Metabolic Adaptations of Azospirillum brasilense to Oxygen Stress by Cell-to-Cell Clumping and Flocculation

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.     

Three-Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga Haematococcus pluvialis

Haematococcus pluvialis is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of H. pluvialis. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%). In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%), in which the total chloroplast volume was markedly decreased (9.7%). Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage.     

Comparing diatom and Alexandrium catenella/tamarense blooms in Thau lagoon: Importance of dissolved organic nitrogen in seasonally N-limited systems

Diatom blooms in Thau lagoon are always related to rain events leading to inputs of inorganic nutrients such as phosphate, ammonium and nitrate through the watershed with time lags of about 1 week. In contrast, blooms of Alexandrium catenella/tamarense can occur following periods of 3 weeks without precipitation and no significant input of conventional nutrients such as nitrate and phosphate. Field results also indicate a significant drop (from 22–25 to 15–16 μM over 3 days) in dissolved organic nitrogen (DON) at the bloom peak, as well as a significant inverse relationship between A. catenella/tamarense cell density and DON concentrations that is not apparent for diatom blooms. Such dinoflagellate blooms are also associated with elevated (6–9 μM) ammonium concentrations, a curious feature also observed by other investigators, possibly the results of ammonium excretion by this organism during urea or other organic nitrogen assimilation.The potential use of DON by this organism represents short cuts in the nitrogen cycle between plants and nutrients and requires a new model for phytoplankton growth that is different from the classical diatom bloom model. In contrast to such diatom blooms that are due to conventional (nitrate, phosphate) nutrient pulses, Alexandrium catenella/tamarense blooms on the monthly time scale are due to organic nutrient enrichment, a feature that allows net growth rates of about 1.3 d⁻¹, a value higher than that generally attributed to such organisms.     

Recruitment of resting stages may induce blooms of Microcystis at low N:P ratios

Some species of cyanobacteria form resting stages at the sedimentsurface when environmental conditions become unfavourable. Asconditions turn more favourable, these resting stages hatchto the water phase, where the cells grow, reproduce, and sometimesform blooms. Since blooms of cyanobacteria have become an increasingthreat to inland and brackish waters, it is important to assessthe mechanisms and processes involved in the initiation of suchblooms. One such mechanism is recruitment from the sedimentsurface. Potential factors regulating the recruitment of restingstages include variations in nutrient concentrations and ratios,as well as variations in grazing. To investigate how the recruitmentof Microcystis responds to different levels of these factors,we performed an enclosure experiment (zooplankton abundanceswere regulated by predation from fish). We found that recruitmentand growth were most pronounced at the second highest nutrientconcentration (average concentrations were 498 µg l^-1of dissolved nitrogen and 134 µg l^-1 of total phosphorus),while no direct response to different grazing levels was detected.We also found that resting stages can be important for initiatingand sustaining blooms. The environmental conditions most importantin regulating the recruitment rate from resting stages correspondedto the requirements of the plankton cells, namely high nutrientaddition and low N:P ratio.     

How does eutrophication affect the role of grazers in harmful algal bloom dynamics?

Population dynamics of harmful algal bloom species are regulated both from the “bottom-up” by factors that affect their growth rate and from the “top-down” by factors that affect their loss rates. While it might seem apparent that eutrophication would have the greatest impact on factors affecting growth rates of phytoplankton (nutrient supply, light availability) the roles of top-down controls, including grazers and pathogens, cannot be ignored in studies of harmful bloom dynamics. Lags between the growth of phytoplankton and zooplankton populations, or disruption of zooplankton populations by adverse environmental conditions may be important factors in the initiation of plankton blooms under eutrophic conditions. Grazers that avoid feeding on harmful species and actively graze on competing species may also play important roles in bloom initiation. Grazers that are not affected by phytoplankton toxins and have growth rates comparable to phytoplankton (e.g. protozoan grazers) may have the potential to control the initiation of blooms. If the inhibition of grazers varies with cell density for blooms of toxic phytoplankton, eutrophication may increase the chances of blooms reaching threshold densities for grazer inhibition. In addition, secondary effects of eutrophication, including hypoxia and change in pH may adversely affect grazer populations, and further release HAB species from top-down control. The Texas brown tide (Aureoumbra lagunensis) blooms provide evidence for the role of grazer disruption in bloom initiation and the importance of high densities of brown tide cells in continued suppression of grazers.     

