Mature Heterocysts Research Articles

Control of Heterocyst Development in the Cyanobacterium Anabaena cylindrica

The differentiation of proheterocysts and heterocysts in Anabaena cylindrica was initiated by the removal of NH4Cl from the medium. The development of both proheterocysts and mature heterocysts was inhibited by rifampicin, chloramphenicol and mitomycin C, at final concentrations of 0.2, 4.0 and 1.0 μg ml−1, respectively. However, lower concentrations than these were sufficient to inhibit mature heterocyst development while permitting proheterocyst formation. The presence of rifampicin (0.2 μg ml−1) during the early stages of the induction of differentiation, just prior to the initial visual detection of proheterocysts, led to an increase in the frequency of heterocysts that subsequently developed. To assess the commitment of cells to differentiation, samples of culture were incubated with inhibitors or NH4Cl, or were transferred to darkness, at different times following the initiation of development. Each treatment produced a characteristic commitment time which differed for proheterocysts and mature heterocysts. The data obtained from these experiments have been used to formulate a model for the control of heterocyst development in A. cylindrica, in which DNA replication serves as a timer mechanism for the process.

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.

Synchronous Akinete Germination and Heterocyst Differentiation in Anabaena PCC 7937 and Nostoc PCC 6720

Isolates of the filamentous cyanobacteria Anabaena PCC 7937 and Nostoc PCC 6720 were selected in which the germling extruded from the akinete coat at the single cell stage and in which heterocyst differentiation occurred at the two or three cell stage after germination in the absence of combined nitrogen. The process of heterocyst differentiation of these germinating akinetes was synchronous and highly reproducible, and it resulted in transient yields of mature heterocysts exceeding 35% of the total cell population.

Estimation of gene expression in heterocysts of Anabaena variabilis by using DNA-RNA hybridization

In the filamentous cyanobacterium Anabaena variabilis, specialized cells called heterocysts occur in a regular pattern along the filament and are the sites of nitrogen fixation. We used two different types of DNA-excess RNA hybridization techniques to estimate the number of genes expressed in recently differentiated, mature heterocysts. In the first, RNA and DNA were incubated in a phosphate buffer at 60 degrees C, and the hybrids were separated from the unhybridized material by hydroxylapatite chromatography. In the second, the nucleic acids were incubated at 50 degrees C in a buffer containing 50% formamide, and the fraction of DNA in duplexes was assayed by S1 nuclease digestion. Both techniques revealed that approximately 65% of the A. variabilis genome was expressed in vegetative cells and 45% of the genome was expressed in heterocysts. Two experiments were conducted to estimate the number of heterocyst-specific mRNA transcripts. In one, hybridization of heterocyst RNA to a null DNA probe (DNA not transcribed in vegetative cells) revealed that heterocyst-specific transcripts were encoded by 25% of the DNA sense strand, representing approximately 1,000 genes (assuming each to be 1,500 nucleotides in length). The second approach, in which total cell DNA was hybridized to a mixture of heterocyst and vegetative cell RNA, indicated that 14.7% of the DNA sense strand, or about 600 genes, was transcribed exclusively in the heterocyst. The remaining 900 to 1,300 transcripts present in the heterocyst appeared to be constitutively produced in both vegetative cells and heterocysts. The heterocyst-specific transcripts were present in abundant copies in the cell, while transcripts that occurred in both cell types were present at much lower frequency.

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.

Localization of ribulose 1,5-bisphosphate carboxylase/oxygenase in the N2-fixing cyanobacteriumAnabaena cylindrica

Monospecific antiserum against ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of Microcystis PCC7820 cross-reacted with cell-free extracts of the heterocystous cyanobacterium Anabaena cylindrica. This antiserum was used in conjunction with colloidal gold to localize RuBisCO protein in filaments of A. cylindrica. Labelling occurred in the vegetative cells where it was associated particularly with carboxysomes in stationary phase cultures and with both carboxysomes and chromatoplasm in continuous cultures, with the proportion of RuBisCO in the chromatoplasm increasing with increasing growth rate. The protein was undetectable in mature heterocysts.

