Early events during the establishment of the Gunnera/Nostoc symbiosis

The symbiosis between Gunnera and Nostoc was reconstituted using G. chilensis Lam. and G. manicata Linden, respectively, and three different Nostoc strains. Six stages characterised by specific modifications in both the cyanobiont and the host were recognised during the infection process. Mucilage-secreting stem glands developed on the Gunnera stems independent of the presence of cyanobacteria (Stage I). Soon after addition of the Nostoc isolates to the plant apices, an abundant differentiation of motile hormogonia commenced. The cyanobacteria accumulated in the mucilage on the surface of the gland (Stage II), and the hormogonia then proceeded into the stem tissue through intercellular channels (Stage III). At the channel bases, Nostoc was detected between the cell walls of small, densely cytoplasmic Gunnera cells and also in elaborate folds of these (Stage IV). The Gunnera cell walls subsequently dissolved adjacent to the cyanobacteria and Nostoc entered the host cells (Stage V). Once the intracellular association was formed, a high proportion of the vegetative Nostoc cells differentiated into heterocysts (Stage VI). Nostoc changed from being rich in inclusions (particularly cyanophycin) while on the gland surface into a comparatively non-storing form during penetration and the early intracellular stages. Bacteria were numerous on the gland surface, fewer in the channels, and were never detected within the Gunnera cells, indicating the existence of specific recognition mechanisms discriminating between conceivable microsymbionts. Mechanisms behind mutual adaptations and interactions between the two symbionts are discussed.The technical assistance of Anette Axen and Gary Wife is gratefully acknowledged. Financial support was provided by the Swedish Natural Science Research Council and the Hierta-Retzius foundations.     

Ultrastructural characterisation of cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium,Trichodesmium spp.

Summary Trichodesmium is the first described example of a filamentous cyanobacterium without heterocysts that contains cells specialised for nitrogen fixation. The ultrastructure of cells with and without nitrogenase were compared using primarilyTrichodesmium tenue Wille, but alsoT. thiebautii Gomont andT. erythraeum Ehrenberg et Gomont. Immunohistochemistry demonstrated that the cytoplasm of certain cells was densely labelled with antibodies against Fe-protein (dinitrogenase reductase). Comparative TEM-image analysis revealed that these cells were also distinguished by a denser thylakoid network, dividing the vacuole-like space into smaller units. The nitrogenase-containing cells also exhibited less extensive gas vacuoles as well as fewer and smaller cyanophycin granules compared to cells which lacked nitrogenase. Carboxysomes were present in both cell types in equal proportion. Longitudinal sections showed that cells with nitrogenase were arranged adjacent to each other, and that groups of cells with and without nitrogenase may coexist in the same trichome. The correlation between modifications in ultrastructure and the presence of nitrogenase suggests a new type of cyanobacterial cell specialisation related to nitrogen fixation. The results obtained also question the systematic affiliation of the genusTrichodesmium.     

The Accumulation and Degradation Dynamics of Cyanophycin in Cyanobacterial Cells Grown in Symbiotic Associations with Plant Tissues and Cells

Five different artificial associations of cyanobacterial cells with the cells or tissues of nightshade and rauwolfia were studied. The associations grown on nitrogen-containing media produced heterocysts. Cyanobacterial cells in the associations retained their ability to take up combined nitrogen from the medium, to store it in the form of cyanophycin granules, and to use them in the process of symbiotic growth. The synthesis and degradation of cyanophycin granules in cyanobacterial cells were more active in the associations than in monocultures. In the symbiotic associations of Chlorogloeopsis fritschii ATCC 27193 with Solanum laciniatum cells and of Nostoc muscorum CALU 304 with the Rauwolfia serpentina callus, heterocysts were produced with a 3- to 30-fold higher cyanophycin content than in pure cyanobacterial cultures. In contrast, in the association of N. muscorum CALU 304 with the Solanum dulcamara callus, heterocysts were produced with a lower cyanophycin content than in the N. muscorum CALU 304 pure culture. The degradation of cyanophycin granules in N. muscorum CALU 304 cells grown in associations with plant tissues or cells was subjected to mathematical analysis. The activation of cyanophycin degradation and heterocyst differentiation in the associations N. muscorum CALU 304–R. serpentinaand C.fritschii–S. laciniatum was accompanied by an enhanced synthesis of the nitrogen-containing alkaloids in plant cells. The data obtained suggest that an integrated system of nitrogen homeostasis can be formed in symbiotic associations. Depending on the growth stage of an association, its plant member can either stimulate the accumulation of combined nitrogen in vegetative cyanobacterial cells in the form of cyanophycin granules, activate their degradation, or initiate the formation of heterocysts independently of the cyanobacterial combined nitrogen deprivation sensing-signaling pathway.     

