CULTURE OF THE TERRESTRIAL CYANOBACTERIUM,NOSTOC FLAGELLIFORME (CYANOPHYCEAE), UNDER AQUATIC CONDITIONS1

Both colonies and free‐living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free‐living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30° C at either 20 or 60 μmol photons·m^?2·s^?1; colonies did not develop at 180 μmol photons·m^?2·s^?1, though this light level resulted in the most rapid growth of the cells. Conditions of 60 μmol photons·m^?2·s^?1 and 25° C appeared to result in the best colonial development and faster growth of the sheath‐held colonies of N. flagelliforme when cultured indoor under aquatic conditions.     

Siderophore production in Azotobacter vinelandii in response to Fe‐, Mo‐ and V‐limitation

Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co‐factors Mo and V. However, regulation of siderophore production by Mo‐ and V‐limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N₂‐fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe‐limitation the production of all siderophores increases, while under Mo‐limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo‐ and V‐limitation. An examination of the potential nitrogen ‘cost’ of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo‐ as opposed to the less efficient V‐nitrogenase.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Energy Transfer from Phycobilisomes to Photosystems of Nostoc flagelliforme Born. et Flah. During the Rewetting Course and Its Physiological Significance

During the non-frost season, the condensation of dew makes Nostocflagelliforme Born. et Flah., a highly drought-tolerant terrestrial cyanobacterium, frequently undergo rehydration-dehydration.Rehydration begins in the dark at night. After rewetting in the dark, photochemical activity and the structure of photosystem (PS)II were not recovered at all; the structure of PSI, energy transfer in phycobilisomes, and energy transfer from phycobilisomes to PSI were recovered within 5 min, as in the light. The recovery of energy transfer from phycobilisomes to PSII was light dependent and energy transfer from phycobilisomes to PSII was only partially recovered in the dark. These results suggest that the two-trigger control (water and light) of photosynthetic recovery may make IV, flagelliforme avoid unnecessary energy consumption and, at the same time, the partial recovery of energy transfer from phycobilisomes to PSII in the dark could help N. flagelliforme accumulate more photosynthetic products during the transient period of rehydration-dehydration.     

Identification of novel smORFs and microprotein acting in response to rehydration of Nostoc flagelliforme

Nostoc flagelliforme, a terrestrial cyanobacterium spread throughout arid and semi-arid areas, has been long known for its outstanding adaptability to extremely dry conditions. This microorganism is able to recover biological activities within hours after months of anhydrobiosis state, attracting investigation through proteomic analysis. Except for canonical proteome, microproteins encoded by small ORFs (smORFs) have recently been regarded as indispensable participants in metabolic processes. However, the involvement of smORFs in N. flagelliforme remains unknown. Here we first constructed a smORF database in N. flagelliforme using bioinformatic prediction, resulting in 6072 novel smORFs. Then LS-MS/MS analysis was applied to identify expression patterns of microproteins and seek smORFs and their encoded microprotein playing a role during rehydration. In total, 18 novel microproteins were mined based on a smORF searching strategy combined with three proteomic assays, of which five were annotated as ribosomal proteins, one as RNA polymerase subunit, and one as acetohydroxy acid isomeroreductase. We also suggested the possible functions of smORFs according to their expression pattern and discovered two neighboring and homologous smORFs. All these results will expand our knowledge of smORFs-encoded microproteins and their relation to the stress response of extremophilic microorganisms.     

Influence of CO2, light and watering on growth of Nostoc flagelliforme mats

The terrestrial blue-green alga (cyanobacterium), Nostoc flagelliforme, was cultured in air at variouslevels of CO₂, light and watering to see theireffects on its growth. The alga showed the highestrelative growth rate at the conditions of highCO₂ (1500 ppm), high light regime (219–414mol m^-2s^-1) and twice daily watering,but the lowest rate at the conditions of low light(58–114 mol m^-2s^-1) and daily twicewatering. Increased watering had little effect ongrowth rate at 350 ppm CO₂, but increased byabout 70% at 1500ppm CO₂ under high lightconditions. It was concluded that enriched CO₂could enhance the growth of N. flagelliformewhen sufficient light and water was supplied.     

