Sustained net CO2 evolution during photosynthesis by marine microorganisms.

BACKGROUND: Many aquatic photosynthetic microorganisms possess an inorganic-carbon-concentrating mechanism that raises the CO2 concentration at the intracellular carboxylation sites, thus compensating for the relatively low affinity of the carboxylating enzyme for its substrate. In cyanobacteria, the concentrating mechanism involves the energy-dependent influx of inorganic carbon, the accumulation of this carbon--largely in the form of HCO3(-)-in the cytoplasm, and the generation of CO2 at carbonic anhydrase sites in close proximity to the carboxylation sites. RESULTS: During measurements of inorganic carbon fluxes associated with the inorganic-carbon-concentrating mechanism, we observed the surprising fact that several marine photosynthetic microorganisms, including significant contributors to oceanic primary productivity, can serve as a source of CO2 rather than a sink during CO2 fixation. The phycoerythrin-possessing cyanobacterium Synechococcus sp. WH7803 evolved CO2 at a rate that increased with light intensity and attained a value approximately five-fold that for photosynthesis. The external CO2 concentration reached was significantly higher than that predicted for chemical equilibrium between HCO3- and CO2, as confirmed by the rapid decline in the CO2 concentration upon the addition of carbonic anhydrase. Measurements of oxygen exchange between water and CO2, by means of stable isotopes, demonstrated that the evolved CO2 originated from HCO3- taken up and converted intracellularly to CO2 in a light-dependent process. CONCLUSIONS: We report net, sustained CO2 evolution during photosynthesis. The results have implications for energy balance and pH regulation of the cells, for carbon cycling between the cells and the marine environment, and for the observed fractionation of stable carbon isotopes.

The cyanobacterial toxin cylindrospermopsin inhibits pyrimidine nucleotide synthesis and alters cholesterol distribution in mice.

The hepatotoxin Cylindrospermopsin, a sulfated-guanidinium alkaloid with substituted dioxypyrimidine (uracil) moiety, was isolated from several cyanobacteria species. Our previous studies on the toxicity of cylindrospermopsin and its derivatives suggested that the uracil moiety is crucial for the toxicity and that such toxicity could partly stem from competitive binding of the toxin to a catalytic site(s) involved in the synthesis of pyrimidine nucleotides (i.e., uridine). In the present study we demonstrated that cylindrospermopsin inhibited in a noncompetitive manner the in vitro activity of uridine monophosphate (UMP) synthase complex (responsible for the conversion of orotic acid to UMP) in a cell free liver extract from mice, with an inhibition constant, KI, of 10 microM. Exposure of mice to cylindrospermopsin at subacute concentrations, via drinking water, only slightly affected the in vitro activity of UMP synthase. The typical metabolic disorder associated with the inhibition of UMP synthase activity, known as "orotic aciduria," was not observed under these conditions, but other anomalous metabolic responses related to cholesterol metabolism were developed.

Enrichment of poultry products with omega3 fatty acids by dietary supplementation with the alga Nannochloropsis and mantur oil.

Experiments were conducted to evaluate the efficiency of the microalga Nannochloropsis sp. (Nanno.), as a supplement to laying hens' diet, for the production of enriched eggs and meat with omega3 fatty acids (FA). Nanno. has a unique FA composition, namely, the occurrence of a high concentration of eicosapentaenoic acid (EPA; 20:5 omega3) and the absence of other omega3 FA. The effect of supplementing diets with Nanno. on omega3 FA levels in eggs, plasma, liver, and thigh muscle was compared to that of mantur oil, high in alpha-linolenic acid (LNA; 18:3 omega3). Nanno. is rich also in carotenoids, which may be useful for egg yolk pigmentation. The observed effect of Nanno. supplementation on yolk pigmentation was dose responsive, in both the rate of coloration and the color intensity. Addition of enzyme preparations (glucanase plus cellulase or glucanase plus pectinase) slightly elevated the yolk color score. The most prominent changes in the level of omega3 FA in egg yolk were evident when the diets were supplemented with 1% Nanno. or mantur lipid extracts. Levels of dietary algal meal (0.1-1.0%) had low and inconsistent effects on the level of yolk omega3 FA. Algal EPA is not accumulated in the liver or in the egg yolk; it is apparently converted and deposited as docosahexaenoic acid (DHA). LNA from mantur oil was partially converted to DHA, and both DHA and LNA were deposited in egg yolks and livers. It is suggested that the absence of DHA and EPA from thigh muscle is due to the small amount of dietary omega3 FA used in this work, compared to other studies, and to the possibility that in laying hens the egg yolk has a priority on dietary FA over that of muscles.

Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin.