Regulation of nitrogenase levels in Anabaena sp. ATCC 33047 and other filamentous cyanobacteria

Incubation in the dark of photoautotrophically grown N2-fixing heterocystous cyanobacteria leads to a loss of nitrogenase activity. Original levels of nitrogenase activity are rapidly regained upon re-illumination of the filaments, in a process dependent on de novo protein synthesis. Ammonia, acting indirectly through some of its metabolic derivatives, inhibits the light-promoted development of nitrogenase activity in filaments of Anabaena sp. ATCC 33047 and several other cyanobacteria containing mature heterocysts. The ammonia-mediated control system is also operative in N2-fixing filaments in the absence of any added source of combined nitrogen, with the ammonia resulting from N2-fixation already partially inhibiting full expression of nitrogenase. High nitrogenase levels, about two-fold higher than those in normal N2-fixing Anabaena sp. ATCC 33047, are found in cell suspensions which have been treated with the glutamine synthetase inhibitor l-methionine-d,l-sulfoximine or subjected to nitrogen starvation. Filaments treated in either way are insensitive to the ammonia-promoted inhibition of nitrogenase development, although this insensitivity is only transitory for the nitrogen-starved filaments, which become ammonia-sensitive once they regain their normal nitrogen status.

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.

Heterocyst differentiation in Cylindrospermum licheniforme: studies on the role of transcription.

Heterocysts of the cyanobacterium Cylindrospermum licheniforme occur at the ends of the filaments. These cells, specialized for aerobic N2 fixation, synchronously differentiate after the fragmentation of filaments grown in a medium free of combined nitrogen. This study has examined the role of transcription during and after heterocyst differentiation. Autoradiography of intact filaments pre-labelled for 3 h with [3H]uracil revealed that RNA synthesis occurred at similar rates in vegetative cells, developing (pro)heterocysts and mature heterocysts. Since mature heterocysts no longer divide and contain a similar amount of DNA as vegetative cells, transcription must continue under conditions where replication no longer occurs. Rifampicin, when added to fragmented filaments at concentrations that reduced RNA synthesis by 95% in all cell types, inhibited proheterocyst formation but not the final morphological step of differentiation (pore plug deposition). However, chloramphenicol blocked all stages of heterocyst differentiation. Since the average mRNA half-life was 29 min, and the rifampicin-insensitive stage lasted 3-6 h, the later stages of differentiation may depend on long-lived mRNAs.

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.

Influence of phosphorus on morphology ofCalothrix parietina(Cyanophyta) in culture

A strain of Calothrix parietina (Durham No. 550) was isolated from an upland stream whose physical and chemical environment is already known in some detail. Morphological changes were followed in batch culture in a medium lacking combined nitrogen and under conditions which eventually led to phosphorus deficiency. Four different stages were recognized for descriptive purposes with light microscopy. Stage I commences with hormogonia and ends with the development of a heterocyst at one end of the trichome. Stage II commences with the formation of a mature heterocyst; this is followed rapidly by a tapering of the trichome, first by a narrowing at the apical end and then by a broadening at the basal end. Under phosphorus-rich conditions the apical part of this tapered trichome can continue to give rise to a gas-vacuolate hormogonium, but with increasing phosphorus deficiency hormogonium synthesis ceases and the terminal cells start to differentiate into a hair. This latter event is recognized as the start of ...

Biosynthesis of the Envelope Glycolipids during Heterocyst Development in Anabaena cylindrica

SUMMARY: Incorporation of sodium [1-14C]acetate into the heterocyst-specific glycolipids and glycerolipids of the cyanobacterium Anabaena cylindrica during induced heterocyst differentiation indicated that (1) the activity of key enzymes involved specifically in the biosynthesis of the very long chain polyhydroxyalkanes and hydroxyfatty acid moieties of the heterocyst glycolipid fraction is stimulated during heterocyst differentiation, and (2) in mature heterocysts, formation of the characteristic very long chain heterocyst glycolipids essentially ceases.

