Anabaena Species Research Articles

Isolation of arginine auxotrophs, cloning by mutant complementation, and sequence analysis of the argC gene from the cyanobacterium Anabaena species PCC 7120.

Arginine auxotrophs of the dinitrogen-fixing cyanobacterium Anabaena species strain PCC 7120 were isolated after ultraviolet light mutagenesis and penicillin enrichment. Two of these auxotrophs were complemented by a cosmid gene library of the wild-type strain established in Escherichia coli that was transferred en masse to the mutants by conjugation. The gene complementing one of those mutants was found to complement an E. coli argC mutant. Sequencing analysis of the gene showed that it encodes a 322-residue polypeptide that is homologous to the ArgC protein of E. coli, Bacillus subtilis and Streptomyces clavuligerus and to the C-terminal moiety of the Saccharomyces cerevisiae ARG5,6 gene product, N-acetylglutamate semialdehyde dehydrogenase. A cysteine residue present in a highly conserved domain in the five proteins is probably located in the active site of the enzyme. Conserved among the ArgC proteins, sequences resembling the primary structure of nucleotide-binding domains are also found. Downstream of the Anabaena argC gene seven nearly perfect repeats of a heptanucleotide (consensus sequence:5'-CTAATGA-3') are found.

Superoxide dismutase in symbiotic, free-living and wild Anabaena and Nostoc (Nostocales, Cyanophyta)

Symbionts and isolates of Anabaena azollae Strasburger (Cyanophyta) from various Azalia species, an Arthrobacter globiformis Conn et Dimmick isolate, Azolla filiculoides Lamarck plants (Pteridophyta), isolates of Nostoc species from cycad coralloids, and free-living Anabaena and Nostoc from the wild were tested for the presence of iron, manganese and iron-manganese superoxide dismutase. The three isoenzymes were found in symbionts and isolates of Anabaena and Nostoc, and in free-living Nostoc species from the wild. Only iron superoxide dismutase was found in Azolla filiculoides, in Arthrobacter globiformis and in free-living Anabaena from the wild. The presence of similar superoxide dismutase isoenzymes in Anabaena azollae and Nostoc isolates suggests the possibility that the Azalia phycobiont may be a species of Nostoc rather than a species of Anabaena.

Contribution of benthic blue‐green algal recruitment to lake populations and phosphorus translocation

SUMMARY1. A two‐season investigation was undertaken to determine the contribution of benthic recruitment to the population development of several species of blue‐green algae.2. Gloevtrichia echinulata populations were shown to be heavily subsidized by benthic recruitment, deriving on average 40% of their planktonic colonies from the benthos.3. Benthic recruitment of Aphanizomenon flos‐aquae, Anabaena flos‐aquae and a second Anabaena species contributed less than 2% to planktonic increases, while Microcystis aeruginosa recruitment was negligible.4. Phosphorus translocation via migrating G. echinulata accounted for most of the phosphorus (P) in the planktonic colonies, and constituted a significant portion of the internal loading of the lake.5. Estimated P translocation via Aph. flos‐aquae was relatively minor, although there was evidence of luxury uptake in the benthos.

Use of membrane filters for soil algal bioassays

A culture technique for eucaryotic and procaryotic soil algae involving the use of membrane filters superimposed on the surface of inorganic nutrient agar is described. Nostoc commune grew exponentially when cultured in this manner. No significant differences in biomass production or nitrogenase activity were detected among culture subsets within replicate experiments run under standard conditions. Estimates of daily growth rates (0.340), culture doubling time (48.9 h), and nitrogenase activity (14.54 nM C2H2 reduced µg−1 chl a h−1) were consistent with laboratory and field estimates reported for several planktonic species of Anabaena and strains of Nostoc commune isolated from diverse terrestrial habitats. Therefore, the filter-culture technique is an alternative which may be superior to traditional liquid culture methodology for studies involving certain soil procaryotic and eucaryotic algae.

Geosmin and 2-Methylisoborneol Production by Cyanobacteria in Fish Ponds in the Southeastern United States

Levels of geosmin and 2-methylisoborneol and composition of phytoplankton communities were determined for water samples collected from 35 fish ponds at Auburn, Alabama, and 34 ponds in west-central Mississippi. In Auburn ponds, geosmin levels ranged from 0.05 to 8.9 µg/l but 2-methylisoborneol was never detected. Highest geosmin concentrations were measured in ponds with plankton blooms dominated by Anabaena species with straight trichomes. In Mississippi ponds, 2-methylisoborneol levels ranged from 0.05 to 76 µg/l. Highest 2-methylisoborneol concentrations were associated with blooms of Oscillatoria cf. chalybea, a cyanobacterium that produces 2-methylisoborneol in unialgal culture. Geosmin was found infrequently in Mississippi pond waters; geosmin levels ranging from 0.05 to 6.25 µg/l could be associated with blooms of Anabaena spiroides, and always concurred with 2-methylisoborneol concentrations ranging from 0.45 to 3.25 µg/l. Neither geosmin nor 2-methylisoborneol at either location could be associated with phytoplankton blooms of Aphanizomenon flos-aquae, Oscillatoria agardhii, Raphidiopsis brookii, or Microcystis aeruginosa.

