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Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and Nostoc (Nostocales, Cyanobacteria).

The heterocytous cyanobacteria form a monophyletic group according to 16S rRNA gene sequence data. Within this group, phylogenetic and morphological studies have shown that genera such as Anabaena and Aphanizomenon are intermixed. Moreover, the phylogeny of the genus Trichormus, which was recently separated from Anabaena, has not been investigated. The aim was to study the taxonomy of the genera Anabaena, Aphanizomenon, Nostoc and Trichormus belonging to the family Nostocaceae (subsection IV.I) by morphological and phylogenetic analyses of 16S rRNA gene, rpoB and rbcLX sequences. New strains were isolated to avoid identification problems caused by morphological changes of strains during cultivation. Morphological and phylogenetic data showed that benthic and planktic Anabaena strains were intermixed. In addition, the present study confirmed that Anabaena and Aphanizomenon strains were not monophyletic, as previously demonstrated. The evolutionary distances between the strains indicated that the planktic Anabaena and Aphanizomenon strains as well as five benthic Anabaena strains in cluster 1 could be assigned to a single genus. On the basis of the 16S rRNA, rpoB and rbcLX gene sequences, the Anabaena/Aphanizomenon strains (cluster 1) were divided into nine supported subclusters which could also be separated morphologically, and which therefore might represent different species. Trichormus strains were morphologically and phylogenetically heterogeneous and did not form a monophyletic cluster. These Trichormus strains, which were representatives of three distinct species, might actually belong to three genera according to the evolutionary distances. Nostoc strains were also heterogeneous and seemed to form a monophyletic cluster, which may contain more than one genus. It was found that certain morphological features were stable and could be used to separate different phylogenetic clusters. For example, the width and the length of akinetes were useful features for classification of the Anabaena/Aphanizomenon strains in cluster 1. This morphological and phylogenetic study with fresh isolates showed that the current classification of these anabaenoid genera needs to be revised.

Anabaena↗

NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene.

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.

Amino Acid Sequence↗

Identification of the region of cyanobacterial plasmid pDU1 necessary for replication in Anabaena sp. strain M-131.

Shuttle vectors based on plasmid pDU1 from Nostoc sp. strain PCC7524 are able to replicate both in Escherichia coli and in strains of Anabaena and Nostoc spp. Derivatives partially deleted in the pDU1 portion were tested for their ability to replicate in Anabaena sp. strain M-131. Plasmid pRL6HE containing a 1.75-kb HindIII-ScaI fragment of pDU1 replicated stably in that cyanobacterium and also in Anabaena sp. strain PCC7120. Plasmid pRL6HC, containing an even smaller HpaI-ScaI fragment (1.3 kb) replicated in Anabaena sp. strain M-131 but not in Anabaena sp. strain PCC7120. Similarly, when the 1.75-kb fragment of pDU1 was transferred from pRL6HE to another vector (pRL40 delta), the resulting plasmids replicated in Anabaena sp. strain M-131 but not in Anabaena sp. strain PCC7120.

Cyanobacteria↗

Nitrogen starvation mediated by DL-7-azatryptophan in the cyanobacterium Anabaena sp. strain CA.

The addition of DL-7-azatryptophan (AZAT), a tryptophan analog, to continuous cultures of Anabaena sp. strain CA grown with 10 mM nitrate as the nitrogen source resulted in the differentiation of heterocysts. Analysis of the intracellular amino acid pools of Anabaena sp. strain CA after the addition of AZAT showed a marked decline in the intracellular glutamate pool and a slight increase in the levels of glutamine. The in vitro activity of glutamate synthase, the second enzyme involved in primary ammonia assimilation in Anabaena spp., was partially inhibited by the presence of AZAT at concentrations which are effective in triggering heterocyst formation (15% inhibition at 10 microM AZAT and up to 85% inhibition at 1.0 mM AZAT). Azaserine, a glutamine analog and potent glutamate synthase inhibitor, had no effect on the triggering of heterocyst development from undifferentiated batch and continuous cultures of Anabaena sp. strain CA. However, the presence of 1.0 microM azaserine significantly decreased the intracellular glutamate pool and increased the glutamine pool. The addition of AZAT also caused a decrease in the C-phycocyanin content of Anabaena sp. strain CA as a result of its proteolytic degradation. AZAT also had an inhibitory effect on the nitrogenase activity of Anabaena sp. strain CA. All these results suggest that AZAT causes a general nitrogen starvation of Anabaena sp. strain CA filaments, triggering heterocyst synthesis.

Acetylene↗

Paracrystalline inclusions in various isolates of the blue-green bacteria Nostoc and Anabaena.