Induction and Differentiation of Heterocysts in the Filamentous Cyanobacterium Cylindrospermum licheniforme

The fragmentation of Cylindrospermum licheniforme filaments resulted in the induction of a synchronous round of heterocyst differentiation. Young heterocysts (proheterocysts) appeared 12 to 15 h after fragmentation, and 12 to 15% of the cells became heterocysts within 24 h. NH4Cl or NaNO3 prevented the formation of new heterocysts, while differentiation was stimulated twofold by the absence of atmospheric N2. The sequence of appearance of heterocyst-specific characteristics was observed following filament fragmentation. Nitrogenase activity increased rapidly between 13 and 26 h, paralleling the increase in heterocyst frequency. Glycolipids unique to the heterocyst envelope were also synthesized between 13 and 26 h, and activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase increased after 10 h and 90 h respectively. Aged medium induced sporulation in intact filaments within 1 d. but sporulation could occur no earlier than 2 d after filament fragmentation, indicating that the induction of sporulation requires the presence of mature (but not necessarily functional) heterocysts.

Cellular differentiation and nitrogenase activity in the cyanobacteriumAnabaena

Nitrogenase activity at periods of differentiation of heterocysts and akinetes was assayed by the acetylene reduction technique. There was no nitrogenase activity in ammoniumgrown, non-heterocystousAnabaena sp.; the activity appeared only after a lag-phase of about 17 – 21 h after the ammonium-grown culture had been transferred to medium free of combined nitrogen. This activity started appearing as the proheterocysts were developing to mature heterocysts. Maximum nitrogenase activity was attained with exponential phase of culture and mature heterocysts. This activity gradually decreased with the differentiation of akinetes. Only insignificant nitrogenase activity was observed in old cultures in which most cells had matured into akinetes.

Heterocyst differentiation and tryptophan metabolism in the cyanobacterium Anabaena sp. CA

Anabaena sp. CA does not synthesize heterocysts or express nitrogenase activity when grown with nitrate as the nitrogen source. Heterocysts and nitrogenase are induced in such cultures by various tryptophan analogs. The effect does not require inhibition of de novo protein synthesis in the culture. It is restricted to tryptophan analogs only, and, more specifically, to those which can be incorporated into proteins. dl-7-Azatryptophan was effective at triggering both heterocysts and nitrogenase when incubated in the culture for only 1–2 h, even though 6–7 h was required for heterocysts to fully mature and nitrogenase activity to be expressed. Chloramphenicol completely negated this effect, supporting the idea that the analogs are either incorporated into protein themselves or trigger the synthesis of proteins which initiate complete development of mature heterocysts. Using toluene-permeabilized cells, we have shown that anthranilate synthetase, the first key enzyme in tryptophan biosynthesis, has glutamine-dependent activity. This activity can be effectively feedback inhibited by the various tryptophan analogs at concentrations which are also effective in triggering heterocyst differentiation. These data provide firm evidence for a link between tryptophan biosynthesis, nitrogenase synthesis, heterocyst differentiation, and primary ammonia assimilation.

Synthesis of nitrogenase and heterocysts by Anabaena sp. CA in the presence of high levels of ammonia.

Anabaena sp. CA fails to synthesize heterocysts and nitrogenase when grown with KNO3 as the nitrogen source. By contrast, both heterocysts and proheterocysts are synthesized in NH4Cl-containing media to a level nearly commensurate with cells grown in the absence of combined nitrogen. The growth rate of the organism in NH4Cl-containing media was similar to that obtained with KNO3 as the nitrogen source and was independent of the presence of N2 in the atmosphere. Thus, our results indicate that the organism assimilated nitrate and ammonium nitrogen equally well to meet the nitrogen requirements for growth. Moreover, in contrast to previous studies with other cyanobacteria, the repressor singal for heterocyst differentiation in Anabaena sp. CA is not derived from the metabolism of ammonia but appears to be involved with nitrate metabolism. Nitrogenase activity was partially expressed in NH4Cl-grown cultures. Increasing the level of nitrogenase activity to a value representative of a N2-grown culture required both the inhibition of ammonia assimilation and de novo protein synthesis. An increase in the number of mature heterocysts was not required. The fact that high levels of exogenous ammonia only partially repress the synthesis of proteins required for the maximum expression of nitrogenase activity in Anabaena sp. CA has important implications.