A Millifluidic Study of Cell-to-Cell Heterogeneity in Growth-Rate and Cell-Division Capability in Populations of Isogenic Cells of Chlamydomonas reinhardtii

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.     

Changes in the localization and levels of starch and lipids in cambium and phloem during cambial reactivation by artificial heating of main stems of Cryptomeria japonica trees

Background and Aims

Cambial reactivation in trees occurs from late winter to early spring when photosynthesis is minimal or almost non-existent. Reserve materials might be important for wood formation in trees. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules in cambium and phloem were examined from cambial dormancy to the start of xylem differentiation in locally heated stems of Cryptomeria japonica trees in winter.

Methods

Electric heating tape was wrapped on one side of the stem of Cryptomeria japonica trees at breast height in winter. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules were determined by image analysis of optical digital images obtained by confocal laser scanning microscopy.

Key Results

Localized heating induced earlier cambial reactivation and xylem differentiation in stems of Cryptomeria japonica, as compared with non-heated stems. There were clear changes in the respective localizations and levels of starch and lipids (as droplets) determined in terms of relative areas on images, from cambial dormancy to the start of xylem differentiation in heated stems. In heated stems, the levels and number of starch granules fell from cambial reactivation to the start of xylem differentiation. There was a significant decrease in the relative area occupied by lipid droplets in the cambium from cambial reactivation to the start of xylem differentiation in heated stems.

Conclusions

The results showed clearly that the levels and number of storage starch granules in cambium and phloem cells and levels of lipids (as droplets) in the cambium decreased from cambial reactivation to the start of xylem differentiation in heated stems during the winter. The observations suggest that starch and lipid droplets might be needed as sources of energy for the initiation of cambial cell division and the differentiation of xylem in Cryptomeria japonica.     

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L⁻¹ D⁻¹. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.     

Behavioral and Physiological Changes during Benthic-Pelagic Transition in the Harmful Alga,Heterosigma akashiwo: Potential for Rapid Bloom Formation

Many species of harmful algae transition between a motile, vegetative stage in the water column and a non-motile, resting stage in the sediments. Physiological and behavioral traits expressed during benthic-pelagic transition potentially regulate the timing, location and persistence of blooms. The roles of key physiological and behavioral traits involved in resting cell emergence and bloom formation were examined in two geographically distinct strains of the harmful alga, Heterosigma akashiwo. Physiological measures of cell viability, division and population growth, and cell fatty acid content were made using flow cytometry and gas chromatography – mass spectrometry techniques as cells transitioned between the benthic resting stage and the vegetative pelagic stage. Video-based tracking was used to quantify cell-level swimming behaviors. Data show increased temperature and light triggered rapid emergence from the resting stage and initiated cell swimming. Algal strains varied in important physiological and behavioral traits, including survivorship during life-stage transitions, population growth rates and swimming velocities. Collectively, these traits function as “population growth strategies” that can influence bloom formation. Many resting cells regained the up-swimming capacity necessary to cross an environmentally relevant halocline and the ability to aggregate in near-surface waters within hours after vegetative growth supporting conditions were restored. Using a heuristic model, we illustrate how strain-specific population growth strategies can govern the timescales over which H. akashiwo blooms form. Our findings highlight the need for identification and quantification of strain-specific physiological and behavioral traits to improve mechanistic understanding of bloom formation and successful bloom prediction.     

