The Nostoc-Nephroma symbiosis: localization, distribution pattern and levels of key proteins involved in nitrogen and carbon metabolism of the cyanobiont

The qualitative distribution and quantitative estimates of nitrogenase (EC 1.7.99.2), glutamine synthetase (EC 6.3.1.2), phycoerythrin and ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were studied in the cyanobacterium Nostoc residing in internal cephalodia of the tripartite lichen Nephroma arcticum L. Polyclonal antisera, raised in rabbit against the proteins, and goat anti-rabbit IgG conjugated to 10 nm gold were used as probes to detect the antigens by transmission electron microscopy. Western blot analyses demonstrated the monospecificity of the antisera. Nitrogenase was localized in heterocysts, with vegetative cells showing a label intensity comparable to the background. Distribution of the antigen within the heterocysts was uniform. Glutamine synthetase labelling was very low, but appeared to be distributed in both cell types. An intense phycoerythrin labelling was associated with the thylakoid region of the vegetative cells, whereas a much lower labelling was observed in the heterocyst. No significant differences were found between cyanobionts in younger and older cephalodia except for the nitrogenase labelling, which was higher in heterocysts of the cyanobiont in younger cephalodia. Most of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) label was present in vegetative cells. The Rubisco label was pronounced in the carboxysomes, whereas the label in the cytoplasm, on a unit area basis, was much lower. Heterocysts showed a label intensity similar to that of the vegetative cell cytoplasm. In Nostoc of the bipartite lichen Peltigera canina L., the Rubisco protein showed a comparable distribution pattern, but the average number of carboxysomes per vegetative cell was about 4 times higher.     

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.     

Purification of iron superoxide dismutase from the cyanobacterium Anabaena cylindrica Lemm. and localization of the enzyme in heterocysts by immunogold labeling

Iron superoxide dismutase (Fe-SOD; EC 1.15.1.1) was isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica Lemm. Polyacrylamide gel electrophoresis separated the purified protein into three closely running, enzymatically active bands. The molecular weight of the enzyme was estimated by gel filtration to be about 40 kDa. Polyclonal antibodies were produced by immunization of rabbits with the isolated enzyme, and were purified on a column of protein A-Sepharose. The Fe-SOD antibody reacted with the purified Fe-SOD and also specifically recognized the protein in extracts of A. cylindrica. In the extracts, anti-Fe-SOD did not cross-react with Mn-SOD, an enzyme which belongs to an SOD class displaying high homology of primary and three-dimensional structure with respect to Fe-SOD. Iron superoxide dismutase was localized in heterocysts by immunogold labeling and transmission electron microscopy. These results are the first in-situ evidence for the presence of SOD in the cells specialized for nitrogenase activity.Abbreviations ELISA enzyme-linked immunosorbent assay - SDS sodium dodecyl sulfate - SOD superoxide dismutase - PAGE polyacrylamide gel electrophoresis - pI isoelectric point This work was supported by a C.N.R. grant. We are grateful to Dr. A. De Martino for technical assistance.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

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     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

Nitrogenase in free-living and symbiotic cyanobacteria: Immunoelectron microscopic localization

Abstract Nitrogenase (Fe-protein) was localized in the free-living cyanobacterium Anabaena cylindrica and in the cyanobionts of Cycas revoluta and Peltigera aphthosa , using colloidal gold as an immunocytochemical marker. The Fe-protein was found to be evenly distributed throughout the heterocyst cytoplasma in A. cylindrica and in both the cyanobionts, including multiple heterocysts of the C. revoluta cyanobiont. No label was observed in the vegetative cells of free-living A. cylindrica or of the cyanobionts, although the cyanobionts apparently live under microaerobic conditions.     

Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

Cells of free-living nitrogen-fixing Nostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycad Macrozamia. were examined for the presence of ornithine carbamoyl transferase (OCT) by native-PAGE/in situ activity stain, and SDS-PAGE/Western immunoblots. Transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of OCT in the Nostoc cells. Moreover, the effects of photoautotrophic and dark heterotrophic growth metabolism on growth, nitrogenase activity and in vivo citrulline synthesis were investigated. PAGE in combination with in situ activity staining demonstrated an in vitro active OCT with a molecular weight of approximately 80 kDa. SDS-PAGE/Western immunoblots revealed that a polypeptide with a molecular weight of approximately 38 kDa was immunologically related to OCT purified from pea (Pisum sativum L. cv. Alaska). Immunolocalization demonstrated that the OCT protein was located both in vegetative cells and heterocysts. Using the particle analysis of an image processor, the labeling associated with the photosynthetic vegetative cells was calculated to be 75.6 (± 5.5) gold particles μm^?2 compared with 62.0 (± 7.5) in the nitrogen-fixing heterocysts. Glucose and fructose stimulated both cyanobacterial growth and nitrogenase activity in light and darkness. Addition of exogenous ornithine decreased nitrogenase activity. In light grown cells, additions of glucose and fructose in combination with ornithine not only stimulated growth and nitrogenase activity but also in vivo citrulline synthesis, measured as ¹⁴CO₂-fixation into [¹⁴C]-citrulline. In darkness no stimulation was observed on in vivo citrulline synthesis. The substantial stimulation of nitrogenase activity by additions of external glucose and fructose, both in the light and in darkness, was not followed by a simultaneous stimulation of in vivo citrulline synthesis.     

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.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Fructose supports glycogen accumulation,heterocysts differentiation,N2 fixation and growth of the isolated cyanobiont Anabaena azollae

Cells of the cyanobiont Anabaena azollae isolated from the water fern Azolla filiculoides were found to take up and utilize fructose in the light for mixotrophic growth. Fructose was favored by the cyanobiont as a substrate over sucrose and glucose. Cell growth in the presence of 8 mM fructose led to glycogen accumulation in the cells which approached 20% of the cell dry weight within 2 to 3 days, followed by reduction of glycogen content during the fourth day. Glucose-6-phosphate dehydrogenase activity was increased 5–6-fold in the fructose grown cells from the third day of growth onwards. The frequency of heterocysts in fructose-grown cells increased from 6 to 18%, and acetylene reduction by nitrogenase was increased 3-fold in the presence of fructose as compared with control cells, with maximum values observed between the third and fifth day of mixotrophic growth. Fructose-supported growth yielded a 2–4-fold increase in cell dry weight over controls.It is suggested that fructose-supported development and growth of the cyanobiont in batch cultures may resemble its mixotrophic growth and development in situ in the leaf cavity of the host fern Azolla.Abbreviation G6PDH glucose-6-phosphate dehydrogenase     

Nitrogen fixation by Anabaena cylindrica

Hydrogen-supported nitrogenase activity was demonstrated in Anabaena cylindrica cultures limited for reductant. Nitrogen-fixing Anabaena cylindrica cultures sparged in the light with anaerobic gases in the presence of the photosynthesis inhibitor DCMU slowly lost their ability to reduce acetylene in the light under argon but exhibited near normal activities in the presence of 11% H₂ (balance argon). The hydrogen-supported nitrogenase activity was half-saturated between 2 and 3% H₂ and was strongly inhibited by oxygen (50% inhibition at about 5–6% O₂). Batch cultures of Anabaena cylindrica approaching stationary growth phase (“old” cultures) lost nitrogenase-dependent hydrogen evolution almost completely. In these old cultures hydrogen relieved the inhibitory effects of DCMU and O₂ on acetylene reduction. Our results suggest that heterocysts contain an uptake hydrogenase which supplies an electron transport chain to nitrogenase but which couples only poorly with the respiratory chain in heterocysts and does not function in CO₂ fixation by vegetative cells.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica

The thylakoids of vegetative cells of the filamentous cyanobacterium, Anabaena cylindrica, are capable of oxygen-evolving photosynthesis and contain both Photosystems I and II (PSI and PSII). The heterocysts, cells specialized for nitrogen fixation, do not produce oxygen and lack Photosystem II activity, the major accessory pigments, and perhaps the chlorophyll a associated with PSII. Freeze-fracture replicas of vegetative cells and of heterocysts reveal differences in the structure of the thylakoids. A histogram of particle sizes on the expolasmic fracture face (E-face, EF) of vegetative cell thylakoids has two major peaks, at 75 and 100 Å. The corresponding histogram for heterocyst thylakoids lacks the 100 Å size class, but has a very large peak at about 55 Å with a shoulder at 75 Å. Histograms of protoplasmic fracture face (P-face, PF) particle diameters show single broad peaks, the mean diameter being 71 Å for vegetative cells and 64 Å for heterocysts. The thylakoids of both cell types have about 5600 particles/μm² on the P-face. On the E-face, the density drops from 939 particles/μm² on vegetative cell thylakoids to 715 particles/μm² on heterocyst thylakoids. The data suggest that the 100 Å E-face particle of vegetative cell thylakoids is a PSII complex. The 55 Å EF particle of heterocysts may be part of the nitrogenase complex or a remnant of the PSII complex. The role of the 75 Å EF particle is unknown. Other functions localized on cyanobacterial thylakoids, such as respiration and hydrogenase activity, must be considered when interpreting the structure of these complex thylakoids.     