Geosmin Production by Cyanobacteria (Blue‐green Algae) in Fish Ponds at Auburn, Alabama

AbstractStudies on production of geosmin, the compound responsible for earthy‐muddy flavor in water and fish, were carried out at the Auburn University Fisheries Research Station. Water was collected from ponds during 44 cyanobacterial bloom periods from April to September. Geosmin (trans‐1, 10‐dimethyl‐trans‐9‐decalol) was detected at levels averaging 4.77 μg/L when Anabaena species with straight trichomes were predominant. Lower geosmin concentrations were associated with blooms of Anabaena species with coiled trichomes, Microcystis aeruginosa, and Aphanizomenon flos‐aquae, and averaged 0.24, 0.12, and 0.07 μg/L, respectively. Study of four Anabaena blooms over a period of 4–8 weeks showed that changes in geosmin were correlated significantly with changes in trichome abundance. Geosmin disappeared from the water in seven days when Anabaena died. Comparison of geosmin concentrations in raw and filtered water showed that, on average, 90% of the geosmin could be associated with the particulate fraction. Dissolved geosmin concentrations ranged from 0.07–0.85 μg/L.

Highly buoyant colonies of the cyanobacterium Anabaena lemmermannii form persistent surface waterblooms

The colonial gas-vacuolate cyanobacterium Anabaena lemmermannii has formed modest populations in Windermere during the months of June and July in recent years. During the summer of 1990 the colonies were dispersed in the well-mixed upper epilimnion of the main lake basins but formed light surface scums along leeward shores and in sheltered bays, including Mitchell Wyke. The gas vesicles of this species were found to be much stronger (p(c)0.93 MPa) than those in other Anabaena species; they would withstand exposure to hydrostatic pressures at depths exceeding 40 m and they would therefore survive deep-mixing events in this lake. The colonies were highly buoyant; they required only 46% of their gas vesicles to render 50% of the colonies neutrally buoyant in one sample. In another sample the buoyant density of colonies with intact gas vesicles was 975 kg m-3 compared with 1022 kg m-3 after gas vesicles collapse, which indicated that only 51% of the gas vesicles were need to give the same density as water. In a third sample comparison of the mean colony floation rate of 181-mu-m s-1 before gas vesicle collapse with the sinking rate of 186-mu-m s-1 after collapse also demonstrated that 51% of the gas vesicles were required for neutral buoyancy. Colonies incubated in two bottles at the lake surface failed to respond to the high irradiance by losing their buoyancy. It was calculated that the colonies would not have been able to accumulate sufficient carbohydrate in a day to overcome the excess buoyancy provided by their gas vesicles; the high critical pressure of the gas vesicles prevented their collapse by turgor pressure rise. The inability of highly buoyant cyanobacteria to lose buoyancy may provide a general explanation for the persistence of cyanobacterial surface water-blooms.

Crystallization and structure determination of 2.5-.ANG. resolution of the oxidized iron-sulfur [2Fe-2S] ferredoxin isolated from Anabaena 7120

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.

Bacterial Origin of a Chloroplast Intron: Conserved Self-Splicing Group I Introns in Cyanobacteria

A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.

Multinuclear magnetic resonance studies of the 2Fe.cntdot.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.

Multinuclear magnetic resonance studies of the 2Fe.cntdot.2S* ferredoxin from Anabaena species strain PCC 7120. 2. Sequence-specific carbon-13 and nitrogen-15 resonance assignments of the oxidized form

Multinuclear two-dimensional NMR techniques were used to assign nearly all diamagnetic 13C and 15N resonances of the plant-type 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120. Since a 13C spin system directed strategy had been used to identify the 1H spin systems [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085], the sequence-specific 1H assignments [Oh, B.-H., & Markley, J. L. (1990) Biochemistry (first paper of three in this issue)] also provided sequence-specific 13C assignments. Several resonances from 1H-13C groups were assigned independently of the 1H assignments by considering the distances between these nuclei and the paramagnetic 2Fe.2S* center. A 13C-15N correlation data set was used to assign additional carbonyl carbons and to analyze overlapping regions of the 13C-13C correlation spectrum. Sequence-specific assignments of backbone and side-chain nitrogens were based on 1H-15N and 13C-15N correlations obtained from various two-dimensional NMR experiments.

Multinuclear magnetic resonance studies of the 2Fe.cntdot.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary characterization of neurotoxic cyanobacteria blooms and strains from Finland

AbstractTwo hundred fifteen cyanobacteria bloom samples collected from different parts of Finland were studied, 35 of which proved to be neurotoxic. Toxicity was determined by mouse bioassay. Anabaena species were present in all neurotoxic samples except one, in which Oscillatoria dominated. The presence of anatoxin‐a in the blooms and in the isolated strains was studied from freeze‐dried materials by gas chromatography/mass spectrometry. The simultaneous occurrence of neurotoxicity and hepatotoxicity in some samples was studied by high performance liquid chromatography and high performance thin layer chromatography. Thirteen out of 30 bloom samples contained anatoxin‐a. In the remaining samples, neurotoxicity was caused by unknown toxin(s). Strains producing anatoxin‐a were isolated from the genera Anabaena, Aphanizomenon, Oscillatoria, and Cylindrospermum. Anatoxin‐a content of the blooms varied from 12 to 4360 μg/g freeze‐dried material. Some strains were able to produce about three times as much anatoxin‐a as was detected in natural blooms. Simultaneous occurrence of neurotoxicity and hepatotoxicity was found in some samples as well as atypical toxic responses in mouse bioassay.