Weak red light plays an important role in awakening the photosynthetic machinery following desiccation in the subaerial cyanobacterium Nostoc flagelliforme

The subaerial cyanobacterium Nostoc flagelliforme can survive for years in the desiccated state and light exposure may stimulate photosynthetic recovery during rehydration. However, the influence of light quality on photosynthetic recovery and the underlying mechanism remain unresolved. Exposure of field collected N. flagelliforme to light intensity ≥2 μmol photons m⁻² s⁻¹ showed that the speed of photosystem II (PSII) recovery was in the following order: red > green > blue ≈ violet light. Decreasing the light intensity showed that weak red light stimulated PSII recovery during rehydration. The chlorophyll fluorescence transient and oxygen evolution activity indicated that the oxygen evolution complex (OEC) was the activated site triggered by weak red light. The damaged D1 protein accumulated in the thylakoid membrane during dehydration and is degraded and resynthesized during dark rehydration. PsbO interaction with the thylakoid membrane was induced by weak red light. Thus, weak red light plays an important role in triggering OEC photoactivation and the formation of functional PSII during rehydration. In its arid habitats, weak red light could stimulate the awakening of dormant N. flagelliforme after absorbing water from nighttime dew or rain to maximize growth during the early daylight hours of the dry season.     

An investigation on the genetic background of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> by similarity analysis of its partial genomic DNA and phylogenetic comparison of deduced related species

Nostoc flagelliforme, which is distributed on arid and semi-arid steppes of northwestern parts of China, has attracted increasing interest for its stress tolerance. In order to gain more insight into the genetic background of N. flagelliforme, we sequenced its partial genomic DNA for similarity analyses against current public databases, followed by phylogenetic comparison of N. flagelliforme and the potentially related species deduced from the similarity analyses. Approximately 430 kb genomic sequence (~5% of genome as a rough estimate) was determined from 106 distinct genomic clones. Nucleotide BLAST showed that ~23.1% of the partial genomic sequence was similar to N. punctiforme genomic DNA and ~12.4% to its plasmid DNA. Similar protein search by online FASTA-protein program showed 46.2% of the similar proteins had their corresponding orthologs in N. punctiforme genome. Furthermore, phylogenetic comparison based on 16S rRNA sequences showed N. flagelliforme and N. punctiforme clustered closer among the deduced related species. These results indicated that N. punctiforme might also be potentially close neighbor species of N. flagelliforme, in addition to the formerly regarded close neighbor species N. commune and N. sphaeroids. In general, these data enriched our recognition of the evolutionary relationship between N. flagelliforme and other Nostoc species, especially N. punctiforme.     

Metal limitation of cyanobacterial N2 fixation and implications for the Precambrian nitrogen cycle

Nitrogen fixation is a critical part of the global nitrogen cycle, replacing biologically available reduced nitrogen lost by denitrification. The redox‐sensitive trace metals Fe and Mo are key components of the primary nitrogenase enzyme used by cyanobacteria (and other prokaryotes) to fix atmospheric N₂ into bioessential compounds. Progressive oxygenation of the Earth's atmosphere has forced changes in the redox state of the oceans through geologic time, from anoxic Fe‐enriched waters in the Archean to partially sulfidic deep waters by the mid‐Proterozoic. This development of ocean redox chemistry during the Precambrian led to fluctuations in Fe and Mo availability that could have significantly impacted the ability of prokaryotes to fix nitrogen. It has been suggested that metal limitation of nitrogen fixation and nitrate assimilation, along with increased rates of denitrification, could have resulted in globally reduced rates of primary production and nitrogen‐starved oceans through much of the Proterozoic. To test the first part of this hypothesis, we grew N₂‐fixing cyanobacteria in cultures with metal concentrations reflecting an anoxic Archean ocean (high Fe, low Mo), a sulfidic Proterozoic ocean (low Fe, moderate Mo), and an oxic Phanerozoic ocean (low Fe, high Mo). We measured low rates of cellular N₂ fixation under [Fe] and [Mo] estimated for the Archean ocean. With decreased [Fe] and higher [Mo] representing sulfidic Proterozoic conditions, N₂ fixation, growth, and biomass C:N were similar to those observed with metal concentrations of the fully oxygenated oceans that likely developed in the Phanerozoic. Our results raise the possibility that an initial rise in atmospheric oxygen could actually have enhanced nitrogen fixation rates to near modern marine levels, providing that phosphate was available and rising O₂ levels did not markedly inhibit nitrogenase activity.     

Nutritional Composition and Assessment of Gracilaria lemaneiformis Bory

The chemical composition, mineral elements, vitamins, free fatty acids and amino acid content of the edible red alga Gracilaria lemaneiformis Bory, grown in the sea near Nan＇ao island, Guangdong Province, were analyzed in the present study. Gracilaria lemaneiformis Bory showed a total sugar content of 14.65%. The protein content was 21%, of which approximately 41% was determined to be essential amino acids （EAA）. The major amino acid components were glutamic acid, leucine, arginine, and alanine. Of the EAA assayed, methionine and cysteine appeared to be the most limiting amino acids compared with the EAA pattern provided by Food and Agricultural Organization of the United Nations. The total lipids content was 0.87% and comprised a high composition of unsaturated fatty acids （61%）, mainly as linoleic acid and oleic acid, and a little amount of polyunsaturated fatty acid; palmitic acid was the main component （39%） of saturated acids. Relatively high levels of vitamin C, iodine, phosphorus, and zinc were also present in G. lemaneiformis. The nutritional composition between G. lemaneiformis and Nostoc flagelliforme, a rare alga that is widely eaten in Chinese society, was compared. The results suggest that N. flagelliforme can be substituted for by G. lemaneiformis, not only because of their similar shape, but also because of their approximate nutritional composition. Gracilaria lemaneiformis may possibly serve as a potential healthy food in human diets in the future.     