The Effects of Anaerobiosis on Nitrogenase Synthesis and Heterocyst Development by Nostocacean Cyanobacteria

Effective nitrogenase synthesis by nostocacean cyanobacteria (including one mutant strain unable to form heterocysts or fix nitrogen aerobically) was induced in the light in the absence of molecular oxygen. Anaerobiosis was maintained during induction by treating the organisms with dichloromethylurea which prevents photosynthetic oxygen production. Under these special conditions both synthesis and activity of nitrogenase were light-dependent, the required ATP being produced by cyclic photophosphorylation. Enzyme synthesis and activity were also dependent on the availability of an organic substrate that could serve both as a general source of carbon and as a source of reductant. The organic requirement could be fulfilled by the intracellular glycogen reserve or, in facultative hetero-trophs, by a utilizable sugar (e.g. glucose). Nitrogenase synthesized anaerobically was highly susceptible to inactivation by molecular oxygen in vivo: exposure of a suspension of anaerobically induced filaments to 20 % (v/v) O2 for 1 h caused total and irreversible destruction of the enzyme. Anaerobic nitrogenase synthesis was not accompanied by the differentiation of mature heterocysts, the morphogenetic process being arrested at an early (proheterocyst) stage. After the gratuitous anaerobic synthesis of nitrogenase, introduction of either N2, nitrate or ammonia to the illuminated, anaerobic suspension resulted in a rapid accumulation of cyanophycin granules in both vegetative cells and proheterocysts. Cyanophycin was randomly deposited in vegetative cells, but localized at the cell poles of the proheterocysts. The bearing of these findings on the role played by the heterocyst in nitrogen fixation is discussed.

Heterocyst Development in Anabaena cylindrica: The Necessity for Light as an Initial Trigger and Sequential Stages of Commitment

An initial light-dependent stage in heterocyst formation in Anabaena cylindrica was observed. The length of the period of illumination, between 3 and 9 h, determined the frequency 0 mature heterocyst production. Sequential stages of commitment to both proheterocyst and heterocyst formation were shown by manipulation of the light period, ammonia, drugs and analogues. The modulated response of potential heterocyst-forming cells in a filament, requiring sustained periods of light and lack of nitrogen source, is described.

Heterocyst and Nitrogenase Development in Anabaena cylindrica

The differentiation of filaments of a continuous culture of Anabaena cylindrica after removal of fixed nitrogen has been examined. Rapid development of proheterocysts (4-5 h) and mature heterocysts (14 h) in an ordered sequence was observed; the development of the latter was concomitant with the onset of nitrogenase activity. Commitment times of 2-3 h (proheterocyst) and 5-0 (heterocyst) were measured. Evidence is presented that shows that heterocyst development, rather than any product of heterocyst function in nitrogen fixation, is responsible for the imposition of the pattern of heterocysts in a filament of vegative cells. Both phycocyanin content and carbon dioxide fixation declined markedly throughout the filament during the early stages of heterocyst development, indicating that all the cells of the filament are involved in the initial stages of the differentiation process. Heterocysts do not evolve oxygen in the light, but do respire. Nitrogenase activity in isolated heterocysts and in intact filaments was stimulated by phosphoenolpyruvate, ATP and dichlorophenolindophenol-ascorbate.

Growth and nitrogen fixation of mutants defective in heterocyst differentiation in the blue‐green alga Cylindrospermum sp.

AbstractThree classes of spontaneous and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (NTG)‐induced mutants of the blue‐green alga Cylindrospermum sp. were isolated in the presence of combined nitrogen. Mutants with complete loss of heterocyst differentiation formed longer filaments than the parent alga and were unable to grow in nitrogen‐free medium and to fix nitrogen. The second type of mutants formed long filaments with terminal heterocysts. In the third type of mutants, paired proheterocyst differentiation occurred at regular intervals in long filaments and these were defective in their further development into mature heterocysts. Mutants with decreased heterocyst frequency due to defective differentiation and development showed less growth and nitrogen fixation in nitrogen‐free medium than the parent alga.