Large scale preparation of pure phycobiliproteins

This paper describes simple procedures for the purification of large amounts of phycocyanin and allophycocyanin from the cyanobacterium Microcystis aeruginosa. A homogeneous natural bloom of this organism provided hundreds of kilograms of cells. Large samples of cells were broken by freezing and thawing. Repeated extraction of the broken cells with distilled water released phycocyanin first, then allophycocyanin, and provides supporting evidence for the current models of phycobilisome structure. The very low ionic strength of the aqueous extracts allowed allophycocyanin release in a particulate form so that this protein could be easily concentrated by centrifugation. Other proteins in the extract were enriched and concentrated by large scale membrane filtration. The biliproteins were purified to homogeneity by chromatography on DEAE cellulose. Purity was established by HPLC and by N-terminal amino acid sequence analysis. The proteins were examined for stability at various pHs and exposures to visible light.Abbreviations A absorbance at wavelength in nanometers - DEAE cellulose diethylamino ethyl cellulose - HPLC high pressure liquid chromatography - UV ultraviolet     

Isolation of photosynthetic catalysts from cyanobacteria.

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.     

Nitrogen fixing cyanobacteria potential uses

The large scale production of nitrogen fixing cyanobacteria is discussed and the use of ammonia excreting mutants ofAnabaena siamensis is described.Gloeotrichia natans is considered for use as a biofertilizer and for the production of phycobiliproteins.     

Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and, of phycocyanin from the cyanobacterium Spirulina maxima(=Arthrospira maxima) strain F3. The - and -subunitgene-coding regions contain 489 bp and 519 bp,respectively. The -subunit gene is upstream from the -subunitgene,with a 111 -bp segment separating them. Similarities between the-subunits of S. maxima and seven othercyanobacteriawere between 63% and 99%, as were those between the -subunits. Themaximumsimilarity between the - and -subunits from S.maxima was 27%.     

Surface charge on thylakoid membranes: regulation of photosynthetic electron transfer in cyanobacteria

The establishment of the steady-state rate of photosynthetic O₂ evolution by cells of Anabaena variabilis and other cyanobacteria was found to be preceded by a lag-phase the duration of which depended on the time of cell preincubation in the dark. Electron acceptors (benzoquinone, N,N,N,N-tetramethyl-p-phenylenediamine, 2,3,5,6-tetramethyl-p-phenylenediamine or 2,6-dichlorophenolindophenol) abolished the lag-phase as well as the inhibitory effect of cyanide on electron transfer. Mono-, di-and trivalent cations added to the cell suspension markedly reduced the lag-phase. As cation concentrations were increased, acceleration and subsequent deceleration of the O₂ evolution rate were observed. The efficient concentrations of cations decreased as their valency increased. The lag-phase and the rate of photosynthetic O₂ evolution by the blue-green algae are suggested to depend on the value of the membrane surface charge governing the electrostatic interaction between unidentified membrane-bound redox components. The combination of valinomycin and nigericin as well as gramicidin D enhanced the duration of the lagphase by deenergization of thylakoid membrane.Abbreviations 9AA 9-aminoacridine - BQ benzoquinone - DAD 2,3,5,6-tetramethyl-p-phenylenediamine - DPIP 2,6-dichlorophenolindophenol - FeCy ferrycyanide - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - MES 2(N-morpholino)ethane sulphonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine - Tris tris(hydroxymethyl)aminomethane     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

The properties of transketolase from photosynthetic tissue

Transketolase (E.C. 2.2.1.1.) has been partially purified from wheat (Triticum aestivum, cv. Sappo) and spinach (Spinacia oleracea) leaves. The fully-active enzyme is a tetramer of relative molecular mass (M_r) of 150 kM_r requiring thiamin pyrophosphate for maximal activity, and dissociating into a 74 kM_r dimer in its absence or in dilute solution. The chloroplastic transketolase (over 75% of the cellular total) is magnesium-stimulated but the cytosolic form is magnesium-insensitive. Both chloroplastic and cytosolic transketolase showed similar broad specificities towards several ketose phosphate substrates including fructose 6-phosphate and sedoheptulose 7-phosphate. Wheat and spinach leaf transketolases are not light-activated and closely resemble the yeast enzyme in many of their properties.Abbreviations Mr relative molecular mass - TPP thiamin pyrophosphate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Effects of water stress on cryptoendolithic cyanobacteria from hot desert rocks

