Unicellular Cyanobacterial Strains Research Articles

16S rRNA 분석에 의한 Subsection IV cyanobacteria 균주들의 다계통성 기원의 증거

Unicellular cyanobacterial strains of Subsections Ⅰ and Ⅱ and filamentous cyanobacterial strains of Subsection Ⅲ have been shown to be polyphyletic, heterocystous strains of Subsections Ⅳ and Ⅴ, both of which were previously reported to be monophyletic. In this study, the small subunit ribosomal RNA (16S rRNA) sequences of 13 strains of cyanobacteria - one strain, Oscillatoria nigro-viridis PCC7112, of the Subsection Ⅲ, 6 strains including genus Anabaena, Nostoc, Tolypothrix, Calothrix and Scytonema of the Subsection Ⅳ, and 6 strains including genus Hapalosiphon, Fischerella and Chlorogloeopsis of the Subsection Ⅴ - were determined. The phylogenetic analysis of cyanobacteria was carried out using the 16S rRNA sequences. The results of the phylogenetic analyses of 16S rRNA sequences, based on Neighbour-joining, maximum-parsimony, and maximum-likelihood methods, indicated that the members of Subsection Ⅳ were not monophyletic but polyphyletic. In addition, the phylogenetic results strongly indicated that the genus Scytonema in Subsection Ⅳ could be a common ancestor of heterocystous cyanobacteria in Subsection Ⅳ and Ⅴ. Furthermore, the phylogenetic analyses revealed that the genus Anabaena could be phylogenetically diverse and that cyanobacterial strains in Subsection Ⅳ might be polyphyletic, whereas those in Subsection Ⅴ could be monophyletic, as reported before. The results for the genus Anabaena indicate that it should be reclassified.

Enhanced photobiological H2 production by the addition of carbon monoxide and hydrogen cyanide in two unicellular N2-fixing cyanobacterial strains isolated from Korean coasts

Photobiological H2 from marine cyanobacterial strains is widely accepted to be an ideal clean and renewable energy source. Using the two Korean N2-fixing unicellular cyanobacterial strains (Cyanothece sp. KNU CB MAL-031 and Cyanothece sp. KNU CB MAL-058) and the Synechococcus sp. Miami strain BG043511 we performed flask-scale experiments to measure the effect of CO and HCN addition on photobiological H2 production. For the test, 1, 5, 10 and 30% v/v of CO in the N2 atmosphere was applied. Enhancement of H2 production was remarkable at 1–5% concentration range of CO addition. At CO concentrations over 5% no further cost-effective enhancement of H2 production was detectable, which suggests to us that 1–5% CO addition should be adopted for practical photobiological H2 production by the cyanobacterial strains. Maximum enhancement of the photobiological H2 production by CO additions was 2–6 times over the control flasks without CO. When 3 ppm of HCN was injected into the cell suspension of BG043511, the enhancement of hydrogen production was 50–60% of that under 5% CO. Present result implies the possible recycling of waste CO and HCN for the enhancement of the photobiological H2 production using marine cyanobacterial strains.

한국 연안산 질소고정 단세포 남세균 종주의 최적 성장 및 수소생산 온도

Photobiological hydrogen production by nitrogen-fixing unicellular cyanobacteria has long been considered to be an environmentally sound and very promising method for the future supply of renewable clean energy. Using six Korean nitrogen-fixing unicellular cyanobacterial strains and the Synechococcus sp. strain Miami BG043511 we performed cultivation experiments to find out the strain-specific optimal temperature for population growth and <TEX>$H_2$</TEX> production. Under <TEX>$20^{\circ}C$</TEX> the population growth of all the tested strains was significantly retarded in contrasts to the faster and higher growth under 25, 30 or <TEX>$35^{\circ}C$</TEX>. The highest growth rates in all the 7 strains were measured under <TEX>$30^{\circ}C$</TEX> while the maximal biomass yields were under <TEX>$30^{\circ}C$</TEX> (strains CB-MAL 026, 054, and 055) or <TEX>$35^{\circ}C$</TEX> (strains 002, 031, 058, and Miami BG043511). The difference between the maximal biomass yields at <TEX>$30^{\circ}C$</TEX> and <TEX>$35^{\circ}C$</TEX> was not greater than 10%. The quantity of photobiologically produced <TEX>$H_2$</TEX> was only slight larger under <TEX>$35^{\circ}C$</TEX> than that under <TEX>$20^{\circ}C$</TEX>. Our result may suggest a two-step process of <TEX>$H_2$</TEX> production which includes rapid and sizable production of biomass at <TEX>$30^{\circ}C$</TEX> and the following high <TEX>$H_2$</TEX> production at <TEX>$20^{\circ}C$</TEX> by the test strains of marine nitrogen-fixing unicellular cyanobacteria.

DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM)

A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with root cells and their visualization under CLSM. DTAF (5-(4,6-dichlorotriazinyl) aminofluorescein) is a fluorescent dye that has been widely used for staining various biological samples for fluorescent microscopy. It reacts with polysaccharides and peptides at ordinary conditions. The possible application and efficiency of DTAF for CLSM studies were examined in various aspects of cyanobacterial-plant interactions. Seedlings of Pisum sativum, Vigna rediata and Triticum aestivum were co-cultivated and stained with DTAF as a fluorochrome. Extracellular and intracellular interactions of cyanobacteria and the plant root surface were observed by CLSM. Results were compared with staining by other commonly used lectins. Advantages of the use of DTAF over other stains are its penetration into root tissues and binding with polysaccharides, mainly the cellulose. The staining was smooth, which clearly showed minute details on the cell of surface and root hairs with higher resolution. The emission wavelength for DTAF is 517 nm, which is highly advantageous as cyanobacteria have auto-fluorescence at 665 nm, and both can be simultaneously used in CLSM by visualizing in different channels. This worked efficiently with all three plants used and with filamentous and unicellular cyanobacterial strains. Cyanobacterial presence was not only clearly observed on the root surface, but also inside the root tissue and epidermal cells. The easy protocol and absence of tissue processing make DTAF a useful probe for studies of cyanobacterial associations with plant roots by CLSM.

The iron-regulated isiA gene of Fischerella muscicola strain PCC 73103 is linked to a likewise regulated gene encoding a Pcb-like chlorophyll-binding protein.

The expression of the chlorophyll a-binding, iron stress-induced protein IsiA is part of the cyanobacterial response to iron deficiency. A new isiA gene from the filamentous heterocystous cyanobacterial strain, Fischerella muscicola PCC 73103, was identified using standard and inverse PCR. While in unicellular cyanobacterial strains isiA is organized in an operon with isiB (encoding flavodoxin), in Fischerella not an isiB gene but another chlorophyll-binding protein encoding gene was identified downstream of isiA, which shows significant similarities to Pcb-like protein encoding genes known from prochlorophytes. The expression of both genes was clearly activated under iron deficiency. Although isiA and pcbC were independently transcribed, the size of the pcbC transcript indicates a large iron-regulated operon. Beside a 10-fold increase of isiA transcript content iron-starved cells of Fischerella showed a blue-shift in the red chlorophyll a absorption peak. In addition, chlorophyll fluorescence at 77 K was dominated by an emission peak at 685 nm. These features are in accordance with the characteristics of IsiA accumulation in iron-starved unicellular cyanobacteria, suggesting identical IsiA function in heterocystous strains in spite of different genetic organization.

The iron-regulated isiA gene of Fischerella muscicola strain PCC 73103 is linked to a likewise regulated gene encoding a Pcb-like chlorophyll-binding protein

The expression of the chlorophyll a-binding, iron stress-induced protein IsiA is part of the cyanobacterial response to iron deficiency. A new isiA gene from the filamentous heterocystous cyanobacterial strain, Fischerella muscicola PCC 73103, was identified using standard and inverse PCR. While in unicellular cyanobacterial strains isiA is organized in an operon with isiB (encoding flavodoxin), in Fischerella not an isiB gene but another chlorophyll-binding protein encoding gene was identified downstream of isiA, which shows significant similarities to Pcb-like protein encoding genes known from prochlorophytes. The expression of both genes was clearly activated under iron deficiency. Although isiA and pcbC were independently transcribed, the size of the pcbC transcript indicates a large iron-regulated operon. Beside a 10-fold increase of isiA transcript content iron-starved cells of Fischerella showed a blue-shift in the red chlorophyll a absorption peak. In addition, chlorophyll fluorescence at 77 K was dominated by an emission peak at 685 nm. These features are in accordance with the characteristics of IsiA accumulation in iron-starved unicellular cyanobacteria, suggesting identical IsiA function in heterocystous strains in spite of different genetic organization.

