Production and regeneration of spheroplasts from the cyanobacterium Spirulina platensis

Abstract The development of a micro-method for the production and regeneration of spheroplasts starting from S. platensis trichomes is presented. The influence of the growth stage along with different treatments and conditions on the efficiency of spheroplast formation and regeneration are analyzed.     

Isolation and characterization of amino acid-analogue-resistant mutants of Spirulina platensis

Amino acid-analogue-resistant mutants of the cyanobacterium Spirulina platensis were isolated using amino acid analogues -2-thienylalanine, p-fluorophenylalanine, ethionine and azetidine-2-carboxylic acid. The growth and other cellular contents in these mutants were less than in the parent. The internal free amino acid pool showed varying amounts. Maximal overproduction occurred of proline whereas overproduction of aspartic acid, alanine and lysine was much less.     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.     

Two tuf genes in the cyanobacterium Spirulina platensis.

Probes derived from the tufA gene of Escherichia coli have been utilized to detect homologous sequences on Spirulina platensis DNA. A 6-kilobase-pair fragment of S. platensis DNA appears to contain two sequences homologous to the E. coli gene. Thus, as reported for gram-negative bacteria, the cyanobacterium presumably contains two tuf genes.     

Production,isolation and preliminary characterization of the exopolysaccharide of the cyanobacterium Spirulina platensis

Summary The soluble exocellular polysaccharide secreted by the filamentous cyanobacteria Spirulina platensis is a primary metabolite. It is formed by ten different types of monomer units including six neutral sugars (xylose, rhamnose, fucose, galactose, mannose and glucose in the proportions 1.3/0.3/0.7/2.7/traces/2), two unidentified sugars, two uronic acids and sulphate groups accounting for 40 % and 5 % respectively of the mass of the molecule. This polysaccharide displays a non Newtonian behaviour and a strong pseudoplastic characteristic that may originate in the polyelectrolytic property of the molecule.     

Chemical stabilization of the phycocyanin from cyanobacterium Spirulina platensis

Phycocyanin (PC) prepared from a cyanobacterium Spirulina platensis by the DEAE-DE52 cellulose column chromatography that was developed by gradient elution of 50-250 mM phosphate buffer (pH 7.0) was stabilized by its subunits cross-linked covalently with formaldehyde. The single blue band that the chemically stabilized PC showed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the stabilized PC still maintained its trimeric aggregate form even after its incubation at 60 degrees C for 3h and at 100 degrees C for 10 min in the denatured buffer containing 5% (w/v) SDS. Moreover, the stabilized PC exhibited similar spectroscopic properties of absorption and fluorescence to those of the native PC, and showed adequate energy coupling with R-phycoerythrin (R-PE) after it was conjugated with R-PE via glutaraldehyde.     

Effective transformation of the cyanobacterium Spirulina platensis using electroporation

Although Spirulina (Arthrospira) is expected to be a suitableorganism for producing recombinant proteins, a gene transfer system hasnot yet been established, due to a lack of suitable vectors and because Spirulina appears refractory to common genetic manipulations. As theinitial stages of the development of recombinant DNA methodology, weexamined the effects on transformation efficiency of electroporationconditions such as electric-field strength (2, 4, 6, 8, 10, 12kV cm^-1) and time constant (2.5, 5 ms). At a time constant of2.5 ms, few transformants were observed regardless of the field strength.The longer time constant of 5.0 ms reproducibly yielded transformants atthe middle field strength of 4 - 8 kV cm^-1, but gave high killingand no transformation at the higher field strength of 10 - 12kV cm^-1. Chloramphenicol acetyltransferase (CAT) activities wereincreased only in the transformants from 2–6 kV cm^-1 and 5.0 ms.The density of the transformants was significantly correlated with therelative value of CAT activity (r = 0.89, n = 11, p < 0.01),suggesting that the chloramphenicol resistance was due to CAT activity. Weconcluded that transformation of S. platensis was most effective at apulse duration 5.0 ms with an electric field of 4 kV cm^-1, and thatforeign genes can be expressed in this organism.     

Photooxidative damage to the cyanobacterium Spirulina platensis mediated by singlet oxygen

Experiments on the specific growth rate, bleaching of pigments, O₂ evolution, lipid peroxidation, and loss of sulfhydryl (-SH) content in response to the varying light intensities (2–28 W/m²) suggested that photodamage to the Spirulina cells was maximum at or beyond the photosynthesis saturating light intensity (12 W/m²). However, photobleaching of the chlorophyll a was relatively higher than carotenoid. The results on the N,N-dimethyl-p-nitrosoaniline (RNO) bleaching in the presence of oxygen radical quenchers exhibited maximum effect of sodium azide and indicated about the generation of singlet oxygen. The chlorophyll a-sensitized production of singlet oxygen by a type II reaction cannot be ruled out because of maximum oxidative damage to the cells at or beyond the photosynthesis saturating light intensity, i.e., 12 W/m², when the availability of triplet chlorophyll is maximum.     