Four strains of Chroococcidiopsis and one Chroococcus, all isolated from extreme arid desert rocks, and one marine Chroococcus, were subjected to water stress using both matric and osmotic control methods. For all Chroococcidiopsis strains, photosynthetic rates decreased with decreasing water potential. After 24h preincubation the decrease was linear but after 72h there was a sharp drop below-3400 kPa (a _w0.976). In contrast, the two Chroococcus strains showed optimum photosynthesis between-3000 and-4000 KPa. It appears, therefore, that Chroococcidiopsis in deserts may have a different survival strategy in response to aridity than Chroococcus (rare in deserts).Absolute rates of ¹⁴CO₂ uptake were higher in matric than in osmotic control systems. It is suggested that, in a matric experimental system, the water status is more representative of the natural conditions in arid environments.The consistent differences between different strains in their response to water stress suggest that this character in Cyanobacteria may be of taxonomic significance.     

Large scale inoculation of actinorhizal plants withFrankia

Summary From 1979 to 1984 more than seven million seedlings of actinorhizal plants were successfully inoculated on an industrial scale withFrankia inoculants. Nodulated seedlings were produced in greenhouses to be used for land reclamation in northern Québec by the Societe d'Energie de la Baie James (SEBJ) and also by the City of Montréal for a revegetation program. Crushed-nodule homogenates andFrankia pure culture formulations were compared for large scale inoculation of green alder. Pure culture inoculant was found to be superior than crushed-nodule homogenates yielding reproducible nodulation of seedlings. Two inoculation methods of theFrankia pure culture inocula were compared: soil injection and spraying with greenhouse watering devices. Both methods resulted in efficient nodulation ofAlnus crispa, A. glutinosa, A. rugosa, Elaeagnus angustifolia, E. commutata, Hippophaë rhamnoides, Myrica gale andShepherdia argentea.     

3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。     

Harvest of Arthrospira platensis from Lake Kossorom (Chad) and its household usage among the Kanembu

In 1997 a survey was conducted among the Kanembu whoharvest Arthrospira (Spirulina) from LakeKossorom in the Prefecture of Lac (Chad). Informationon the amount of Arthrospira harvested and thepreparation and use of dihé was obtained byinterviewing the women who daily gather around thelake for the harvesting. Dihé is obtained byfiltering and sun drying the algal biomass on thesandy shores of the lake. The semi-dried dihé is then cut into small squares and taken tothe villages, where the drying is completed on mats inthe sun. Dihé is mainly used to prepare la souce, a kind of fish or meat and vegetable broth.Part of the harvest is sold to local consumers or towholesalers, who trade the product in the markets ofMassakori, Massaquet and N'Djamena and also across theborder of the country. The local trading valueof the dihé annually harvested from LakeKossorom (about 40 t) amounts to more than US$100,000, which represents an important contributionto the economy of the area.     

Daphnia vertical distribution and the presence of toxic cyanobacteria

Daphnia can alter its vertical position in the water column in response to chemical cues from predators. In this study we tested the hypothesis that a Daphnia pulex clone with little evolutionary exposure to cyanobacteria would move away from patches of cyanobacteria (Anabaena affinis and A. flos-aquae) which contain potent endotoxins. Daphnia was censused at 2 h intervals for 6 h in laboratory columns in which there was a steep vertical gradient of cyanobacteria. Data were analyzed by repeated-measures ANOVA. Control (no Anabaena) and experimental columns showed no significant differences in Daphnia distributions. Daphnia in experimental columns frequently moved into areas with dense concentrations of Anabaena and stayed there for long periods of time. Our results show that this D. pulex clone does not exhibit a rapid (within 6 h) avoidance response to toxic Anabaena.     