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O₂ production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.     

Sub-cellular Localization of Calcium in Azolla-Anabaena Symbiosis by Chlortetracycline,ESI and EELS

The subcellular localization of calcium in cells of symbiotic partners located within leaf cavities of Azolla was investigated by using chlorotetracycline, ESI and EELS analysis. Loosely membrane-bound calcium was evidenced by using CTC or EGTA and CTC, in cytoplasmic regions of Azolla hair cells and in cytoplasm of the cyanobiont. Tightly membrane-bound calcium revealed by CTC, and ESI and EELS analysis, was observed in cyanophycin granules and carboxysomes of the cyanobiont. A third calcium type, revealed by ESI and EELS analysis, was localized at the level of cell walls of simple and branched Azolla hairs, in the envelope of heterocysts, and in the cell walls of the cyanobiont.     

Developmental patterns related to nitrogen fixation in theNostoc-Gunnera magellanica Lam. symbiosis

Developmental patterns related to nitrogen fixation in the heterocystous cyanobacteriumNostoc harboured in distinct colonies along the stem ofGunnera magellanica Lam. plantlets were examined using successive plant sections. Pronounced morphological, physiological and biochemical alterations in the cyanobacterium were demonstrated. Close to the growing apex the cyanobacterial biomass, contained in smallGunnera cells, was low and consisted mostly of vegetative cells showing a high density of different storage structures except for cyanophycin granules. In contrast, both the total and specific nitrogenase activity and the relative nitrogenase protein level were at maximum within this part; while the frequency of heterocysts increased from zero to 30% within the same area. The nitrogenase protein was localized only in the heterocysts throughout the plant. Further down theGunnera stem there was a progressive increase in both the cyanobacterial biomass and the heterocyst frequency, which finally constituted about 60% of the cyanobacterial cell population. Throughout this part of the stem, cyanophycin granules were frequent in the vegetativeNostoc cells. At the base of the stem, degeneratedNostoc cells dominated and the nitrogenase activity was close to zero, although the nitrogenase protein remained. Degeneration of theNostoc cells and leaf shedding coincided. Both intact plants (approx. 20 mm in height) and plant stem sections (2 mm in length) showed substantial nitrogenase activity, although sectioning caused a 30% reduction in total nitrogenase activity.     

Ultrastructural analysis of the rehydration of desiccatedNostoc commune HUN (cyanobacteria) with particular reference to the immunolabelling of NifH

Summary Vegetative cells and heterocysts of the filamentous desiccation-tolerant cyanobacteriumNostoc commune HUN retain their ultrastructural organisation and the integrity of their intra- and extracellular membranes after two years of desiccation and subsequent rehydration. Immunogold-labelling of thin sections demonstrated the presence of NifH (Fe protein of nitrogenase) in vegetative cells and heterocysts within five minutes of the rehydration of dried colonies. Immunogold label accumulated in discrete areas vegetative cells within 5 minutes of the rewetting of cells, and after 30 minutes a conspicuous association of NifH protein with heterocyst ribosomes was detected. After longer periods of rehydration, the deposition of gold particles became more random within both cell types but occurred with a greater frequency in heterocysts. Up to 24 hours after the rewetting of cells, two morphologically-distinct forms of heterocyst could be discerned. NifH protein was detected through Western blotting (subunit M_r=33,800) in protein extracts from samples ofNostoc commune, collected in different parts of the world and including some which had been desiccated for periods of up to 10 years. The results are discussed in relation to the sequential recovery of metabolic functions, particularly nitrogen fixation, which occurs upon the rehydration of cells after their prolonged storage in the air-dry state.     

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.     

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains

The occurrence activity and localization of calmodulin in three heterocystous cyanobacteria of the genus Anabaena were studied. Boiled crude extracts caused a Ca2+-dependent stimulation of NAD kinase. Such a stimulation was blocked by EGTA and chlorpromazine, SDS-PAGE and Western blot analysis using antiserum against eukaryotic spinach calmodulin, revealed a polypeptide of about 17 kDa. Immunogold localization of calmodulin gave a dense gold label in both vegetative cells and heterocysts. The label was mainly confined to the centroplasm in vegetative cells, while it was evenly distributed in the cytoplasm of mature heterocysts.