spreading of the heterocyst pattern. Whereas HetC and HetP appear to have linked functions that allow heterocyst differentiation to progress, PatC may have a role in selecting sites of differentiation, suggesting that these closely positioned genes may be functionally related.