A new natural derivative of the sulfated guanidinium zwitterionic toxin cylindrospermopsin, 7-epi-cylindrospermopsin, was recently isolated from the cyanobacterium Aphanizomenon ovalisporum (Forti). The toxicity of the molecule (LD50 ip 5 d), estimated by mouse bioassay, was 200 microg/kg mouse, a value similar to that of cylindrospermopsin. Treatment of cylindrospermopsin with chlorine solution or chlorine-related oxidants produced two new derivatives. The chemical structure of these products was elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques and toxicity was determined. In the first derivative, the vinylic proton at position 5 of the pyrimidine ring was substituted by chlorine to yield 5-chlorocylindrospermopsin. The other product is a truncated one, where C-6 of the pyrimidine ring was oxidized to a carboxylic acid. A trivial name, cylindrospermic acid, was given to this compound. Both products showed no toxic effects even at doses 50 times higher than the LD50 of cylindrospermopsin (10 mg/kg mouse ip). Based on these results, the pyrimidine ring is postulated as the molecule component essential for the toxicity of cylindrospermopsin.

Oral toxicity of the cyanobacterial toxin cylindrospermopsin in mice: long-term exposure to low doses.

The hepatotoxin cylindrospermopsin, a sulfated-guanidinium alkaloid with substituted dioxypyrimidine (uracil) moiety, was isolated from several cyanobacteria species. The acute toxicity of cylindrospermopsin was well established based on intraperitoneal and oral exposure; however, only a few long-term subacute exposure studies were performed to permit a reliable guideline value for cylindrospermopsin in drinking water. In the study reported herein, female and male mice were exposed to cylindrospermopsin in their drinking water. Cylindrospermopsin-containing, Aphanizomenon ovalisporum (cyanobacterium)-free medium was provided as the only source of drinking water, whereas a control group was given a fresh medium for cyanobacteria as drinking water. Over a period of 42 weeks, experiment groups were exposed to cylindrospermopsin concentration, gradually increased from 100 to 550 microg L(-1) (daily exposure ranged between 10 and 55 microg kg(-1) day(-1)). Body and organ weights were recorded, and serum and hematology analyses were performed 20 and 42 weeks after the beginning of the experiment. The most pronounced effect of cylindrospermopsin was elevated hematocrit levels in both male and female mice after 16 weeks of exposure to cylindrospermopsin. The observed changes in the hematocrit level were accompanied by deformation of red blood cells, which were changed into acanthocyte. Based on these results, a daily cylindrospermopsin dose of 20 microg kg(-1) day(-1) (equivalent to 200 microg L(-1)) is proposed as the lowest-observed-adverse-effect level for both male and female mice.

Ecological implications of the emergence of non-toxic subcultures from toxic Microcystis strains.

Two toxic, microcystin-producing, Microcystis sp. strains KLL MG-K and KLL MB-K were isolated as single colonies on agar plates from Lake Kinneret, Israel. Two non-toxic subcultures, MG-J and MB-J spontaneously succeeded the toxic ones under laboratory conditions. Southern analyses showed that MG-J and MB-J are lacking at least 34 kb of the mcy region, encoding the microcystin synthetase. Analyses of the 16S rRNA genes, the intergenic spacer region between cpcB and cpcA and the patterns of the polymerase chain reaction products of randomly amplified polymorphic DNA and highly iterated palindrome, and presence of mobile DNA elements did not allow unequivocal distinction between toxic and non-toxic subcultures. Laboratory and field experiments indicated an advantage of the toxic strain over its non-toxic successor. When grown separated by a membrane, which allowed passage of the media but not the cells, MG-K severely inhibited the growth of MG-J. Furthermore, when MG strains were placed in dialysis bags in Lake Kinneret during the season in which Microcystis is often observed, cells of MG-J lysed, whereas MG-K survived. Mechanisms whereby the non-toxic subcultures emerged and prevailed over the corresponding toxic ones under laboratory conditions, as well as a possible role of microcystin under natural conditions, are discussed.

Algal autoflocculation--verification and proposed mechanism.

Biomass autoflocculation in outdoor algal cultures was found to be associated with increases of culture pH levels, due to CO(2) consumption by the algal photosynthetic activity. Under these alkaline conditions, some medium chemical ions precipitated together with the algal biomass. The chemical substances involved with the process and its dependence on pH value were studied by simulation of autoflocculation in laboratory experiments. Proper concentrations of calcium and orthophosphate ions in the medium are important for autoflocculation and, in order to attain it within the pH range 8.5-9.0, the culture should contain 0.1mM-0.2mM orthophosphate and 1.5mM-2.5mM calcium prior to raising the pH level. Calcium phosphate precipitates are considered as the flocculating agent which reacts with the negatively charged surface of the algae and promotes aggregation and flocculation.

Dietary lipids from marine unicellular algae enhance the amount of liver and blood omega-3 fatty acids in rats.

