Yukiko Taniuchi

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.

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).

Abundance of picophytoplankton in the halocline of a meromictic lake, Lake Suigetsu, Japan

Numerous (0.5 to 4.8 × 105 cells/ml), small phytoplankton (smaller than 0.5–1 × 1–2 μm in cell size, picophytoplankton) were distributed in the halocline (depth 2–12 m, 4–14 practical salinity units) of the saline meromictic lake, Lake Suigetsu (35°35′ N, 135°52′ E), located in the central part of the coast of Wakasa Bay along the Japan Sea in Fukui Prefecture, Japan. Vertical distribution of phytoplankton revealed that the maximum number of picophytoplankton was always observed near or a little deeper than the oxic-anoxic boundary layer (depth 5–6 m); they were dominant phytoplankton in the water layer deeper than the oxic-anoxic boundary from July to late September 2005. Spectral analysis of autofluorescence emitted from the particle fractions smaller than 5 μm measured with a spectrofluorometer and from individual cells measured with a microscope photodiode array detector revealed that the major component of picophytoplankton was phycoerythrin-rich, unicellular cyanobacteria (picocyanobacteria). Eukaryotic phytoplankton about 2.5 μm in diameter were also found, but the numbers were low. Fluorescence intensity of chlorophyll a at 685 nm (room temperature) emitted from the particle fractions smaller than 5 μm was increased by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These observations indicated that at least some picophytoplankton had a functional photosystem II in the halocline where sulfide, the potential inhibitor of oxygenic photosynthesis, was always present. The large abundance together with their physiological potency suggest that picophytoplankton are one of the important primary producers in the halocline of Lake Suigetsu.

Relation between nitrogenase synthesis and activity in a marine unicellular diazotrophic strain, Gloeothece sp. 68DGA (Cyanophyte), grown under different light/dark regimens

The relationship between the abundance of nitrogenase and its activity was studied in the marine unicellular cyanobacterium Gloeothece sp. 68DGA cultured under different light/dark regimens. The Fe‐ and MoFe‐protein of nitrogenase and nitrogen (N2)‐fixing (acetylene reduction) activity were detected only during the dark phase when the cells were grown under a 12 h light/12 h dark cycle (12L/12D). Nitrogenase activity appeared about 4 h after entering the dark phase. Maximum nitrogenase activity occurred at around the middle of the dark phase, and the activity rapidly decreased to zero before the start of the light phase. The rapid decrease of nitrogenase activity and the Fe‐protein of nitrogenase near the end of the dark phase in 12L/12D were partly recovered by the addition of l‐methionine‐sulfoximine, an inhibitor of glutamine synthetase. Diurnal oscillation of the abundance of nitrogenase was maintained in the first subjective dark phase (i.e. the period corresponding to the dark phase) after the cells were transferred from 12L/12D to continuous illumination. However, enzyme activity was detected only when photosynthetic oxygen (O2) evolution was completely suppressed by reducing the light intensity or by the addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Nitrogenase always appeared in the cells about 16 h after starting the light phase, even when the 12L/12D cycle was modified by the addition or subtraction of a single 6 h period of light or dark. These results suggest the following: (i) N2‐fixation by Gloeothece sp. 68DGA is primarily regulated by an endogenous circadian oscillator at the level of nitrogenase synthesis. (ii) The endogenous circadian rhythm resets on a shift of the timing of the light phase. (iii) Nitrogenase activity is not always reflected in the presence of nitrogenase. (iv) The activity of nitrogenase is negatively regulated by fixed nitrogen and the concentration of ambient O2.

Seasonal dynamics of the phytoplankton community in Sendai Bay, northern Japan

Sendai Bay is located on the Pacific coast of northern Japan and suffered serious damage following the 2011 off the Pacific coast of Tohoku earthquake and tsunami in March 2011. To assess the impact on the marine ecosystem, information was needed on the phytoplankton communities and their seasonal variation. However, such information was limited. Therefore, an intensive monitoring of the phytoplankton was carried out from March 2012 to April 2014. Seasonal variation of the phytoplankton community was similar at coastal and offshore stations. Total phytoplankton biomass, based on Chl a concentration, peaked in spring and then decreased to a minimum in summer, before gradually increasing during early winter and peaking again in the following spring. This seasonal pattern was consistent with previous studies conducted before the earthquake and tsunami. Also, size structure of the phytoplankton community and its four main groups was estimated from the size-fractioned samples of Chl a. Our results also showed that the spring bloom consisted of large diatoms, with their growth ceasing due to nitrogen depletion. The bloom was followed by a summer period where cyanobacteria and picoeukaryote became dominant, with high cell densities in spite of low nutrient concentrations. In addition, sporadic environmental changes, such as those following typhoons, were observed. These resulted in large increases/decreases in individual phytoplankton groups.