Physiological and genomic analysis of newly-isolated polysaccharide synthesizing cyanobacterium Chroococcus sp. FPU101 and chemical analysis of the exopolysaccharide.

A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated. The isolate, named Chroococcus sp. FPU101, grew between 20 and 30°C and at light intensities between 10 and 80 µmol m-2 s-1. Purified EPS from Chroococcus sp. FPU101 had a molecular size of 5.9 × 103 kDa and contained galactose, rhamnose, fucose, xylose, mannose, glucose, galacturonic acid, and glucuronic acid at a molar ratio of 17.2:15.9:14.1:11.0:9.6:9.5:13.0:9.7. The EPS content significantly increased when the NaCl concentration in the medium was increased from 1.7 to 100 mM. However, high NaCl concentrations did not significantly affect the molecular size or chemical composition of the EPS. The genes wza, wzb, wzc, wzx, wzy, and wzz that are involved in EPS synthesis were conserved in the genome of Chroococcus sp. FPU101, which was sequenced in this study. These results suggest that the Wzy-dependent pathway is potentially involved in EPS production in this organism.

Formation of prolamellar-body-like ultrastructures in etiolated cyanobacterial cells overexpressing light-dependent protochlorophyllide oxidoreductase in Leptolyngbya boryana.

Protochlorophyllide (Pchlide) reduction is the penultimate step of chlorophyll (Chl) biosynthesis, and is catalyzed by two evolutionarily unrelated enzymes: dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). Because LPOR is the sole Pchlide reductase in angiosperms, dark-grown seedlings of angiosperms become etiolated. LPOR exists as a ternary complex of Pchlide-NADPH-LPOR to form paracrystalline prolamellar bodies (PLBs) in etioplasts. Because LPOR is distributed ubiquitously across oxygenic phototrophs including cyanobacteria, it would be important to determine whether cyanobacterial LPOR has the ability to form PLBs. We isolated a DPOR-less transformant ΔchlL/LPORox, carrying a plasmid to overexpress cyanobacterial LPOR in the cyanobacterium Leptolyngbya boryana. The transformant did not produce Chl in the dark and became etiolated with an accumulation of Pchlide and LPOR. Novel PLB-like ultrastructures were observed in etiolated cells, which disappeared during the early stage of the light-dependent greening process. However, the rate of Chl production in the greening process of ΔchlL/LPORox was almost the same as that observed in the control cells, which carried an empty vector. An in vitro LPOR assay of extracts of dark-grown ΔchlL/LPORox cells suggested that the PLB-like structures are deficient in NADPH. Low-temperature fluorescence emission spectra of membrane fractions of the etiolated cells indicated the absence of the photoactive form of Pchlide, which was consistent with the inefficiency of the greening process. Cyanobacterial LPOR exhibited an intrinsic ability to form PLB-like ultrastructures in the presence of the co-accumulation of Pchlide; however, the PLB-like structure differed from the authentic PLB regarding NADPH deficiency.

Physiological properties and genetic analysis related to exopolysaccharide (EPS) production in the fresh-water unicellular cyanobacterium Aphanothece sacrum (Suizenji Nori).

The clonal strains, phycoerythrin(PE)-rich- and PE-poor strains, of the unicellular, fresh water cyanobacterium Aphanothece sacrum (Suringar) Okada (Suizenji Nori, in Japanese) were isolated from traditional open-air aquafarms in Japan. A. sacrum appeared to be oligotrophic on the basis of its growth characteristics. The optimum temperature for growth was around 20°C. Maximum growth and biomass increase at 20°C was obtained under light intensities between 40 to 80 µmol m-2 s-1 (fluorescent lamps, 12 h light/12 h dark cycles) and between 40 to 120 µmol m-2 s-1 for PE-rich and PE-poor strains, respectively, of A. sacrum . Purified exopolysaccharide (EPS) of A. sacrum has a molecular weight of ca. 104 kDa with five major monosaccharides (glucose, xylose, rhamnose, galactose and mannose; ≥85 mol%). We also deciphered the whole genome sequence of the two strains of A. sacrum. The putative genes involved in the polymerization, chain length control, and export of EPS would contribute to understand the biosynthetic process of their extremely high molecular weight EPS. The putative genes encoding Wzx-Wzy-Wzz- and Wza-Wzb-Wzc were conserved in the A. sacrum strains FPU1 and FPU3. This result suggests that the Wzy-dependent pathway participates in the EPS production of A. sacrum.

