Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica

Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.

Cloning and expression of lipoxygenase gene from Anabaena sp. PCC 7120 and purification, characterization of the recombinant enzyme / 生物工程学报

We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.

A toxic cyanobacterial bloom in an urban coastal lake, Rio Grande do Sul state, Southern Brazil / Uma floração de cianobacterias em um lago costeiro urbano, Rio Grande do Sul, Brasil

Reports of cyanobacterial blooms developing worldwide have considerably increased, and, in most cases, the predominant toxins are microcystins. The present study reports a cyanobacterial bloom in Lake Violão, Torres, Rio Grande do Sul State, in January 2005. Samples collected on January 13, 2005, were submitted to taxonomical, toxicological, and chemical studies. The taxonomical analysis showed many different species of cyanobacteria, and that Microcystis protocystis and Sphaerocavum cf. brasiliense were dominant. Besides these, Microcystis panniformis, Anabaena oumiana,Cylindrospermopsis raciborskii, and Anabaenopsis elenkinii f. circularis were also present. The toxicity of the bloom was confirmed through intraperitoneal tests in mice, and chemical analyses of bloom extracts showed that the major substance was anabaenopeptin F, followed by anabaenopeptin B, microcystin-LR, and microcystin-RR.

O número de relatos de ocorrências de florações de cianobactérias em todo o mundo vem aumentando consideravelmente e na maioria desses episódios, as toxinas dominantes são as microcistinas. O presente estudo relata a ocorrência de floração na Lagoa do Violão, município de Torres, RS, em janeiro de 2005. As amostras coletadas em 13/01/2005 foram submetidas a estudos taxonômicos, toxicológicos e químicos. O exame microscópico do fitoplancton mostrou a dominância das espécies Microcystis protocystis e Sphaerocavum cf. brasiliense; foram observadas, também, Microcystis panniformis, Anabaena oumiana,Cylindrospermopsis raciborskii e Anabaenopsis elenkinii f. circularis. A toxicidade da floração foi confirmada através de ensaio intraperitonial em camundongos e a análise química de extratos obtidos da biomassa liofilizada mostrou que a substância majoritária era a anabaenopeptina F, seguida por anabaenopeptina B, microcistina-LR e microcistina-RR.

Iron induced metabolic changes in the diazotrophic cyanobacterium Anabaena PCC 7120.

Iron induced changes in growth, N2-fixation, CO2 fixation and photosynthetic activity were studied in a diazotrophic cyanobacterium Anabaena PCC 7120. Iron at 50 microM concentration supported the maximum growth, heterocyst frequency, CO2 fixation, photosystem I (PS I), photosystem II (PS II) and nitrogenase activities in the organism. Higher concentration of iron inhibited these processes. Chl a and PS II activities were more sensitive to iron than the protein and PS I activity.

Screening of Cyanobacteria (Blue-Green algae) from Rice Paddy Soil for Antifungal Activity against Plant Pathogenic Fungi

Soil cyanobacteria isolated from the rice paddy fields of 10 different locations across Korea were evaluated by agar plate diffusion test for antifungal activity. Aqueous, petroleum ether, and methanol extracts from one hundred and forty two cyanobacterial strains belonging to the 14 genera were examined for antifungal properties against seven phytopathogenic fungi causing diseases in hot pepper (Capsicum annuum L). Of total cyanobacteria, nine cyanobacteria (6.34%) exhibited antifungal effects. The nine cyanobacteria selected with positive antifungal activities were two species of Oscillatoria, two of Anabaena, three of Nostoc, one of Nodularia, and one of Calothrix. Alternaria alternata and Botrytis cinerea were inhibited by nine and eight species of cyanobacteria, respectively. Rhizopus stolonifer was suppressed by only methanol extract of Nostoc commune FK-103. In particular, Nostoc commune FK-103 and Oscillatoria tenuis FK-109 showed strong antifungal activities against Phytophthora capsici. Their antifungal activity at the late exponential growth phase is related to the growth temperature and not associated with the growth parameters such as cell biomass and chlorophyll-alpha concentration. The high inhibition levels of antibiotics were 22.5 and 31.8 mm for N. commune FK-103 and O. tenuis FK-109, respectively. The optimal temperature for antibiotic productivity was 35degrees C.

Studies on relationship between the expression of hTNF-alpha gene and photosynthesis in Anabaena sp. IB02 / 生物工程学报

The effects of illumination on growth of Anabaena sp. IB02 and hTNF-alpha expression were studied. Photosynthetic activity, PS I and PS II activity of Anabaena sp. IB02 were assayed. Illumination enhanced the growth of Anabaena sp. IB02 and hTNF-a expression. Some relations were observed between hTNF-alpha expression and ture photosynthesis activity, PS I, PS II activity of Anabaena sp. IB02. Significant differences of the photosynthetic activity of host were detected simultaneously when hTNF-a expressed: the respiration rate increased (-68%), the light saturation point descended (+66%), all these suggested that the metabolic charge of host were increased and grow faster than wild type under low illumination.