Toxicity of parathion-methyl to cells, akinetes and heterocysts of the cyanobacterium Cylindrospermum, sp. and the probit analysis of toxicity

The toxicity of a commercial formulation of the insecticide parathion‐methyl to the N2‐fixing filamentous cyanobacterium (blue‐green alga) Cylindrospermum, sp. was studied. A concentration of parathion‐methyl of 0.5 ppm caused growth increase in liquid growth media. The minimum inhibitory concentration of parathion‐methyl for both types (N₂, fixing and nitrate supplemented) of liquid and solid media was 1.0 ppm. LC₅₀ values were: 4.4 ppm (liquid, N₂, fixing), 5.5 ppm (liquid, nitrate supplemented), 3.3 ppm (agar, N₂‐fixing) and 4.0 ppm (agar, nitrate supplemented). LC100 values for N₂‐fixing liquid and both types of agar media were 10.0 ppm, while for the liquid nitrate supplemented medium the LC₁₀₀ was 12.0 ppm. Both akinete (spore) formation and germination were inhibited below the highest permissive concentration of 8.0 ppm, with the insecticide incorporated in the agar media. In soil, the LC50 and LC100 values for parathion‐methyl were 13.6 and 30 ppm, respectively. Both the dehydrogenase activity of heterocysts (monitored by 2,3,5‐triphenyl tetrazolium chloride reduction) and the nitrogen concentration of cultures (estimated by the micro‐Kjeldahl method) were affected by the insecticide, but the latter (N₂‐fixation) was more sensitive. The Kruskal‐Wallis H test on the numbers of vegetative cells in the filaments revealed that the insecticide significantly affected the division of vegetative cells. The cyanobacterium could detoxify the growth medium containing high levels (30 and 40 ppm) of the insecticide in short‐term exposures at the expense of cell viability.     

Nitrate assimilation in rice seedlings as affected by mineral nutrition

Deficiencies of each macronutrient (N, P, K, Ca, Mg and Fe)in the culture solution depressed the specific activities ofnitrate reductase (NR) and nitrite reductase (NiR) from riceseedlings. Nitrate and potassium deficiencies especially loweredNR induction, whereas phosphorus deficiency caused the leastdecrease in enzyme induction. On the other hand the activityof NiR was decreased most by deficiencies of nitrate and phosphorus.Potassium deficiency was not as effective in suppressing theinduction of NiR. Sulfur deficiency slightly promoted the inductionof both NR and NiR. Generally, micronutrient deficiencies didnot affect either enzyme. NR induction was slightly decreasedby B, Zn, Cu and Mo deficiencies, and increased by Mn deficiency;whereas NiR activity was slightly increased by B and Cu deficiencies,and was not affected by other micronutrients. Nitrate contentwas decreased by deficiencies of N, P, K, Ca, and micronutrients,and unaffected by Mg, Fe and S deficiencies. Glutamic acid dehydrogenase(GDH) activity was increased by N, Fe and P deficiencies, anddecreased by Mo and Zn deficiencies, and unaffected by othernutrient treatments. (Received August 25, 1976; )     

Symbiosis constraints: Strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa—a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N₂‐fixing cyanobacteria (Nostoc sp.)—as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N‐fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ¹³C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO₂ among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth.     

NUTRIENT REQUIREMENTS OF THE DINOFLAGELLATE PERIDINIUM GATUNENSE1

Optimum nutrient conditions for growth and photosynthesis of Peridinium gatunense (Nygaard) (Peridinium cinctum fa. westii) were investigated using axenic clones in batch cultures. Selenium (Se) had previously been found to be an indispensable growth factor for P. gatunense. Optimal, suboptimal, and supraoptimal concentrations of HCO₃^?, N, Ca, Cl, Mg, P, K, S, Si, EDTA-Na, Fe, Mo, Zn, Mn, Co, Se, B, Br, I, and various trace element mixtures were determined by measuring biomass development, growth rates, ¹⁴C uptake, and/or oxygen production at various concentration gradients of these elements. The general characteristics of the best formulation, medium-L 16, relative to other media, are its high content of NaHCO₃ (1 meq · L^?1) and Mo (0.2 μM) but low concentrations of NO_3-N (150 μM), PO_4-P (10 μM), and Fe (0.4 μM), in addition to its content of Se. The total content of trace metals, except for Se, may be reduced to one-fourth of that in medium-L 16 without altering the major growth-promoting properties of the medium. Medium-L 16 deviated considerably from Lake Kinneret (Israel) water, being much lower in macroelements except for N and P. The pH (8.1–8.4) was in the same range, but the values of conductivity (140 μS · cm^?1), alkalinity (1 meq · L^?1) and NaCl (200 μM) were > 8, 2, and 30 times higher, respectively, in the lake water. Selenium deficiency may limit the growth of P. gatunense in this lake.     