The accumulation and degradation dynamics of cyanophycin in cyanobacteria grown in symbiotic associations with plant tissues and cells

Five different artificial associations of cyanobacterial cells with the cells or tissues of nightshade and rauwolfia were studied. The associations grown on nitrogen-containing media produced heterocysts. Cyanobacterial cells in the associations retained their ability to take up bound nitrogen from the medium, to store it in the form of cyanophycin granules, and to use them in the process of symbiotic growth. The synthesis and degradation of cyanophycin granules in cyanobacterial cells were more active in the associations than in monocultures. In the symbiotic associations of Chlorogloeopsis fritschii ATCC 27193 with Solanum laciniatum cells and of Nostoc muscorum CALU 304 with the Rauwolfia serpentina callus, heterocysts were produced at 3- to 30-fold higher cyanophycin contents than in cyanobacterial monocultures. In contrast, in the association of N. muscorum CALU 304 with the Solanum dulcamara callus, heterocysts were produced at lower cyanophycin contents than in the N. muscorum CALU 304 monoculture. The degradation of cyanophycin granules in N. muscorum CALU 304 cells grown in associations with plant tissues or cells was subjected to mathematical analysis. The activation of cyanophycin degradation and heterocyst production in the associations N. muscorum CALU 304-R. serpentina and C. fritschii-S. laciniatum was accompanied by an enhanced synthesis of the nitrogen-containing alkaloids in plant cells. The data obtained suggest that an integrated system of nitrogen homeostasis can be formed in symbiotic associations. Depending on the growth stage of an association, its plant member can either stimulate the accumulation of bound nitrogen in vegetative cyanobacterial cells in the form of cyanophycin granules, or activate their degradation, or initiate the formation of heterocysts independently of the cyanobacterial sensory-signalling system.     

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     

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     

Sub-Cellular Element Analysis of a Cyanobacterium (Nostoc sp.) in Symbiosis with Gunnera manicata by ESI and EELS

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free-living heterocyst-forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free-living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.     

Immunocytochemical localization of carbamyl phosphate synthetase in the filamentous heterocystous cyanobacteriumNostoc PCC 73102

Summary Free-living nitrogen-fixingNostoc PCC 73102 cells, a filamentous heterocystous cyanobacterium originally isolated from the cycadMacrozamia, were grown without or with the addition of either citrulline or ornithine and examined for the presence of carbamyl phosphate synthetase (CPS) by SDS-PAGE and Western immunoblots. Transmission electron microscopy and immunocytochemical labelling were used to study the cellular and subcellular distribution of CPS in theNostoc cells.Western immunoblots revealed that a polypeptide with a molecular weight of approximately 130 kDa was immunologically related to CPS purified fromE. coli. Nitrogen-fixingNostoc 73102 cultures grown without or with the addition of either citrulline or ornithine showed no differences in their CPS-polypeptide levels, indicating no regulatory effect on the CPS-protein level by these two amino acids. Immunolocalization demonstrated that the CPS protein was located both in vegetative cells and heterocysts, subcellularly evenly distributed over the two cell-types. Using the particle analysis of an image processor and cells grown both without or with addition of either citrulline or ornithine, about 2.5 times more CPS-gold labelling per cell area were observed in the photosynthetic vegetative cells compared to the nitrogen-fixing heterocysts.Abbreviations CPS carbamyl phosphate synthetase - IgG immunoglobulin G - OCT omithine carbamyl transferase     

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     

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.     