Activity of glycosidases from freshwater heterotrophic microorganisms on the degradation of extracellular polysaccharide produced by Anabaena spiroides (Cyanobacteria)

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41 per center do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59 per center). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-a-D- e 4-MUF-b-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-a-L-ramnopiranosídeo, MUF-b-D-galactosídeo, MUF-a-D-manopiranosídeo, MUF-b-D-fucosídeo, MUF-b-D-manopiranosídeo, MUF-a-L-arabinopiranosídeo, e MUF-b-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.

Cyanobacterial N2 fixation in presence of nitrogen fertilizers.

Anabaena oryzae ARM 570 was examined for its growth (chlorophyll and protein), heterocyst frequency, nitrogenase (acetylene reduction) activity, ammonia excretion, and glutamine synthetase and nitrate reductase in response to two levels of urea-N vis-à-vis N2-N. Growth of cyanobacterium increased with duration of incubation. Reduction in heterocyst frequency (40%) was observed at 30 ppm of urea-N, whereas at 60 ppm of urea-N, filaments were completely devoid of heterocysts and no nitrogenase activity was observed. Maximum excretion of ammonia occurred at 30 ppm of urea-N, which was concomitant with minimum glutamine synthetase activity. These results suggested that A. oryzae could be effectively utilized in cyanobacterial biofertilizer programme even in the presence of combined nitrogen, for improving N-budget in rice cultivation.

Crecimiento, producción de pigmentos y exopolisacáridos de la cianobacteria anabaena sp. PCC 7120 en función del pH y CO2 / Effect of pH and CO2 on growth, pigments and exopolysaccharides production from cianobacteria anaebaena sp. PCC 7120

El crecimiento producción de clorofila a, ficocianina, carotenoides y exopolisacáridos (EPS) de la cianobacteria filamentosa Anabaena sp. PCC 7120 fueron analizados en función del pH (5,5-10,5) con o sin soluciones amortiguadoras en cultivos discontinuos y en función del CO2 (0,03 y 5,0 por ciento) en cultivos semicontinuos a una tasa de renovación del 10 por ciento. El crecimiento de la cianobacteria se analizó por turbidez a 750nm y recuento celular, y la actividad fotosintética mediante uso de un electrodo de O2. Los cultivos, por triplicado, se mantuvieron con aireación constante, a 28 ñ 2ºC y con iluminación continua o fotoperíodo según el experimento. El pH y tampón utilizado influyeron en el crecimiento. El mayor crecimiento y contenido de EPS se alcanzaron, respectivamente, a pH 8,0-9,0 y 10,0. Sin embargo, los cultivos no tamponados resultaron con mayor contenido de clorofila a y ficocianina. El crecimiento, la actividad fotosintética y los carotenoides no variaron con la adición de CO2. Los cultivos semicontinuos con bajo CO2 produjeron los valores más elevados de clorofila a, ficocianina y exopolisacáridos, con 25,9 ñ 1,69, 2010 ñ 22,61 y 2286,0 ñ 42,76 µg.ml-1, respectivamente. El contenido de ficocianina y de EPS fue de 2,7 y 4 veces superior al obtenido a altos niveles de CO2. El pH y el sistema semicontinuo constituyen herramientas importantes para modular el crecimiento y contenido de pigmentos y de EPS de Anabaena sp. PCC 7120

Physiological and biochemical alterations in Anabaena 7120 under iron stress.

Various physiological and biochemical process like growth, NO3- -uptake, nitrate reductase, glutamine synthetase and ATPases (Mg2+ and Ca2+ dependent) in the cyanobacterium Anabaena 7120 were observed under iron stress. Growth was found to be maximum in 50 microM Fe3+ added cells however, 20 microM Fe3+ (the Fe3+ concentration generally used for routine culturing of cyanobacterial cell in Chu 10 medium) incubation resulted in lower growth. Fe3+ starvation on the other hand showed very poor growth up to 4th day but once the growth started it reached at significant level on 7th day. Higher Fe3+ concentration reflected reduced growth with lethality at 500 microM Fe3+. Chlorophyll a fluorescence under Fe3+ stress reflected almost the similar results as in case of growth. However, the pigment was found to be more sensitive as compared to protein under Fe3+ stress. Similar results have been observed in case of NO3-uptake with only 80% reduction in nutrient uptake in 500 microM Fe3+ incubated cells. Nitrate reductase activity was lower in Fe3+ starved cells as compared to significant enzyme activity in 20 and 50 microM Fe3+ incubated cells. Similar to nitrate reductase, glutamine synthetase also showed maximum level in 50 microM Fe3+ added cells, however, higher Fe3+ concentration (300-500 microM ) resulted in reduced enzymatic activity. Glutamine synthetase activity was less sensitivity as compared to nitrate reductase activity under Fe3+ stress. ATPase (Mg2+ and Ca2+ dependent) always showed higher level with increasing Fe3+ concentration.