Biological crust of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> (cyanobacteria) on sand bed materials

Nostoc flagelliforme in liquid culture was cultivated on sand bed materials as inoculum and formed a biological crust. The biological crust had no common appearance of wild N. flagelliforme thallus in naked eye observation, but it had natural morphology of wild N. flagelliforme thallus in microscopic observation. By examining the photosynthetic activity, N. flagelliforme cells on sand were found to be heat resistant. The sand grain size had some influence on N. flagelliforme growth, and the growth rate on fine sand was higher than on coarse sand. It is necessary to consider the characteristics of sand particle size to prevent desertification. N. flagelliforme cells could form the algal biological crust, which indicates that the biological crust has potential application in soil desertification and was extremely important to improve desertification.     

PHOSPHATE UPTAKE AND GROWTH KINETICS OF TRICHODESMIUM (CYANOBACTERIA) ISOLATES FROM THE NORTH ATLANTIC OCEAN AND THE GREAT BARRIER REEF,AUSTRALIA1

We compared inorganic phosphate (P_i) uptake and growth kinetics of two cultures of the diazotrophic cyanobacterium Trichodesmium isolated from the North Atlantic Ocean (IMS101) and from the Great Barrier Reef, Australia (GBRTRLI101). Phosphate‐limited cultures had up to six times higher maximum P_i uptake rates than P‐replete cultures in both strains. For strain GBRTRLI101, cell‐specific P_i uptake rates were nearly twice as high, due to larger cell size, but P‐specific maximum uptake rates were similar for both isolates. Half saturation constants were 0.4 and 0.6 μM for P_i uptake and 0.1 and 0.2 μM for growth in IMS101 and GBRTRLI101, respectively. Phosphate uptake in both strains was correlated to growth rates rather than to light or temperature. The cellular phosphorus quota for both strains increased with increasing P_i up to 1.0 μM. The C:P ratios were 340–390 and N:P ratios were 40–45 for both strains under severely P‐limited growth conditions, similar to reported values for natural populations from the tropical Atlantic and Pacific Oceans. The C:P and N:P ratios were near Redfield values in medium with >1.0 μM P_i. The North Atlantic strain IMS101 is better adapted to growing on P_i at low concentrations than is GBRTRLI101 from the more P_i‐enriched Great Barrier Reef. However, neither strain can achieve appreciable growth at the very low (nanomolar) P_i concentrations found in most oligotrophic regimes. Phosphate could be an important source of phosphorus for Trichodesmium on the Great Barrier Reef, but populations growing in the oligotrophic open ocean must rely primarily on dissolved organic phosphorus sources.     

PHYSIOLOGICAL DIFFERENCES BETWEEN TWO SPECIES OF CYANOBACTERIA IN RELATION TO PHOSPHORUS LIMITATION1

In order to gain a better understanding of the factors responsible for changes in cyanobacterial species composition in the Alberche River (central Spain), which is characterized by spatiotemporal N and P variability, we undertook P‐limitation studies on two isolated N₂‐fixing species that are well represented in the river: Calothrix elenkinii (Kosinskaja) UAM 225 and Nostoc punctiforme (Kützing) UAM 220. We examined differences in phosphorus‐storage capacity, phosphate uptake, and phosphatase activity between the two isolates grown under N₂‐fixing conditions. Although they shared a similar pattern of phosphate uptake, C. elenkinii cultures had a greater phosphorus storage capacity and greater phosphatase activity than those of N. punctiforme. The former showed a stronger response when cultured under low P concentrations. The two species showed specific differences in the rates of intracellular polyphosphate granule utilization and phosphatase activity. Our results suggest that C. elenkinii might be particularly well suited to river environments in which nutrient concentrations are generally low, but which experience episodic pulses of high P supply (inorganic and/or organic), favoring “luxury uptake” and storage strategies.     

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES,CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH₂PO₄) or Pi‐sufficient (100 μM NaH₂PO₄) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH₂PO₄ decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The K_m value of Pi uptake rate was decreased by incubation with 1 μM NaH₂PO₄ and the decrease in K_m value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.