Physico-chemical parameters influencing DNase activity of the cyanobacterium Spirulina platensis

The effects of temperature, Mg2+, EDTA concentration and rinsing on extra- and intra-cellular DNase activity of Spirulina platensis strain SSP-14, were investigated. The results indicate that the tested strain contains very high extra- and intracellular DNase activity, which actually hinders the transfer of foreign gene(s) to S. platensis, a cyanobacterium with multiple economic potentials. The extracellular DNase activity could easily be removed by rinsing the cells with Zarrouk medium more than once. The intracellular DNase activity could also be inhibited by (1) removal of Mg2+, (2) maintaining EDTA concentration above 1 mmol l(-1), and (3) manipulating below 0-4 degrees C, during all the incubation procedures. We suggest that, by using one or more of, or combining, all those experimental conditions, the chances of foreign DNA attempted to be introduced into S. platensis without being digested would be increased.     

A glucan from the cell wall of the cyanobacterium Spirulina platensis

A polysaccharide was isolated from the cell wall of the cyanobacterium Spirulina platensis. Hydrolysis of the polysaccharide only yielded glucose. Curie-point pyrolysis mass spectrometry of the polysaccharide resulted in a spectrum very similar to that of -1,2-glucan. Probably the glucan originates from the fibrillar inner layer of the cell wall.     

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.Publication No. 34 from the Micro-Algal Biotechnology Lab.     

Intracellular phosphorus pool in a culture of the cyanobacterium Spirulina platensis

An intracellular phosphorus pool in a monoculture of the cyanobacterium Spirulina platensis was assessed using radioactive and nonradioactive phosphorus. The derived dependence of specific growth rate on the intracellular content of mineral phosphorus can be presented in the form of the Droop equation. It was found that the stage of replenishment of the intracellular phosphorus pool may affect the phosphorus turnover estimation in aquatic environments from the results of short-term measurements of phosphorus uptake.     

Modelling of growth conditions for cyanobacterium Spirulina platensis in microcosms

The influence of cultivation conditions on the growth of the cyanobacterium Spirulina platensis was investigated by using two types of photobioreactors. In a rotative photobioreactor the doubling time (t _d) was 3.54 days. The better value found for t _d in an aerated photobioreactor by changing the initial nitrogen concentration (NaNO₃) at 0.003, 0.015, 0.030 and 0.060M was 2.5 days. A factorial experimental design was performed in order to estimate the contributions of initial nitrogen concentration, inoculum and cultivation time as well as their interactions. All three factors and their interactions proved to be significant in influencing the cellular concentration of S. platensis.     

Molecular characterization of an adenylate cyclase gene of the cyanobacterium Spirulina platensis

A cyaA gene, encoding an adenylate cyclase, was isolated from a filamentous cyanobacterium, Spirulina platensis, by functional complementation of a cya mutant of Escherichia coli, defective in adenylate cyclase activity. The predicted gene product of cyaA contains a signal peptide-like domain, a putative sensor domain similar to the gene product of vsrA of Pseudomonas solanacearum, a putative membrane-spanning domain and an adenylate cyclase-like catalytic domain. Two other positive clones that complemented the E. coli mutant were isolated from the same cyanobacterium, suggesting that several cya genes are functioning in S. platensis.     

Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis.

The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.     

Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis.

The gene for beta-isopropylmalate dehydrogenase (EC 1.1.1.85) of Spirulina platensis (leuB) was cloned from a lambda EMBL3 genomic library by heterologous hybridization using the Nostoc UCD 7801 leuB gene as a probe. The sequence of the entire leuB coding region was determined as well as 645 bp of 5' flanking region and 956 bp of 3' flanking region. DNA sequencing revealed an open reading frame of 1065 nucleotides capable of encoding a polypeptide of 355 amino acids. Homologies between the amino acid sequence deduced from the nucleotide sequence of the S. platensis leuB gene and the amino acid sequences published for corresponding proteins either from bacteria or yeasts are 45% or more. Northern hybridization analysis indicated that the S. platensis leuB gene is transcribed as a single monocistronic RNA of approximately 1200 bases.