A cyanobacterial recombination study, involving an efficient N 2-fixing non-heterocystous partner

Many filamentous cyanobacteria fix atmospheric nitrogen under natural conditions in specialized anaerobic compartments, heterocysts, interspersed between vegetative cells, which provide protection to the O 2-sensitive nitrogenase. A few unicellular cyanobacterial strains are also known to fix nitrogen aerobically at a slower rate. Filamentous cyanobacteria lacking heterocysts are not known so far to fix nitrogen. We describe the isolation and purification of a non-heterocystous filamentous cyanobacterium from the fronds of the water-fern Azolla, fixing nitrogen at 18.7 ± 0.2 n moles ethylene μg Chi. a -1 h -1 when grown in nitrogen-free medium at a low level of oxygen between two layers of agar. This strain of Anabaena azollae has been designated as het - nif + (nonheterocystous and nitrogen-fixing), and is found to be easily and effectively preserved in nitrogen-free medium in standard synthetic cyanobacterial nutrient medium (pH 8.5) at a continuous light intensity of 2800 lx at 25 ± 1 °C. This het - nif + strain is an effective donor of the nif + marker to a het + nif - strain of another cyanobacterium, Nostoc muscorum, when both are grown together in a recombination study.

Unusual ultrastructural features in three strains of Cyanothece (cyanobacteria).

Three unicellular cyanobacterial strains (PCC 7425, PCC 8303, PCC 9308) assigned to the genus Cyanothece Komárek 1976, which showed an unusually high content of light refractile inclusions when viewed by phase-contrast microscopy, were characterized by confocal laser scanning microscopy and transmission electron microscopy. All strains had concentric cortical thylakoids and a compact central nucleoid. Frequently, the two innermost thylakoid membranes protruded to form circular enclosures containing cytoplasm or electron-transparent granules, or both. The largest granules were partially immersed in the nucleoid region, but they remained attached to the inner cortical thylakoids by a single narrow connection. The pattern of binary cell division in strain PCC 7425 was different than that in strains PCC 8303 and PCC 9308. In the former, all cell wall layers invaginated simultaneously, whereas in the latter the invagination of the outer membrane was delayed compared to that of the cytoplasmic membrane and the peptidoglycan layer. Thus, prior to completion of cell division, the new daughter cells of strains PCC 8303 and PCC 9308 were transiently connected by a thick septum, which was not observed in strain PCC 7425. Nucleoid partitioning coincided with initiation of cell division in all three strains and was unlike that reported in other bacteria and in archaea, in which separation of the nucleoids precedes cell division. Based on the common morphological and ultrastructural features, the three strains of Cyanothece examined constitute a distinct cluster, which might deserve independent generic status.

Detection of seven major evolutionary lineages in cyanobacteria based on the 16S rRNA gene sequence analysis with new sequences of five marine Synechococcus strains.

Although molecular phylogenetic studies of cyanobacteria on the basis of the 16S rRNA gene sequence have been reported, the topologies were unstable, especially in the inner branchings. Our analysis of 16S rRNA gene phylogeny by the maximum-likelihood and neighbor-joining methods combined with rate homogeneous and heterogeneous models revealed seven major evolutionary lineages of the cyanobacteria, including prochlorophycean organisms. These seven lineages are always stable on any combination of these methods and models, fundamentally corresponding to phylogenetic relationships based on other genes, e.g., psbA, rbcL, rnpB, rpoC, and tufA. Moreover, although known genotypic and phenotypic characters sometimes appear paralleled in independent lineages, many characters are not contradictory within each group. Therefore we propose seven evolutionary groups as a working hypothesis for successive taxonomic reconstruction. New 16S rRNA sequences of five unicellular cyanobacterial strains, PCC 7001, PCC 7003, PCC 73109, PCC 7117, and PCC 7335 of Synechococcus sp., were determined in this study. Although all these strains have been assigned to "marine clusters B and C," they were separated into three lineages. This suggests that the organisms classified in the genus Synechococcus evolved diversely and should be reclassified in several independent taxonomic units. Moreover, Synechococcus strains and filamentous cyanobacteria make a monophyletic group supported by a comparatively high statistical confidence value (80 to 100%) in each of the two independent lineages; therefore, these monophylies probably reflect the convergent evolution of a multicellular organization.

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains.

Microcystis aeruginosa (Synechocystis) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains

Microcystis aeruginosa (Synechocystis) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.

Proton gradients in intact cyanobacteria.

The internal pH values of two unicellular cyanobacterial strains were determined with electron spin resonance probes, over an external pH range of 6 to 9, in the light and in the dark. The slow growing, thylakoid-lacking Gloeobacter violaceus was found to have a low capacity for maintaining a constant internal pH. The distribution pattern of weak acid and amine nitroxide spin probes across the cell membranes of this organism, in the light and in the dark, was consistent with the assumption that it contains a single intracellular compartment. At an external pH of 7.0, intracellular pH was 6.8 in the dark and 7.2 in the light. The cells of Agmenellum quadruplicatum, a marine species, were found to contain two separate compartments; in the dark, the pH of the cytoplasmic and the intrathylakoid spaces were calculated to be 7.2 and 5.5, respectively. Upon illumination, the former increased and the latter decreased by about 0.5 pH units.