Interaction Effects of Ambient UV Radiation and Nutrient Limitation on the Toxic Cyanobacterium <Emphasis Type="Italic">Nodularia spumigena</Emphasis>

Nodularia spumigena is one of the dominating species during the extensive cyanobacterial blooms in the Baltic Sea. The blooms coincide with strong light, stable stratification, low ratios of dissolved inorganic nitrogen, and dissolved inorganic phosphorus. The ability of nitrogen fixation, a high tolerance to phosphorus starvation, and different photo-protective strategies (production of mycosporine-like amino acids, MAAs) may give N. spumigena a competitive advantage over other phytoplankton during the blooms. To elucidate the interactive effects of ambient UV radiation and nutrient limitation on the performance of N. spumigena, an outdoor experiment was designed. Two radiation treatments photosynthetic active radiation (PAR) and PAR +UV-A + UV-B (PAB) and three nutrient treatments were established: nutrient replete (NP), nitrogen limited (−N), and phosphorus limited (−P). Variables measured were specific growth rate, heterocyst frequency, cell volume, cell concentrations of MAAs, photosynthetic pigments, particulate carbon (POC), particulate nitrogen (PON), and particulate phosphorus (POP). Ratios of particulate organic matter were calculated: POC/PON, POC/POP, and PON/POP. There was no interactive effect between radiation and nutrient limitation on the specific growth rate of N. spumigena, but there was an overall effect of phosphorus limitation on the variables measured. Interaction effects were observed for some variables; cell size (larger cells in −P PAB compared to other treatments) and the carotenoid canthaxanthin (highest concentration in −N PAR). In addition, significantly less POC and PON (mol cell⁻¹) were found in −P PAR compared to −P PAB, and the opposite radiation effect was observed in −N. Our study shows that despite interactive effects on some of the variables studied, N. spumigena tolerate high ambient UVR also under nutrient limiting conditions and maintain positive growth rate even under severe phosphorus limitation.     

Prorocentrum minimum (Pavillard) Schiller: A review of a harmful algal bloom species of growing worldwide importance

Prorocentrum minimum (Pavillard) Schiller, a common, neritic, bloom-forming dinoflagellate, is the cause of harmful blooms in many estuarine and coastal environments. Among harmful algal bloom species, P. minimum is important for the following reasons: it is widely distributed geographically in temperate and subtropical waters; it is potentially harmful to humans via shellfish poisoning; it has detrimental effects at both the organismal and environmental levels; blooms appear to be undergoing a geographical expansion over the past several decades; and, a relationship appears to exist between blooms of this species and increasing coastal eutrophication. Although shellfish toxicity with associated human impacts has been attributed to P. minimum blooms from a variety of coastal environments (Japan; France; Norway; Netherlands; New York, USA), only clones isolated from the Mediterranean coast of France, and shellfish exposed to P. minimum blooms in this area, have been shown to contain a water soluble neurotoxic component which killed mice. Detrimental ecosystem effects associated with blooms range from fish and zoobenthic mortalities to shellfish aquaculture mortalities, attributable to both indirect biomass effects (e.g., low dissolved oxygen) and toxic effects. P. minimum blooms generally occur under conditions of high temperatures and incident irradiances and low to moderate salinities in coastal and estuarine environments often characterized as eutrophic, although they have been found under widely varying salinities and temperatures if other factors are conducive for growth. The physiological flexibility of P. minimum in response to changing environmental parameters (e.g., light, temperature, salinity) as well as its ability to utilize both inorganic and organic nitrogen, phosphorus, and carbon nutrient sources, suggest that increasing blooms of this species are a response to increasing coastal eutrophication.     

Growth, Nitrogen Fixation, and Nodularin Production by Two Baltic Sea Cyanobacteria

