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.     

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     

Measurement of the Cyanophycin Granule Polypeptide Contained in the Blue-Green Alga Anabaena cylindrica

An assay was developed to determine the amount of cyanophycin granules in blue-green algae. The amount of this polypeptide in cells of Anabaena cylindrica was measured as a function of culture age and was compared with changes in other proteinaceous cellular components. The data presented support the notion that the cyanophycin granule is a cellular nitrogen reserve.     

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.     

The fine structure of akinete formation and germination in Cylindrospermum

Summary Akinete formation and germination were studied in a species of Cylindrospermum using the electron microscope. The differentiation of a vegetative cell into an akinete is characterized by cell enlargement, sheath condensation, deposition of several spore envelope layers, including a dense fibrillar layer and deposition of large cyanophycin granules. The mature akinete in addition to the multilayered envelope retains internally a large number of cyanophycin granules, a photosynthetic thylakoid system, polyhedral bodies, lipid deposits and nucleoplasmic regions. Germination of the akinete can take place in several modes differing in detail. Most frequently the spore envelope remains intact and the germling which may or may not have divided emerges through a pore at one end of the envelope. The photosynthetic thylakoid system appears to increase by the fusion of small vesicles found in the cytoplasm. Alpha-granules are numerous and cyanophycin is nearly absent in the germling.     

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.     

Ammonium content, nitrogenase activity and heterocyst frequency within the leaf cavities of Azolla filiculoides Lam

Abstract Ion selective new microelectrodes have been used to measure the ammonium concentrations within the various leaf cavities from the apical to the basal ones, of Azolla filiculoides Lam. Ammonium is present in solution within all leaf cavities, and its concentration varies considerably from the apex to the base. Median leaf cavities, which have the highest rate of nitrogenase activity, contain 0.6–0.8 mM of ammonium and exhibit numerous cyanophycin granules accumulated within the Anabaena vegetative cells. Basal cavities contain 6 mM ammonium, the lowest nitrogenase activity and lowe cyanophycin granules in Anabaena . The mechaniems involved in ammonium accumulation in the basal leaf cavities are discussed.     

Variation in the fixation image of “Structured granules” in Anabaena

Summary The varied appearance of so-called structured granules in vegetative cells and akinetes of Anabaena sp. is dependent upon the length of primary glutaraldehyde fixation, the type and duration of secondary fixation, and the temperature during both fixations as well as possible changes under electron bombardment. The image produced may be one of an open hole, an area of low to medium electron opacity or that of a dense structured body and may include all intergradations. These structured granules represent the cyanophycin granules of light microscopy. Various cytochemical tests did not establish their chemical composition.     

DEVELOPMENT AND GERMINATION OF AKINETES OF APHANIZOMENON FLOS-AQUAE1

Flakes of Aphanizomenon flos-aquae collected from an ice-covered lake were found to contain all developmental stages from vegetative cells to mature akinetes. Changes during development include increase in cell size, gradual disappearance of gas vacuoles (clusters of gas vesicles), narrowing of intrathylakoidal spaces, and increase in cytoplasmic density. Development of akinetes is accompanied by proliferation of ribosomes, including polyribosomes, cyanophycin granules (structured, granules), and glycogen granules. The lipid bodies of vegetative cells are reduced in size and number in mature akinetes. Akinetes may occur singly or as multiples in sequence in a filament, either terminal or intercalary. Loss of flotation by increase in cytoplasmic density permits filaments to sink and overwinter in bottom sediments. The sequence was found to be reversed during germination of akinetes. Cyanophycin granules are reduced in size and staining density in the sporelings, and very few glycogen granules are seen. Gas vesicles reappear and increase in number, and intrathylakoidal spaces become wider. These changes then would permit the sporelings to rise from the bottom and begin another season's bloom.     

Effect of sulfur starvation on the morphology and ultrastructure of the cyanobacterium Gloeothece sp. PCC 6909

Gloeothece sp. PCC 6909 is a unicellular, nitrogen-fixing cyanobacterium that accumulates sulfate in its sheath. An ultrastructural study of sulfate-deficient and normal Gloeothece sp. PCC 6909 cells was carried out. The physiological alterations, caused by sulfur starvation, were related to important morphological alterations in the cell: a structureless sheath, accumulation of cyanophycin, polyhydroxybutyrate and glycogen granules, and disintegration of thylakoidal membranes. Most of these changes were reversed by the addition of sulfate to the culture medium. The important role of sulfate in the sheath structure was demonstrated.     

Expression of Synechocystis sp. PCC6803 cyanophycin synthetase in Lactococcus lactis nisin-controlled gene expression system (NICE) and cyanophycin production

Cyanophycin is a natural source of polypetide consisting of aspartic acid as a backbone and arginine as its side chain. After the removal of arginine, the remaining poly-aspartate can be served in numerous industrial and biomedical applications. The synthesis of cyanophycin is catalyzed by cyanophycin synthetase. In this study, we used lactic acid bacteria to produce cyanophycin by nisin-controlled gene expression system (NICE). The cyanophycin synthetase gene cphA of Synechocystis sp. strain PCC6803 was cloned to the vector pNZ8149 followed by transformation into Lactococcus lactis subsp. cremoris NZ3900. The effects of nisin concentrations and the amounts of supplemented aspartic acid and arginine were examined for the production of cyanophycin. Alterations of the terminus of cphA gene were also conducted in an attempt to increase the yield of cyanophycin. An optimal cyanophycin production was noted under a culture condition of log phase induced at 250 ng/mL nisin in M17L medium supplemented with 20 mM arginine and 10 mM aspartic acid. An insertion of glycine residue at the C terminus of cyanophycin synthetase resulted in a yield of 20% of dry cell weight, a 10-fold increase when compared with the wild type. The results showed that recombinant lactic acid bacteria, a GRAS system, could provide an alternative approach of producing cyanophycin suitable for agricultural and biomedical applications.     