Mitotic spindle formation in Triparma laevis NIES-2565(Parmales, Heterokontophyta).

The parmalean algae possess a siliceous wall and represent the sister lineage of diatoms; they are thought to be a key group for understanding the evolution of diatoms. Diatoms possess well-characterized and unique mitotic structures, but the mitotic apparatus of Parmales is still unknown. We observed the microtubule (MT) array during interphase and mitosis in Triparma laevis using TEM. The interphase cells had four or five centrioles (∼80 nm in length), from which MTs emanated toward the cytoplasm. In prophase, the bundle of MTs arose at an extranuclear site. The position of centrioles with respect to an MT bundle changed during its elongation. Centrioles were observed on the lateral side of a shorter MT bundle (∼590 nm) and on either side of an extended MT bundle (∼700 nm). In metaphase, the spindle consisted of two types of MTs-MT bundle that passed through a cytoplasmic tunnel in the center of the nucleus and single MTs (possibly kinetochore MTs) that extended from the poles into the nucleus. The nuclear envelope disappeared only at the regions where the kinetochore MTs penetrated. In telophase, daughter chromosomes migrated toward opposite poles, and the MT bundle was observed between segregating chromosomes. These observations showed that MT nucleation does not always occur at the periphery of centrioles through cell cycle and that the spindle of T. laevis has a similar configuration to that of diatoms.

Diversity and oceanic distribution of the Parmales (Bolidophyceae), a picoplanktonic group closely related to diatoms.

Bolidomonas is a genus of picoplanktonic flagellated algae that is closely related to diatoms. Triparma laevis, a species belonging to the Parmales, which are small cells with a siliceous covering, has been shown to form a monophyletic group with Bolidomonas. We isolated several novel strains of Bolidophyceae that have permitted further exploration of the diversity of this group using nuclear, plastidial and mitochondrial genes. The resulting phylogenetic data led us to formally emend the taxonomy of this group to include the Parmales within the Bolidophyceae, to combine Bolidomonas within Triparma and to define a novel species, Triparma eleuthera sp. nov. The global distribution of Bolidophyceae was then assessed using environmental sequences available in public databases, as well as a large 18S rRNA V9 metabarcode data set from the Tara Oceans expedition. Bolidophyceans appear ubiquitous throughout the sampled oceans but always constitute a minor component of the phytoplankton community, corresponding to at most ~4% of the metabarcodes from photosynthetic groups (excluding dinoflagellates). They are ~10 times more abundant in the small size fraction (0.8-5 µm) than in larger size fractions. T. eleuthera sp. nov. constitutes the most abundant and most widespread operational taxonomic unit (OTU) followed by T. pacifica, T. mediterranea and the T. laevis clade. The T. mediterranea OTU is characteristic of Mediterranean Sea surface waters and the T. laevis clade OTU is most prevalent in colder waters, in particular off Antarctica.

Defense mechanisms of sargassacean species against the epiphytic red alga Neosiphonia harveyi.

Flora diversity and abundance of epiphytes are specific to their basiphyte species and may relate to variations in the defensive abilities of basiphytes. Thus, investigating the interactions between epiphytes and basiphytes is useful for a better understanding of the biological impact of epiphytism and the survival strategies of basiphytes. We examined the epiphyte density on five sargassacean species at six locations between two study sites, which showed that the epiphytic red alga Neosiphonia harveyi was remarkably less abundant on Sargassum siliquastrum at all locations. To assess its defense mechanism against N. harveyi, we performed bioassays of phlorotannins, which are considered effective in deterring fouling, by culturing sargassacean blades with N. harveyi carpospores and observed the process by which sargassacean blades remove epiphytes. When the carpospores were incubated with various concentrations of dissolved phlorotannins, settlement and germination were inhibited only at the highest concentrations (>0.1 g · L(-1) ), and this effect did not significantly differ among the five sargassacean species. When the carpospores were combined with blades from the five species, many of the spores attached and germinated on every blade. Because N. harveyi penetrated rhizoids into basiphyte tissues, cuticle peeling observed in all five sargassacean species could not remove this epiphyte after germination. However, in S. siliquastrum, the blade tissues around the germlings became swollen and disintegrative, and were removed together with the germlings. The spores normally grew on the dead blades, suggesting that the tissue degradation of S. siliquastrum is triggered by the infection of N. harveyi.