The nutritional effect of omega-3 (omega 3) polyenoic fatty acids, originating from marine unicellular algae or from fish oil, on the liver and blood lipids was studied in weanling rats fed for 2 weeks on control or experimental diets. Isolipid experimental diets containing either 10% marine microalgae or algal lipids or fish (capelin) oil substituting part (40%) or all of the soybean oil of the control diet. The algae employed were Nannochloropsis sp. or Isochrysis galbana, which are rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. Cell disruption improved the digestibility of the Nannochloropsis biomass. Diets containing algal meal significantly reduced the relative abundance of arachidonic acid (AA) in the blood and liver lipids and caused a significant increase in the percentage of the omega 3 polyunsaturated fatty acids (PUFA). Feeding Nannochloropsis lipids resulted in a similar effect on the plasma and liver fatty acid pattern as that of a diet containing disrupted cells of Nannochloropsis biomass. In comparison, the response of the plasma and liver lipids to capelin oil was characterized by a further reduction in the abundance of AA and a significant elevation in the percentage of EPA and DHA. These differences are probably due to the variations in the fatty acid composition and not to the fact that omega 3 fatty acids are associated with different lipid classes in these lipid sources. Based on the present study, it is postulated that certain marine unicellular algae can be used as a nutritional source for omega 3 PUFA.

Energy transfer in the light-harvesting complex II of Dunaliella tertiolecta is unusually sensitive to Triton X-100.

Triton X-100, a detergent commonly used to solubilize higher plant thylakoid membranes, was found to be deleterious to Dunaliella LHC II. It disrupted the transfer of excitation energy from chlorophyll b to chlorophyll a. Based on analysis of pigments and immunoassays of LHC II apoproteins from sucrose density gradient fractions, Triton X-100 caused aggregation of the complex, but apparently did not remove chlorophyll b from the apoprotein. Following solubilization with Triton X-100 only CPI could be resolved by electrophoresis. In contrast, solubilization of Dunaliella thylakoids with octyl-ß-D-glucopyranoside preserved energy transfer from chlorophyll b to chlorophyll a. This detergent also effectively prevented aggregation on sucrose gradients and preserved CPI oligomers, as well as LHCP1 and LHCP3 on non-denaturing gels. Solubilization with Deriphat gave similar results. We propose that room temperature fluorescence excitation and emission spectroscopy be used in conjunction with other biophysical and biochemical probes to establish the effects of detergents on the integrity of light harvesting chlorophyll protein complexes. Methods used here may be applicable to other chlorophytes which prove refractory to protocols developed for higher plants.

Potential enhancement of photosynthetic energy conversion in algal mass culture.

Actual laboratory data obtained from steady-state Dunaliella tertiolecta cultures grown under a wide range of photon flux densities were used in a simple model to calculate daily production in a conventional algal mass culture system. In spite of large physiological and biochemical variations between low-light- (LL) and high light- (HL) adapted cultures, the overall calculated daily productivity is almost identical for both strains grown at optimal conditions. When production of fine biochemicals is considered, however, a hypothetical HL strain, which cannot shade adapt, is advantageous. Based on biochemical and biophysical analysis of D. tertiolecta responses to growth irradiance levels, specific targets are defined for genetic manipulation to enhance productivity in algal mass culture systems. The targets identified are (1) amplification of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase relative to the electron transport complexes, which should increase photosynthetic capacity at light saturation, and (2) enlargement of the light-harvesting complexes by varying their pigment composition in order to increase light harvesting at low photon flux densities.

7-Epicylindrospermopsin, a toxic minor metabolite of the cyanobacterium Aphanizomenon ovalisporum from lake Kinneret, Israel.

A toxic minor metabolite, 7-epicylindrospermopsin (1), was isolated from a culture of the cyanobacterium Aphanizomenon ovalisporum isolated from Lake Kinneret in Israel. Homonuclear and inverse-heteronuclear 2D NMR techniques, as well as HRMS and comparison of the NMR data with model compounds, enabled the structure determination of the new compound. Four polymethoxy-1-alkenes, 3-6, were isolated from the lipophilic extract of the cyanobacterium as well.

Adaptation of the Photosynthetic Apparatus to Irradiance in Dunaliella tertiolecta: A Kinetic Study.

The time course of adaptation from a high to a low photon flux density was studied in the marine chlorophyte Dunaliella tertiolecta. A one-step transition from 700 to 70 micromole quanta per square meter per second resulted in a reduction of doubling rate from 1.1 to 0.4 per day within 24 hours, followed by a slower accumulation of photosynthetic pigments, light harvesting antenna complexes, Photosystem II reaction centers and structural lipids that constitute the thylakoid membranes. Photoregulated changes in the biochemical composition of the thylakoid proteins and lipids were functionally accompanied by decreases in the minimal photosynthetic quantum requirement and photosynthetic capacity, and an increase in the minimal turnover time for in vivo electron transport from water to CO(2). Analysis of de novo synthesis of thylakoid membranes and proteins indicates that a high light to low light transition leads to a transient in carbon metabolism away from lipid biosynthesis toward the synthesis of the light harvesting antenna protein complexes, accompanied by a slower restoration rate of reaction centers and thylakoid membranes. This pattern of sequential synthesis of light harvesting complexes followed by reaction centers and membranes, appears to optimize light harvesting capabilities as cells adapt to low photon flux densities.

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophyll a/c-containing protein from the chrysophyte Isochrysis galbana: implications for evolution of the cab gene family.

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyte Isochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophyll a/c-binding light-harvesting protein (FCP) of I. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chl a/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity between I. galbana FCP and other cab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related to cab genes encoding LHC I, CP 29, and CP 24 of higher plants than to cab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chl a/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.