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.     

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.     

Arginine and nitrogen mobilization in cyanobacteria

Cyanobacteria have evolved mechanisms to adapt to environmental stress and nutrient availability, including accumulation of storage compounds in inclusions and granules. As arginine is a key building block of cyanophycin, a dynamic nitrogen reservoir in many cyanobacteria, arginine metabolism plays a key role in cyanobacterial nitrogen storage and remobilization. Recently, an arginine dihydrolase AgrE/ArgZ was identified as a major arginine‐degrading enzyme in nondiazotrophic Synechocystis, which catalyzes the conversion of arginine into ornithine and ammonia. The N‐terminal domain of AgrE/ArgZ is responsible for arginine dihydrolase activity. Burnat et al. (2019) identified the arginine catabolic pathway in diazotrophic Anabaena, which starts with the reaction catalyzed by AgrE/ArgZ. Moreover, this study identified the C‐terminal domain of AgrE/ArgZ as an ornithine cyclodeaminase that catalyze the conversion of ornithine to proline. The results demonstrated that arginine is catabolized to generate glutamate by the concerted action of AgrE/ArgZ and bifunctional proline oxidase PutA in the vegetative cells of Anabaena. These findings expand our knowledge on nitrogen mobilization and redistribution in Anabaena under nitrogen‐fixation conditions. AgrE/ArgZ is widely present in many diazotrophic cyanobacteria and may be important for their contribution to marine nitrogen fixation. AgrE/ArgZ may have potential applications in metabolic engineering and biotechnology.     

Interrelation between cyanophycin synthesis, L-arginine catabolism and photosynthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

Ultrastructural and immunocytochemical investigations gave evidence that cyanophycin (multi-L-arginyl-poly-L-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on L-arginine or L-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of L-arginine or L-asparagine with nitrate only caused minor cyanophycin accumulation. Growth of Synechocystis PCC 6803 on L-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on L-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. L-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on L-arginine as sole N-source. In both cells types the L-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on L-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of L-arginine to the total nitrogen pool, and the intracellular L-arginine concentration is greatly influenced by the activity of the L-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, L-arginine catabolism, and in addition photosynthesis in Synechocystis PCC 6803.     

Accumulation of Cyanophycin Granules as a Result of Phosphate Limitation in Agmenellum quadruplicatum

Phosphate-limited growth of the blue-green alga Agmenellum quadruplicatum resulted in the accumulation of cyanophycin granule polypeptide (CGP), which is a 1:1 co-polymer of aspartic acid and arginine. The progressive accumulation of CGP began after depletion of phosphate from the medium. CGP increased in concentration much faster than the increase in cell number. Electron microscopy indicated that both the number of cyanophycin granules per cell section and the diameter of each granule increased as phosphate starvation progressed. A marked decrease in the electron density of the inter-thylakoidal areas took place concurrently with the accumulation of CGP. At the same time a progessive decrease in the pigment concentration of cells and in the rate of nitrate uptake was observed. Thirty-two hours after phosphate depletion from the medium up to 28% of total cellular nitrogen was found in CGP.     

Mutation of a Gene Encoding a Putative Glycoprotease Leads to Reduced Salt Tolerance, Altered Pigmentation, and Cyanophycin Accumulation in the Cyanobacterium Synechocystis sp. Strain PCC 6803

The salt-sensitive mutant 549 of the cyanobacterium Synechocystis sp. strain PCC 6803 was genetically and physiologically characterized. The mutated site and corresponding wild-type site were cloned and partially sequenced. The genetic analysis revealed that during the mutation about 1.8 kb was deleted from the chromosome of mutant 549. This deletion affected four open reading frames: a gcp gene homolog, the psaFJ genes, and an unknown gene. After construction of mutants with single mutations, only the gcp mutant showed a reduction in salt tolerance comparable to that of the initial mutant, indicating that the deletion of this gene was responsible for the salt sensitivity and that the other genes were of minor importance. Besides the reduced salt tolerance, a remarkable change in pigmentation was observed that became more pronounced in salt-stressed cells. The phycobilipigment content decreased, and that of carotenoids increased. Investigations of changes in the ultrastructure revealed an increase in the amount of characteristic inclusion bodies containing the high-molecular-weight nitrogen storage polymer cyanophycin (polyaspartate and arginine). The salt-induced accumulation of cyanophycin was confirmed by chemical estimations. The putative glycoprotease encoded by the gcp gene might be responsible for the degradation of cyanophycin in Synechocystis. Mutation of this gene leads to nitrogen starvation of the cells, accompanied by characteristic changes in pigmentation, ultrastructure, and salt tolerance level.     

Ammonia effects in cultures of normal and transformed 3T3 cells

3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH₃ z NH₄⁺) added as NH₄C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.