In late summer, nitrogen-fixing cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae form blooms in the open Baltic Sea. N. spumigena has caused several animal poisonings, but Baltic A. flos-aquae is not known to be toxic. In this laboratory study, performed with batch cultures, the influences of environmental conditions on the biomass and nitrogen fixation rate of N. spumigena and A. flos-aquae were compared and the toxin (nodularin) concentration produced by N. spumigena was measured. Several differences in the biomasses and nitrogen fixation rates of N. spumigena and A. flos-aquae were observed. A. flos-aquae preferred lower irradiances, salinities, and temperatures than N. spumigena. The biomass of both species increased with high phosphate concentrations and with accompanying bacteria and decreased with unnaturally high inorganic nitrogen concentrations. Nodularin concentrations in cells and growth media, as well as nitrogen fixation rates, were generally highest under the conditions that promoted growth. Intracellular nodularin concentrations increased with high temperature, high irradiance, and high phosphate concentration and decreased with low and high salinities and high inorganic nitrogen concentrations. Nodularin concentrations in growth media increased with incubation time, indicating that intracellular nodularin was released when cells lysed. The different responses of A. flos-aquae and N. spumigena to changes in salinity, irradiance, and temperature may explain the different spatial and temporal distribution of these species in the Baltic Sea. According to the results, toxic N. spumigena blooms may be expected in late summer in areas of the Baltic Sea with high phosphorus concentrations and moderate salinity.     

A Quantification of the Significance of Assimilatory Starch for Growth of Arabidopsis thaliana L. Heynh

These studies use starch synthesis mutants to quantify the contribution of assimilatory starch to whole plant growth and form. Arabidopsis thaliana (L.) Heynh plants were used with null plastid phosphoglucomutase (T Caspar, SC Huber, CR Sommerville, [1986] Plant Physiol 79; 1-7) or 7% of wild-type ADP-glucose pyrophosphorylase (T-P Lin, T Caspar, CR Sommerville, J Preiss [1988] Plant Physiol 88; 1175-1179). The daily turnover of starch and the rate of biomass increase in the mutants and the wild type were investigated during growth in a 14 hour light/10 hour dark cycle in high irradiance (600 micromoles per square meter per second) and nitrogen (6 millimolar NH₄NO₃), in high irradiance and low nitrogen (0.1 millimolar NH₄NO₃) or in low irradiance (80 micromoles per square meter per second) and high nitrogen. There is some variability in the data, but the following conclusions can be drawn. Growth was slow in the absence of starch turnover. In high nitrogen conditions, about 1 mole of carbon per gram dry weight per day was incorporated additionally into structural biomass for every one mole of carbon turned over as starch per gram dry weight per day. In low nitrogen, the gain was much lower. This indicates that temporary storage of photosynthate is important for rapid growth in high nitrogen, but not in low nitrogen when carbohydrate is in excess. Starch-deficient plants showed the usual decrease of the shoot/root ratio in low nitrogen and increase of the ratio in low light. This shows that adjustment of plant form to nitrogen nutrition and irradiance is not mediated via regulation of photosynthate partitioning in the leaf. Starch deficient plants had lower shoot/root ratios than the wild type and the nitrogen concentration in their leaves was increased. It is discussed how interactions between carbohydrate allocation, respiration and growth at the organ and whole plant level generate these changes. We conclude that mutants with a decreased capacity to carry out a particular partial process provide a powerful tool to disect complex mutually interacting systems, and define and quantify causal interactions at the level of whole plant growth.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

Squalene-based oil-in-water emulsion adjuvants perturb metabolism of neutral lipids and enhance lipid droplet formation

Oil-in-water emulsions are used as vaccine adjuvants, but the mechanism of action remains unknown. In this paper we used phagocytes (monocytes, macrophages, dendritic cells) and non-phagocytic cells (fibroblasts, skeletal muscle cells) to study internalization of emulsions in vitro, and to characterize the influence of emulsion uptake on cellular metabolism of neutral lipids. We found that all tested cell types endocytose the emulsion droplets, and that the uptake leads to an acute accumulation of neutral lipids in the form of cytoplasmic lipid droplets. The accumulated lipids comprise not only the delivered squalene, but also cholesteryl esters, triacylglycerols, fatty acids, and diacylglycerols. Lipid metabolism and innate immunity are closely linked, and accumulation of lipids in non-adipose tissues is known to induce inflammatory conditions. We propose that one aspect of o/w emulsion adjuvanticity could depend on their ability to rapidly change lipid metabolism of the target cells.     