Effects of silicon-limitation on growth and morphology of Triparma laevis NIES-2565 (Parmales, Heterokontophyta).

The order Parmales (Heterokontophyta) is a group of small-sized unicellular marine phytoplankton, which is distributed widely from tropical to polar waters. The cells of Parmales are surrounded by a distinctive cell wall, which consists of several siliceous plates fitting edge to edge. Phylogenetic and morphological analyses suggest that Parmales is one of the key organisms for elucidating the evolutionary origin of Bacillariophyceae (diatoms), the most successful heterokontophyta. The effects of silicon-limitation on growth and morphogenesis of plates were studied using a strain of Triparma laevis NIES-2565, which was cultured for the first time in artificial sea water. The cells of T. laevis were surrounded by eight plates when grown with sufficient silicon. However, plate formation became incomplete when cells were cultured in a medium containing low silicate (ca. <10 µM). Cells finally lost almost all plates in a medium containing silicate concentrations lower than ca. 1 µM. However, silicon-limitation did not affect growth rate; cells continued to divide without changing their growth rate, even after all plates were lost. Loss of plates was reversible; when cells without plates were transferred to a medium containing sufficient silicate, regeneration of shield and ventral plates was followed by the formation of girdle and triradiate plates. The results indicate that the response to silicon-limitation of T. laevis is different from that of diatoms, where cell division becomes inhibited under such conditions.

Morphological, phylogenetic and physiological studies of pico-cyanobacteria isolated from the halocline of a saline meromictic lake, Lake Suigetsu, Japan.

Small cyanobacteria (<2 µm, pico-cyanobacteria) are abundant in waters deeper than the oxic-anoxic zone in the halocline of a saline meromictic lake, Lake Suigetsu, Fukui, Japan. We have isolated 101 strains that were grouped into six groups by means of the phycobiliprotein composition and sequence homology of the intergenic spacer between the 16S and 23S rRNA genes. Significant growth was observed under weak green light (1.5 µmol m⁻² s⁻¹, approx. 460 to 600 nm), whereas the cells died under white light at even moderate intensities. The isolates grew in a wide range of salinities (0.2 to 3.2%). Tolerance to sulfide varied: four groups grew in medium containing sulfide, however, two groups did not. None of the isolates were capable of anoxygenic photosynthetic (PS-II independent photosynthetic) growth using sulfide as an electron donor. All groups were included within fresh and brackish water of Synechococcus/Cyanobium clade, but they were not monophyletic in the 16S rRNA gene-based phylogenetic tree. The physiological properties of pico-cyanobacteria showed that they had the ability to survive in unique physicochemical environments in the halocline of this saline meromictic lake.

Isolation and characterization of the unicellular diazotrophic cyanobacterium Group C TW3 from the tropical western Pacific Ocean.

A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5-3.0 µm in width and 4.0-6.0 µm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h(-1) when cultured at 30°C and 140 µmol m(-2) s(-1) of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light-dark cycle, was 7.31 × 10(-15) mol N cell(-1) day(-1) . Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.

ISOLATION AND CHARACTERIZATION OF PARMALES (HETEROKONTA/HETEROKONTOPHYTA/STRAMENOPILES) FROM THE OYASHIO REGION, WESTERN NORTH PACIFIC(1).