Inhibition of nitrogen fixation by nitrite inAnabaena variabilis

Addition of nitrite to rapidly growing, nitrogen-fixing filaments ofAnabaena variabilis caused an immediate drop in nitrogenase activity. This was followed by a transient induction of nitrite reductase, recovery of nitrogen fixation and cyanobacterial growth. The experiments with isolated heterocysts and a partially purified nitrogenase preparation from heterocysts showed that nitrite primarily exerted its inhibitory effect by inactivating nitrogenase irreversibly, rather than interfering with photosynthetic energy conservation.Abbreviations ATCC American type culture collection - Chl chlorophyll - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - Tes 2-{[2 hydroxy-1,1-bis(hydroxymethyl)ethyl] amino} ethane sulfonic acid     

Lichens toGunnera — with emphasis onAzolla

Summary N₂-fixing cyanobacteria occur in symbiotic associations with fungi (ascomycetes) as lichens and with a few green plants. The associated cyanobacterium is always a species ofNostoc orAnabaena. Only a small number of plant genera are involved but there is a remarkable range of host diversity. Associations occur with several bryophytes (e.g.Anthoceros, Blasia, Cavicularia), a pteridophyte (Azolla), cycads (nine genera includingMacrozamia andEncephalartos) and an angiosperm (Gunnera). Except forGunnera, where the cyanobacterium penetrates the plant cells, the cyanobacteria are extracellular with specialized morphological modifications and/or structures of the host plant organs providing an environment which facilitates interaction with the prokaryote.Salient aspects of current knowledge pertaining to the establishment, perpetuation, and functioning of the individual symbioses are summarized. Where possible this includes information concerning recognition and specificity, mode(s) of infection, morphological modifications/adaptations of the host plant and a synopsis of morphological, physiological and biochemical changes common to the symbiotic cyanobacteria. The latter encompasses heterocyst frequencies, enzymes involved in ammonia assimilation, photosynthetic capability and metabolic interaction with the host.TheAzolla-Anabaena symbioses, which have potential agronomic significance as an alternative nitrogen source and maintain continuity with the endophyte through the sexual cycle, are emphasized.     

Multiple Roles of Soluble Sugars in the Establishment of Gunnera-Nostoc Endosymbiosis