Cloning and characterization of c-phycocyanin operon from the cyanobacterium <Emphasis Type="Italic">Arthrospira platensis</Emphasis> FACHB341

By using in vitro PCR method, C-phycocyanin operon of Arthrospira platensis FACHB341 was cloned and characterized. The operon consists of 427 bp ussB, 519 bp cpcB gene, 111 bp igsB-A region, 489 bp cpcA gene, 184 bp ussH region and 357 bp cpcH gene. Promoter prediction and signal scan show that there are putative promoter sequences and regulatory elements in ussB and ussH sequences.     

Spectral and functional characterization of membrane fragments from the facultative photosynthetic bacterium Rhodopseudomonas blastica

The cytochromes of photosynthetically grown Rhodopseudomonas blastica have been thermodynamically characterized using the technique of redox titrations. Six cytochromes were present; two cytochromes c, E _m7= +295mV, E _m7=+345mV; and four cytochromes b, E _m7=+290mV, E _m7=+130mV, E _m7=+60mV, E _m7=-4mV. These cytochromes were tightly bound except for cytochrome c with E _m7 of+345mV which was mostly present in the soluble cell extracts.The effects of cyanide on both the cytochrome c oxidase activity and the NADH-dependent respiration, revealed the presence of a branched respiratory chain, one branch leading to a cyanide-resistant oxidase containing pathway and the other including the cyanide-sensitive cytochrome c-oxidase.The effects of antimycin A, myxothiazol and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) on the steadystate NADH-dependent respiration were also studied. Antimycin A and myxothiazol appeared to act at the level of the ubiquinol-cytochrome c oxidoreductase while UHDBT drastically affected both respiratory branches.Absorption spectra of chromatophore photopigments resulted to be similar to those reported in many species of facultative photosynthetic bacteria although carotenoid absorption maxima were blue-shifted by 5 nm.The light-induced oxygen reduction performed by chromatophores from R. blastica suggested a strict interaction between photosynthetic and respiratory apparatuses.     

Large scale selection of aluminum-resistant mutants from plant cell culture: expression and inheritance in seedlings

Summary A large number of aluminum-resistant variants, selected from non-mutagenized homozygous diploid cell cultures of Nicotiana plumbaginifolia Viv., are characterized. Of 115 variants cloned and reselected from single cells, 67 retained Al resistance in callus cultures after 6–9 months of growth in the absence of Al. There was no association between Al resistance and callus growth in the absence of Al, suggesting that the Al-resistant phenotype is not detrimental in the absence of Al challenge and that Al resistance is not the result of increased vigor. Plants regenerated from initially resistant callus lines that subsequently lost their resistance failed, with one exception, to transmit resistance to their seedling progeny. Fertile plants were regenerated from 40 of the 67 variants that retained stable Al resistance in callus culture. All 40 transmitted Al resistance to their seedling progeny (selfed and backcrossed) in segregation ratios expected for a single dominant mutation. The selfed progeny of many variants also segregated for recessive lethal mutations which were attributed to independent mutations that occurred during cell culture.     

Bioactivity of intra and extracellular substances from cyanobacteria and lactic acid bacteria on “wood blue stain” fungi

Cyanobacteria (photoautotrophic prokariota) have potential for the control of pathogenic bacteria and fungi. The effect of intra and extracellular products from cyanobacterial strains on the growth of fungi isolated from “wood blue stain,” was tested. Extracellular products were obtained by concentration and sterilization of the culture medium where cyanobacteria were grown. Cyanobacterial substances promoted or inhibited fungal growth according to the fungal and cyanobacterial strains tested. Extracellular products from Nostoc muscorum 79a and the methanolic extract from Microchaete tenera 84b biomass inhibited growth of Sphaeropsis sapinea 2157 (64.7 and 775.6%, respectively). Extracellular products of Nostoc piscinale 59 and biomass methanolic extract from N. muscorum 79a produced the highest growth promotion of Trichoderma boningii 452 (105.0%) and T. viride 993 (136.7%). Extracellular products of the heterotrophic lactic acid bacterium Streptococcus termophilus were also tested and strongly inhibited (64–92%) all the fungal strains. The tested fungi have different sensitivity to the bioactive substances present in the biomass and/or the culture medium of the studied cyanobacteria and lactic acid bacterium. N. muscorum 79a, M. tenera 84b, and S. termophilus have potential to control the wood blue stain fungi by a friendly environmental alternative.