Effect of butachlor on growth and nitrogen fixation by Anabaena sphaerica.

Present study was carried out to examine the effect of Butachlor on growth and nitrogen fixation by Anabaena sphaerica. The increased concentration of the pesticide did not have any adverse effect on the alga. Rather it accelerated the algal contribution in terms of biomass and nitrogen fixation.

Biodiversity of Anabaena azollae isolates from different Azolla cultures.

The random amplified polymorphic DNA (RAPD) profile of A. azollae strains isolated from four different Azolla cultures was studied by using different primers. The objective of this study was to determine whether polymerase chain reaction (PCR) with different primers could differentiate the isolated A. azollae strains from one another. The primers amplified specific sequences of the isolates and generated fingerprinting pattern characteristic of each isolate. Clear polymorphism was noticed among all the strains which depends on the primer sequence.

Influence of clay minerals on selenium toxicity to algae

In this study, selenite was found to be acutely toxic to the fresh water algae Anabaena constricta and Chlorella ellipsoidea. However, the latter was more tolerant. Severe reduction in chlorophyll a content was significant reaching 65.2%, 61.4% for Anabaena and Chlorella respectively at 30mg L[-1] Se after 24 hrs incubation. The EC50 values were: 138 mg SeL-1, 141 mg Se L[-1] at one day; 120 mg Se L[-1], 126 mg Se L[-1] at 3 days; 91 mg Se L[-1], 100 mg Se L[-1] at 5 days and 67 mg Se L[-1], 70 mg Se L[-1] at 7 days for Anabaena and Chlorella, respectively. Addition of the clay minerals kaolinite and bentonite decreased selenite toxicity considerably, bentonite was more effective. The EC50 values for combined effect of selenium and either kaolinite or bentonite in reducing growth after 7 days incubation were 76,83 mg Se L[-1] for Anabaena and 73, 81 mg Se L[-1] for Chlorella. Phosphorus uptake by Chlorella was enhanced and went parallel with a concomitant elevation in pH values of the medium with increasing selenite concentrations, and observation which was controversely to that exhibited in Anabaena cultures. Acid and alkalize phosphatase activity decreased in both organisms with increasing Se concentrations

Potency of nitrogen fixation, nitrogenase and nitrate reductase activities in anabaena oryzae and nostoc muscorum, as influenced by some pesticides

Exposure of Anabaena oryzae and Nostoc muscorum cultures to different rates of some common pesticides [dimethoate, dursban, Karathane and trifluralin] resulted generally in pronounced disturbances in nitrogen metabolism, stimulated the process of nitrogen fixation and nitrate reductase activities in both cyanobacteria. Elevation in total fixed nitrogen by either organism was always consistent with respective rise in the amounts of total nitrogen released to the media and total nitrogen contents of both species. Nitrogenase activity in Nostoc muscorum and Anabaena oryzae tended to floculate following pesticides application, depending on the type of the pesticide, its concentration and also incubation period. The increase in potency of nitrogen fixation of both organisms was not accompanied, in some treatments by respective rise in nitrogenase activity. A possible explanation relevant to such contradiction may lay on the interaction of several physical, chemical and biological factors, which can influence the metabolism of the pesticide, altering respiratory and enzymatic potentialities

Amino acid content and transaminases activities in anabaena oryzae and nostoc muscorum as affected by some pesticides

Dimethoate and Karathane induced statistically highly -significant decreases in GOT activities of Nostoc and Anabaena. Dursban was less effective, whereas trifluralin encouraged the enzyme activity in Anabaena, but hardly affected it in Nostoc. GPT, behaved differently in both organisms, as a result of pesticides application. Since it was stimulated in dimethoate treated cultures of either cyanobacterium. Dursban, Karathane, and trifluralin, although favored the enzyme activity in Anabaena, yet they inhibited or even totally arrested its potentiality in Nostoc. The moderate doses of dimethoate, Karathane and trifluralin attenuated synthesis of all individual amino acids in both organisms, though valine was hardly affected. Dursban on the other hand, encouraged the biosynthesis of all individual amino acids in Nostoc and Anabaena; an effect that led subsequently to significant elevation in total amino acid content of the two cyanobacterial biomasses. The applied pesticides, at all levels, accelerated heterocyst development in Anabaena, but were of no detectable effects on its differentiation in Nostoc

Influence of trace elements, tungsten and vanadium, on the in vivo nitrate reduction in azoola-anabaena symbiosis

Molybdenum [Mo] is an essential microelement for the induction of nitrate reductase in Azolla-plants. In Mofree cultures, tungsten [W] or vanadium [V] could not replace Mo. for nitrate reductase activity. Although- vanadium, up to a concentration of 0.01 ppm, enhanced the activity of nitrate reductase in Mo-containing cultures, tungsten showed an inhibitory effect. The suppression of nitrate reductase activity may, be related to the replacement of Mo co-factor or inhibition of the NADH-linked enzyme