A small siliceous species of marine phytoplankton, order Parmales (Heterokonta), was isolated and characterized for the first time with the aid of a fluorescent silicon tracer 2-(4-pyridyl)-5-([4-(2-dimethylaminoethylaminocarbamoyl)-methoxy]phenyl)oxazole (PDMPO). This dye was easily detected by clear fluorescence in newly produced silica cell plates. Our isolate was surrounded by eight smooth plates without any ornamentation, suggesting a similarity to Triparma laevis B. C. Booth. TEM observation showed the typical ultrastructure of photosynthetic heterokontophytes; with two chloroplast endoplasmic reticulate membranes, a girdle lamella, three thylakoid lamellae, and mitochondrion with tubular cristae. Molecular phylogenetic analyses of SSU rDNA and rbcL genes showed that the parmalean alga was within the bolidophycean clade of autotrophic naked flagellates and a sister group of diatoms. HPLC analysis detected chl a, c1 + c2 , and c3 ; fucoxanthin; and diadinoxanthin as major photosynthetic pigments, and a composition that is shared with Bolidophyceae and diatoms. Together, these data indicate a close evolutionary relationship between Parmales, Bolidophyceae, and diatoms. The PDMPO-staining procedure should accelerate isolation of other Parmales species, helping to establish their diversity and aiding quantitative study of their role in oceanic processes.

Diazotrophy under continuous light in a marine unicellular diazotrophic cyanobacterium, Gloeothece sp. 68DGA.

Nitrogenase is extremely sensitive to molecular oxygen (O(2)), and unicellular diazotrophic cyanobacteria separate nitrogen (N(2))-fixation and photosynthesis to protect nitrogenase from O(2) produced by photosynthesis. When grown under 12 h light/12 h dark cycles (LD), the marine unicellular diazotrophic cyanobacterium Gloeothece sp. 68DGA expressed the nitrogenase protein and its activity (acetylene reduction activity) only during the dark phase. However, this strain was able to grow diazotrophically under continuous light (CL). To determine whether nitrogenase synthesis and N(2)-fixation are temporally separated from photosynthesis in the Gloeothece cells that have fully acclimated to CL, the proportion of cells containing nitrogenase (the Fe-protein of nitrogenase) in the culture was measured using an immunocytochemical technique. Cells were grown in a continuous-culture device to maintain constant cell density. Under LD, the cells showed diurnal oscillation of nitrogenase activity, photosynthesis, respiration and the expression and the abundance of the Fe-protein. The oscillation was gradually reduced after the transfer of the cells to CL, and was lost after 23-25 days of cultivation under CL. In CL-acclimated cultures, the Fe-protein was always detected in about 94 % of the cells, although the nitrogenase activity was about one-third of the maximum activity in LD-acclimated cultures. These results suggest that synthesis of nitrogenase proceeds without diurnal oscillation in the CL-acclimated cells of Gloeothece sp. 68DGA. As the respiration rate in CL-acclimated culture was as high as the maximum rate observed in LD-acclimated culture, O(2)-uptake mechanism(s) may have been upregulated to maintain low intracellular pO(2).

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE(1).

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form-genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form-genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form-genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form-genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form-genus Cyanothece were observed to be in a close relationship with our isolates.

On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature.

The fluorescence emitted at 710 nm by Phaeodactylum tricornutum (F(710)) was characterized. Development of F(710) was found to be regulated by the quality of light needed for algal growth: weak red light absorbed mainly by Chl a induced its development, and weak blue-green light absorbed mainly by fucoxanthin and Chl c suppressed it. The difference spectra between cells grown under the two light conditions revealed two Chl a forms, absorption peaks of which were located at 692 nm (Chl a(692)) and at 703 nm (Chl a(703)), respectively, in red-light-grown cells. During cell growth under red light, the appearance and intensification of the emission correlated well with development of Chl a(692) and Chl a(703) suggesting that the two forms of Chl a are involved in the energy flow to F(710). A clear induction phenomenon characteristic of the PSII fluorescence was observed not only with the emission at 680 nm but also with F(710), indicating that F(710) is emitted by PSII Chl a. Development of F(710) under red light was sensitive to cycloheximide, indicating that the development of the energy flow to F(710) requires protein synthesis and that the emitter is installed in a protein encoded in the nuclear genome like the light-harvesting complex (LHC). Centrifugal fractionation of pigment-protein complexes revealed F(710) to be located at fractions slightly heavier than the major LHC. Development of F(710) was also found in red-light-grown cells of the diatom Nitzschia closterium.