Using Activated Carbon to Remove Toxicity From Drinking Water Containing Cyanobacterial Blooms

Toxic blooms of cyanobacteria, including Anabaena species, which are neurotoxic, and Microcystis, which is hepatotoxic, are becoming increasingly common in potable water sources. Normal flocculation and chlorination processes do not remove the toxicity. The laboratory and pilot‐plant experiments described in this article showed that the toxicity can be removed by both powdered and granular activated carbon, with and without chlorination, alum flocculation, and polyelectrolyte addition.

The isolation of genomic DNA from cyanophage infecting Nostoc and Anabaena species of cyanobacteria

SUMMARYThe preparation of genomic DNA from a recently isolated group of cyanophage which infect species of Nostoc and Anabaena (cyanobacteria) has been investigated. Factors which affect the yield of DNA, including culture density on infection, divalent cation requirements, NaCl tolerance and desorption from cell debris, have been assessed. These factors have been incorporated into optimized procedures for the isolation of genomic DNA from five cyanophage. The DNA isolated from the rive cyanophage species was identified using restriction endonuclease fragment patterns.

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.

Relationship between Sodium Influx and Salt Tolerance of Nitrogen-Fixing Cyanobacteria

The relationship between sodium uptake and cyanobacterial salt (NaCl) tolerance has been examined in two filamentous, heterocystous, nitrogen-fixing species of Anabaena. During diazotrophic growth at neutral pH of the growth medium, Anabaena sp. strain L-31, a freshwater strain, showed threefold higher uptake of Na than Anabaena torulosa, a brackish-water strain, and was considerably less salt tolerant (50% lethal dose of NaCl, 55 mM) than the latter (50% lethal dose of NaCl, 170 mM). Alkaline pH or excess K (>25 mM) in the medium causes membrane depolarization and inhibits Na influx in both cyanobacteria (S. K. Apte and J. Thomas, Eur. J. Biochem. 154:395-401, 1986). The presence of nitrate or ammonium in the medium caused inhibition of Na influx accompanied by membrane depolarization. These experimental manipulations affecting Na uptake demonstrated a good negative correlation between Na influx and salt tolerance. All treatments which inhibited Na influx (such as alkaline pH, K above 25 mM, NO(3), and NH(4)), enhanced salt tolerance of not only the brackish-water but also the freshwater cyanobacterium. The results indicate that curtailment of Na influx, whether inherent or effected by certain environmental factors (e.g., combined nitrogen, alkaline pH), is a major mechanism of salt tolerance in cyanobacteria.

Algal Competition for Phosphorus: The Influence of Zooplankton and Fish

Interspecific competition for phosphorus between the blue-green alga Anabaena flos-aquae and the green alga Staurastrum luetkemuelleri was studied in enclosure experiments. Both algal populations increased in number upon introduction of fish, but addition of herbivorous zooplankton led to an increase solely in the Staurastrum population. This could not be attributed to grazing by zooplankton, chemical or physical conditions (light, pH, inorganic carbon), or the total supply or rate of supply of phosphorus. The observed differences were apparently due to the patchy release of phosphorus by the fish, and the more homogenous release of this nutrient by the zooplankton. This would allow the Anabaena species, which was capable of rapid uptake of temporary pulses of phosphate, to coexist with Staurastrum in the enclosures with fish.

Morphological and ultrastructural variability of planktonic cyanophyceae in relation to seasonal periodicity.

Vegetative cells, heterocysts and akinetes in Anabaena solitaria are described under natural conditions by following wild populations of the alga over two successive field seasons. Ultrastructure of the vegetative cells and heterocysts is similar to that elucidated for other species of Anabaena in culture. Akinete development resembles that previously described for other members of the Nostocaceae except that the envelope is formed in two asymmetrical parts. Unlike the remainder of the envelope, fibres at the junction are oriented in one plane only, rendering it responsive to mechanical stress during germination. The need for a comparative study of natural and cultured material is emphasized by a demonstration of the morphological variability of akinete formation in Anabaena solitaria maintained in culture for ten years.

The form resistance of sinking algal chains

The relationship between sinking velocity and chain length was investigated in a species of Anabaena and with physical models constructed from glass beads. The sinking velocities of glass bead chains from one to 30 beads long were measured in tanks of glycerol, ensuring low Reynolds numbers, with the chains orientated in both horizontal and vertical directions. The coefficient of form resistance (⊘) for each length and attitude was calculated as a correction factor in the Stokes equation. Average sinking velocities of the Anabaena chains were found to fall within the range of velocities predicted from the Stokes equation with the appropriate values of ⊘ determined from measurements with the physical models.