Gunnera plants have the unique ability to form endosymbioses with N₂-fixing cyanobacteria, primarily Nostoc. Cyanobacteria enter Gunnera through transiently active mucilage-secreting glands on stems. We took advantage of the nitrogen (N)-limitation-induced gland development in Gunnera manicata to identify factors that may enable plant tissue to attract and maintain cyanobacteria colonies. Cortical cells in stems of N-stressed Gunnera plants were found to accumulate a copious amount of starch, while starch in the neighboring mature glands was nearly undetectable. Instead, mature glands accumulated millimolar concentrations of glucose (Glc) and fructose (Fru). Successful colonization by Nostoc drastically reduced sugar accumulation in the surrounding tissue. Consistent with the abundance of Glc and Fru in the gland prior to Nostoc colonization, genes encoding key enzymes for sucrose and starch hydrolysis (e.g. cell wall invertase, α-amylase, and starch phosphorylase) were expressed at higher levels in stem segments with glands than those without. In contrast, soluble sugars were barely detectable in mucilage freshly secreted from glands. Different sugars affected Nostoc’s ability to differentiate motile hormogonia in a manner consistent with their locations. Galactose and arabinose, the predominant constituents of polysaccharides in the mucilage, had little or no inhibitory effect on hormogonia differentiation. On the other hand, soluble sugars that accumulated in gland tissue, namely sucrose, Glc, and Fru, inhibited hormogonia differentiation and enhanced vegetative growth. Results from this study suggest that, in an N-limited environment, mature Gunnera stem glands may employ different soluble sugars to attract Nostoc and, once the cyanobacteria are internalized, to maintain them in the N₂-fixing vegetative state.Nitrogen (N) is an essential element for plant growth, but availability of reduced N in the soil is often limiting. Representatives from a wide range of land plants have evolved the ability to form associations with N₂-fixing microbes (Franche et al., 2009). Associations between rhizobia and legume plants are well-characterized examples of plant-bacterial N₂-fixing symbioses. Unlike rhizobia, which generally exhibit narrow host ranges (Kistner and Parniske, 2002), N₂-fixing cyanobacteria are able to form productive associations with a broad range of plants, including bryophytes (hornworts and liverworts), ferns (Azolla), gymnosperms (cycads), and angiosperms (Gunnera; for review, see Rai et al., 2000; Adams et al., 2006). Free-living cyanobacteria within the genus Nostoc can fix N in specialized microoxic cells called heterocysts. The ability of Nostoc to fix N independent of a host environment may facilitate the formation of symbioses with a wide range of plants. Understanding the physiological conditions that enable a plant host to enter into symbiotic associations with cyanobacteria may allow us to extend the benefit of biological N fixation to crops outside the legume family.Nostoc has the ability to differentiate not only into filaments bearing heterocysts but also into transiently motile filaments, known as hormogonia, which enable the cyanobacteria to enter plants (Meeks and Elhai, 2002). Nostoc can be induced to form hormogonia by different environmental stimuli and by a hormogonia-inducing factor released from N-stressed host plants (Meeks and Elhai, 2002; Adams et al., 2006). The attraction of hormogonia to plants is much less specific than that of rhizobia. Hormogonia are attracted to root extracts from either host or nonhost plants and even to certain simple sugars, such as Ara, Glc, and Gal (Nilsson et al., 2006). After entering a plant host, hormogonia revert back to filaments with N₂-fixing heterocysts. Inside the host, further hormogonia formation is suppressed, and heterocysts appear at a frequency of about 30% to 40%, 3- to 4-times higher than that found in free-living Nostoc (Meeks and Elhai, 2002). Although free-living Nostoc species can support N₂ fixation through photosynthesis, under symbiotic conditions they rely on photosynthate from the host plant. In general, the sugars (Suc, Glc, and Fru) known to support heterotrophic growth in the dark by free-living cyanobacteria coincide with those that support nitrogenase activity in Nostoc-plant associations (Meeks and Elhai, 2002). However, the Nostoc-Gunnera association may be exceptional; only Glc and Fru have been shown to sustain nitrogenase activities (Man and Silvester, 1994; Wouters et al., 2000), although Suc anddextrin were able to keep Nostoc alive without light (Wouters et al., 2000). It is evident from cyanobacterial studies that the plant hosts have evolved the ability to regulate cyanobacterial growth and differentiation during symbiotic associations (Meeks and Elhai, 2002).However, because most studies of plant-cyanobacterial associations have focused on the cyanobacterial partner (e.g. Wang et al., 2004; Ekman et al., 2006), the mechanisms through which plant hosts attract, internalize, and maintain cyanobacteria remain to be elucidated (Adams et al., 2006).The Nostoc-Gunnera association is an ideal system with which to study plant-cyanobacteria symbioses, not only because Gunnera is the only genus of angiosperms known to form endosymbioses with N₂-fixing cyanobacteria but also because the association between the two can be readily established in the laboratory (Bergman et al., 1992; Chiu et al., 2005). Nostoc hormogonia enter Gunnera plants through specialized glands located on the stem. As the gland matures, it secretes polysaccharide-rich mucilage that attracts cyanobacteria (Nilsson et al., 2006), supports their growth on the gland surface (Towata, 1985; Chiu et al., 2005), and permits further hormogonia differentiation (Rasmussen et al., 1994). From there, hormogonia enter the gland and penetrate cells near the base of the gland in the stem (Bonnett, 1990; Bergman et al., 1992). Although each gland is only transiently capable of accepting cyanobacteria, new glands continue to form on the stem at the base of each new leaf.In contrast to the development of nodules in legumes, which requires a complex exchange of signals between the two symbiotic partners (Cooper, 2007), stem gland development in Gunnera takes place in the absence of cyanobacteria (Bonnett, 1990). N limitation, however, is a prerequisite for stem gland development (Chiu et al., 2005), as it is for nodulation (Barbulova et al., 2007). We have taken advantage of the N-deficiency-induced gland development in G. manicata to identify factors that enable Gunnera to form endosymbiosis with cyanobacteria. This study investigated changes in the carbohydrate metabolism during Gunnera gland development and discovered that tissue in the mature glands accumulated high levels of soluble sugars prior to the arrival of cyanobacteria. In agreement with this finding, several key genes encoding enzymes for starch/Suc hydrolysis were expressed at higher levels in the gland compared to the stem. Furthermore, we found that various sugars cyanobacteria may encounter as they approach Gunnera glands as opposed to those they would encounter within plant cells differentially affected Nostoc’s ability to form motile hormogonia.     

Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and Nostoc PCC7936

Two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested with regard to their metal removal capability by using copper as model metal. The experiments, carried out with the sole cyanobacterial biomass suspended in distilled water and confined into small dialysis tubings, showed that C. capsulata biomass is characterized by the best efficiency in metal removal, with a q_max (maximum amount of copper removed per biomass unit) of 96 ± 2 mg Cu(II) removed per g of protein in comparison with the value of 79 ± 3 of Nostoc PCC7936 biomass. The experimental data obtained with both cyanobacterial biomass best fit the Langmuir sorption isotherm. The sorption of copper started from the first minutes of contact with the metal and attained the equilibrium state, when no more copper removal was evident, after 5 and 6 hours, for C. capsulata and Nostoc PCC7936, respectively. The best efficiency in Cu(II) removal was obtained at pH 6.1–6.2, while the presence of Mg²⁺ or Ca²⁺ reduced copper removal capability of both species to 60–70% of their q_max. The results showed that the biomass of C. capsulata and Nostoc PCC7936 possesses a high affinity and a high specific uptake for copper, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use the capsulated trichomes of the two cyanobacteria for the bioremoval of heavy metals from polluted water bodies.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O₂. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H₂ had about half the nitrogen-fixation activity expected on the basis of intact filaments. H₂ did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0°C under N₂.     

Diurnal and seasonal variations of nitrogen fixation and photosynthesis in cyanobacterial mats

In situ measurements of nitrogenase activity and photosynthesis were performed simultaneously in cyanobacterial mats of intertidal sand flats of the Southern North Sea. Two types of cyanobacterial mats, which differed in species composition and biomass content, were investigated. The measurements were done monthly during 3 years to detect seasonal variations of nitrogen fixation and photosynthesis. Diurnal variations were investigated as well. The results showed that (i) freshly colonized sediment with the cyanobacteriumOscillatoria limosa as the dominant organism revealed the highest specific nitrogenase activities (ii) nitrogenase activities were highest in spring and summer, when mat development was initiated and (iii) diurnal fluctuations of nitrogenase activity indicated that it occurred temporally separated from oxygenic photosynthesis.     

Nitrogen metabolism and ultrastructure inAnabaena cylindrica

Nitrogen starvation, effected by incubating a culture ofAnabaena cylindrica in a medium free from combined nitrogen and under an atmosphere of 1% CO₂ in argon, leads to rapid and characteristic changes in the appearance, structure and function of the alga. Change of colour, due apparently to a decrease in the amounts of nitrogenous pigments, is accompanied by a structural transformation of vegetative cells: cyanophycin granules and polyhedral bodies disintegrate, lipid and glycogen accumulate, and large membrane-bound spaces form by means of thylakoid swelling and vesiculation. The rate of heterocyst differentiation and nitrogenase activity is increased. These changes are fully reversed on addition of ammonia to the culture. It appears that thylakoids reform by coalescence of small vesicles assembled in the intrathylakoidal space. Rapid ammonia assimilation is indicated by ample formation of cyanophycin granules in vegetative cells and of “plugs” in the heterocysts.     

Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd

The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO₂; in the absence of CO₂ dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO₂. Dark CO₂ fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO₂ in the dark; in the lichen most dark CO₂ fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of ¹⁴CO₂ in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO₂ in the gas phase. Exogenous NH ₄ ⁺ inhibited nitrogenase activity by cephalodia in the dark especially in the absence of CO₂ but had no effect in the light. The overall data suggest that in the lichen dark CO₂ fixation by the fungus may provide carbon skeletons which accept NH ₄ ⁺ released by the cyanobacterium and that in the absence of CO₂, NH ₄ ⁺ directly, or indirectly via a mechanism which involves glutamine synthetase, inhibits nitrogenase activity.Abbreviations CP carbamoyl phosphate - EDTA ethylenedi-amine tetraacetic acid - PEP phosphoenolpyruvate - RuBP ribulose 1,5 bisphosphate     

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.