A cyanophycin synthetase from <Emphasis Type="Italic">Thermosynechococcus elongatus</Emphasis> BP-1 catalyzes primer-independent cyanophycin synthesis

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires l-aspartic acid (Asp), l-arginine (Arg), ATP, Mg²⁺, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg²⁺ or Mn²⁺ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 ± 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.     

Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19

The biosynthesis and accumulation of cyanophycin in the thermophilic cyanobacterium Synechococcus sp. MA19 were studied. By growing the cells in a 80-l closed tubular photobioreactor under controlled conditions, the cells accumulated cyanophycin amounting up to 3.5% of the dry cell matter. The cyanophycin was purified and chemical analysis showed that it was composed of arginine and aspartic acid occurring at a molar ratio of 1:0.9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a broad distribution of the apparent molecular masses ranging from 20 to 130 kDa with a maximum at 50 kDa. During a three-step purification procedure involving ion exchange chromatography and gel filtration, the cyanophycin synthetase from strain MA19 was purified 144-fold to electrophoretic homogeneity. It consisted of only one single type of subunit exhibiting an apparent molecular mass of 130 kDa. The enzyme catalyzed the polymerization of arginine and aspartate at elevated temperatures and was even active at 80 degrees C.     

Cyanophycin production from feather hydrolysate using biotechnological methods

Abstract

Cyanophycin is a bacterial storage polymer for carbon, nitrogen and energy with emerging industrial applications. As efficient cyanophycin production is enhanced by peptone, but commercial peptones are very expensive, thereby increasing the overall production cost, an enzymatically produced feather hydrolysate (FH) is assessed as a cheap replacement of peptone to lower the costs and make cyanophycin production more economically feasible. Keratinase production using feather as the sole carbon/nitrogen source by S.pactum 40530 at 30-L fermentation scale was achieved within 93?h with degradation rate of 96.5%. A concentration of 60?g/L of FH, generated by keratinolytic activity (8?×?10³?U?g^?1L^?1d^?1) within 24?h, was used as the main carbon/peptone source to produce cyanophycin. The growth performances of E. coli DapE/L using FH was compared to that of casamino acids (CA) and up to 7.1?±?0.4 and 5.3?±?0.3?g/L of cell mass were obtained after 72?h from FH and CA, respectively. Cyanophycin production yielded 1.4?±?0.1g/L for FH with average molecular mass of 28.8 and 1.4?±?0.2 for CA with average molecular mass of 35.3, after 60?h. For the first time, FH generated by biotechnological methods from environmentally problematic, abundant and renewable feather bioresource was successfully used for cyanophycin biopolymer production.     

Effects of light and chloramphenicol stress on incorporation of nitrogen into cyanophycin in Synechocystis sp. strain PCC 6308

(1)H NMR spectroscopy was used to compare the uptake of nitrogen into cyanobacterial cyanophycin from two sources: from the breakdown of intracellular proteins and amino acids, and directly from the external growth medium. Cells grown initially in medium containing (14)N-nitrate were transferred to (15)N-nitrate medium in the presence of chloramphenicol in both low (4 microE m(-2) s(-1)) and normal (100 microE m(-2) s(-1)) light, and in low light alone. Cyanophycin was separated from cells and analyzed by (1)H NMR spectroscopy. Cyanophycin is synthesized both from (14)N (degradation of cellular proteins) and from (15)N in the medium, the latter at a faster rate and to a greater extent under all conditions. SDS-PAGE showed that cyanophycin synthesis takes place by addition of monomers to already synthesized polymer.     

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.     

Nutritional control of the frequency of MNNG-induced true branching habit in Anabaena doliolum

Summary MNNG survival and mutagenesis were studied in Anabaena doliolum Bharadwaja. The survival curves of germinating spores were exponential while those of resting spores and filament-fragments were sigmoidal. Blue mutants with much higher phycocyanin contents than the parent were recovered in varying frequencies; the frequency (1.1%) was the highest with 2 mg/ml MNNG for 15 min. Some of the blue mutants sporulated normally; while others did not. A nonsporulating blue mutant (M16) showed branched filaments in liquid cultures. In nitrogen free medium. M16 had a high frequency (70%) of branched filaments during the early phase of growth. In some filaments, the branch arose from a heterocyst showing three polar nodules. There was no other difference between the parent and the mutant in cell morphology and cellular differentiation. High light intensity adversely affected phycocyanin and chlorophyll a pigments and nitrogen fixation in the mutant. Frequency of mutant branched character appears to beta factor of inorganic nitrogen nutrition.Dedicated to the memory of my revered teacher the late Prof. R. N. Singh     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

Cyanophycin and glycogen synthesis in a cyanobacterial Scytonema species in response to salt stress

The response to salinity of a Scytonema species isolated from the central Australian desert was studied. Under nitrogen-fixing conditions the addition of increasing concentrations of salt (NaCl) caused progressive inhibition of growth, with growth ceasing at 150 mM NaCl. This correlated with a progressive loss of nitrogenase activity, a low level of activity being retained at 150 mM NaCl. The inhibition of growth was overcome when KNO₃ (10 mM) was added to the growth medium. In response to the salt stress, cells accumulated the reserve compounds cyanophycin and glycogen. Time course experiments showed that they were steadily synthesized over 48 h, after which the concentrations stabilized. Cyanophycin synthesis was enhanced in salt-stressed cells grown in nitrate. When cells were restored to their normal growth medium the content of these substances decreased towards control levels.     

Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria

Heterocyst-forming filamentous cyanobacteria are true multicellular prokaryotes, in which heterocysts and vegetative cells have complementary metabolism and are mutually dependent. The mechanism for metabolite exchange between cells has remained unclear. To gain insight into the mechanism and kinetics of metabolite exchange, we introduced calcein, a 623-Da fluorophore, into the Anabaena cytoplasm. We used fluorescence recovery after photobleaching to quantify rapid diffusion of this molecule between the cytoplasms of all the cells in the filament. This indicates nonspecific intercellular channels allowing the movement of molecules from cytoplasm to cytoplasm. We quantify rates of molecular exchange as filaments adapt to diazotrophic growth. Exchange among vegetative cells becomes faster as filaments differentiate, becoming considerably faster than exchange with heterocysts. Slower exchange is probably a price paid to maintain a microaerobic environment in the heterocyst. We show that the slower exchange is partly due to the presence of cyanophycin polar nodules in heterocysts. The phenotype of a null mutant identifies FraG (SepJ), a membrane protein localised at the cell-cell interface, as a strong candidate for the channel-forming protein.     

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.     

Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

N(2)O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N(2)O uptake and conversion of (15)N-N(2)O into (15)N-N(2). Free-living cells of USDA110 showed N(2)O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N(2)O reductase activity. When detached soybean nodules formed with USDA110 were fed with (15)N-N(2)O, they rapidly emitted (15)N-N(2) outside the nodules at a ratio of 98.5% of (15)N-N(2)O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N(2)O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N(2)O (0.34 ppm). These results indicate that the conversion of N(2)O to N(2) depends exclusively on the respiratory N(2)O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N(2)O.     

HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2-FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)

The process of N₂ fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin-containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.     

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.     

Effect of nitrogen source on cyanophycin synthesis in Synechocystis sp. strain PCC 6308

Experiments were carried out to examine the effects of nitrogen source on nitrogen incorporation into cyanophycin during nitrogen limitation and repletion, both with or without inhibition of protein synthesis, in cyanobacteria grown on either nitrate or ammonium. The use of nitrate and ammonium, 14N labeled in the growth medium and 15N labeled in the repletion medium, allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance spectroscopy. The data suggest that nitrogen from both the breakdown of cellular protein (14N) and directly from the medium (15N) is incorporated into cyanophycin. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells and to the possible expression of cyanophycin synthetase during nitrogen starvation.     

Microbial production of cyanophycin: From enzymes to biopolymers

Cyanophycin is an attractive biopolymer with chemical and material properties that are suitable for industrial applications in the fields of food, medicine, cosmetics, nutrition, and agriculture. For efficient production of cyanophycin, considerable efforts have been exerted to characterize cyanophycin synthetases (CphAs) and optimize fermentations and downstream processes. In this paper, we review the characteristics of diverse CphAs from cyanobacteria and non-cyanobacteria. Furthermore, strategies for cyanophycin production in microbial strains, including Escherichia coli, Pseudomonas putida, Ralstonia eutropha, Rhizopus oryzae, and Saccharomyces cerevisiae, heterologously expressing different cphA genes are reviewed. Additionally, chemical and material properties of cyanophycin and its derivatives produced through biological or chemical modifications are reviewed in the context of their industrial applications. Finally, future perspectives on microbial production of cyanophycin are provided to improve its cost-effectiveness.     

Myxoxanthophyll is required for normal cell wall structure and thylakoid organization in the cyanobacterium Synechocystis sp. strain PCC 6803

Myxoxanthophyll is a carotenoid glycoside in cyanobacteria that is of unknown biological significance. The sugar moiety of myxoxanthophyll in Synechocystis sp. strain PCC 6803 was identified as dimethyl fucose. The open reading frame sll1213 encoding a fucose synthetase orthologue was deleted to probe the role of fucose and to determine the biological significance of myxoxanthophyll in Synechocystis sp. strain PCC 6803. Upon deletion of sll1213, a pleiotropic phenotype was obtained: when propagated at 0.5 micromol photons m(-2) s(-1), photomixotrophic growth of cells lacking sll1213 was poor. When grown at 40 micromol photons m(-2) s(-1), growth was comparable to that of the wild type, but cells showed a severe reduction in or loss of the glycocalyx (S-layer). As a consequence, cells aggregated in liquid as well as on plates. At both light intensities, new carotenoid glycosides accumulated, but myxoxanthophyll was absent. New carotenoid glycosides may be a consequence of less-specific glycosylation reactions that gained prominence upon the disappearance of the native sugar moiety (fucose) of myxoxanthophyll. In the mutant, the N-storage compound cyanophycin accumulated, and the organization of thylakoid membranes was altered. Altered cell wall structure and thylakoid membrane organization and increased cyanophycin accumulation were also observed for deltaslr0940K, a strain lacking zeta-carotene desaturase and thereby all carotenoids but retaining fucose. Therefore, lack of myxoxanthophyll and not simply of fucose results in most of the phenotypic effects described here. It is concluded that myxoxanthophyll contributes significantly to the vigor of cyanobacteria, as it stabilizes thylakoid membranes and is critical for S-layer formation.     

NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain PCC 6308

1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy has been used to characterize cyanophycin, a multi-l-arginyl-poly-[l-aspartic acid] polypeptide from the cyanobacterium Synechocystis sp. strain PCC 6308. 1H, 13C and 15N chemical shifts and 1JHN and 1JCN coupling constants were measured in isolated 15N-labeled cyanophycin, and showed chemical shift values and J-couplings consistent with the reported polypeptide structure. 15N enrichment levels were determined from the extent of 1H-15N J-coupling in 1H NMR spectra of cyanophycin. Similar experiments using 13C-15N coupling in 13C NMR spectra were not useful in determining enrichment levels.