Positive feedbacks between bottom-up and top-down controls promote the formation and toxicity of ecosystem disruptive algal blooms: A modeling study

Harmful algal blooms that disrupt and degrade ecosystems (ecosystem disruptive algal blooms, EDABs) are occurring with greater frequency and severity with eutrophication and other adverse anthropogenic alterations of coastal systems. EDAB events have been hypothesized to be caused by positive feedback interactions involving differential growth of competing algal species, low grazing mortality rates on EDAB species, and resulting decreases in nutrient inputs from grazer-mediated nutrient cycling as the EDAB event progresses. Here we develop a stoichiometric nutrient–phytoplankton–zooplankton (NPZ) model to test a conceptual positive feedback mechanism linked to increased cell toxicity and resultant decreases in grazing mortality rates in EDAB species under nutrient limitation of growth rate. As our model EDAB alga, we chose the slow-growing, toxic dinoflagellate Karenia brevis, whose toxin levels have been shown to increase with nutrient (nitrogen) limitation of specific growth rate. This species was competed with two high-nutrient adapted, faster-growing diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii) using recently published data for relationships among nutrient (ammonium) concentration, carbon normalized ammonium uptake rates, cellular nitrogen:carbon (N:C) ratios, and specific growth rate. The model results support the proposed positive feedback mechanism for EDAB formation and toxicity. In all cases the toxic bloom was preceded by one or more pre-blooms of fast-growing diatoms, which drew dissolved nutrients to low growth rate-limiting levels, and stimulated the population growth of zooplankton grazers. Low specific grazing rates on the toxic, nutrient-limited EDAB species then promoted the population growth of this species, which further decreased grazing rates, grazing-linked nutrient recycling, nutrient concentrations, and algal specific growth rates. The nutrient limitation of growth rate further increased toxin concentrations in the EDAB algae, which further decreased grazing-linked nutrient recycling rates and nutrient concentrations, and caused an even greater nutrient limitation of growth rate and even higher toxin levels in the EDAB algae. This chain of interactions represented a positive feedback that resulted in the formation of a high-biomass toxic bloom, with low, nutrient-limited specific growth rates and associated high cellular C:N and toxin:C ratios. Together the elevated C:N and toxin:C ratios in the EDAB algae resulted in very high bloom toxicity. The positive feedbacks and resulting bloom formation and toxicity were increased by long water residence times, which increased the relative importance of grazing-linked nutrient recycling to the overall supply of limiting nutrient (N).     

Modulation and adaptation of carbonic anhydrase activity in Microcystis spp. under different environmental factors

The carbonic anhydrase (CA) activities were determined in three cyanobacterial species, namely Microcystis aeruginosa Kütz., Microcystis viridis (A.Br.) Lemm, and Microcystis wesenbergii (Kom.) Kom, which were dominant in a lake (Dianchi Lake) subject to major blooms. In more detailed experiments on M. aeruginosa, the effects of inorganic carbon, pH, temperature, nitrogen/phosphorus ratio, glucose, and light intensity on CA activity were also investigated. Because of the relatively alkaline pH value of the culture media for the optimum growth of algal cells, bicarbonate ions were the main form of exogenous inorganic carbon. The results showed that the CA activity of M. aeruginosa was influenced dramatically by the concentration of bicarbonate. Consequently, it was suggested that bicarbonate ions were the main form of exogenous inorganic carbon that M. aeruginosa could utilize. Cultures grown in the dark exhibited CA activity six times higher than that of cells cultured mixotrophically with the addition of glucose. Features of eutrophic water bodies promoted an increase in CA activity, and the resulting higher CA activity would accelerate the utilization of inorganic carbon and favor the growth and blooming of Microcystis spp. in eutrophic lakes. Although the experiments were carried out under controlled experimental conditions, they could provide some basic data that would prove useful for the control of cyanobacterial blooms in nature.     

Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme

Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophylic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen-fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), α-tocopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids, whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location, these structures represent a novel sub-organelle in cyanobacteria.     

Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka''s factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.