A number of different crystalline inclusions were observed in various isolates of Anabaena and Nostoc. Membrane-limited crystalline bodies were observed in 7 of 20 isolates of Anabaena and 19 of 29 isolates of Nostoc. These are spherical, single membrane-limited bodies from 0.6 to 0.1 micron in diameter. In most of the isolates they contained needle-like crystals 20 A in thickness and up to 80 nm in length. In 9 of the isolates the inclusions contained granular and fibrillar material. The number of bodies per cell varied in the different isolates from only a few, observed in many sections, up to 5 in a single section of A. subtropica (B1618). Crystalloids were observed in the cytoplasm of Anabaena sp. (1551), N. calcicola (B382), Nostoc sp. (588), and N. punctiforme (1629). In Anabaena sp. (1551) the roughly cuboidal inclusions 0.6 micron in diameter were composed of 100 A thick osmiophilic striations spaced to produce a 150 A periodicity. In Nostoc sp. (588) the elongate, 0.1 micron by 2.5 micron, crystalloids were composed of 100 A thick osmiophilic striations spaced to produce a 200 A periodicity. N. punctiforme (1629) and N. calciola (B382) contained intrathylakoidal crystalloids which consisted of short curved segments with 100 A thick osmiophilic striations producing a 200 A periodicity. Granular areas were observed in 2 isolates of Anabaena and 5 of Nostoc. These bodies found in various locations in the cells, were interpreted to be elongate structures 0.2 micron thick, 1.2 micron long and about 5 micron in depth. These inclusions were composed of 15 nm diameter granules which in some section planes appeared in rows spaced 20 nm apart. Spherical bodies up to 0.7 micron in diameter and of medium electron density were observed in 4 isolates of Anabaena and 2 of Nostoc. Convoluted inclusions were found in N. calcicola (B382) and Anabaena sp. (1551). These roughly spherical bodies up to 0.8 micron in diameter contain lighter swirled areas.

Crystallization↗

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119.

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.

Amino Acids↗

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120.

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.

Amino Acid Sequence↗

Effects of lindane on the photosynthetic apparatus of the cyanobacterium Anabaena: fluorescence induction studies and immunolocalization of ferredoxin-NADP+ reductase.

INTENTION, GOAL, SCOPE, BACKGROUND: Cyanobacteria have the natural ability to degrade moderate amounts of organic pollutants. However, when pollutant concentration exceeds the level of tolerance, bleaching of the cells and death occur within 24 hours. Under stress conditions, cyanobacterial response includes the short-term adaptation of the photosynthetic apparatus to light quality, named state transitions. Moreover, prolonged stresses produce changes in the functional organization of phycobilisomes and in the core-complexes of both photosystems, which can result in large changes in the PS II fluorescence yield. The localization of ferredoxin-NADP+ reductase (FNR) at the ends of some peripheral rods of the cyanobacterial phycobilisomes, makes this protein a useful marker to check phycobilisome integrity. OBJECTIVE: The goal of this work is to improve the knowledge of the mechanism of action of a very potent pesticide, lindane (gamma-hexaclorociclohexane), in the cyanobacterium Anabaena sp., which can be considered a potential candidate for bioremediation of pesticides. We have studied the effect of lindane on the photosynthetic apparatus of Anabaena using fluorescence induction studies. As ferredoxin-NADP+ reductase plays a key role in the response to oxidative stress in several systems, changes in synthesis, degradation and activity of FNR were analyzed. Immunolocalization of this enzyme was used as a marker of phycobilisome integrity. The knowledge of the changes caused by lindane in the photosynthetic apparatus is essential for rational further design of genetically-modified cyanobacteria with improved biorremediation abilities. METHODS: Polyphasic chlorophyll a fluorescence rise measurements (OJIP) have been used to evaluate the vitality and stress adaptation of the nitrogen-fixing cyanobacterium Anabaena PCC 7119 in the presence of increasing concentrations of lindane. Effects of the pesticide on the ultrastructure have been investigated by electron microscopy, and FNR has been used as a marker of phycobilisome integrity. RESULTS AND DISCUSSION: Cultures of Anabaena sp. treated with moderate amounts of lindane showed a decrease in growth rate followed by a recovery after 72 hours of pesticide treatment. Concentrations of lindane below 5 ppm increased the photosynthetic performance and activity of the cells. Higher amounts of pesticide caused a decrease in these activities which seems to be due to a non-competitive inhibition of PS II. Active PS II units are converted into non-QA reducing, so called heat sink centers. Specific activity and amount of FNR in lindane-treated cells were similar to the values measured in control cultures. Release of FNR from the thylakoid after 48 hours of exposure to 5 ppm of lindane towards the cytoplasm was detected by immunogold labeling and electron microscopy. CONCLUSIONS: From these results, we conclude that the photosynthetic performance and activity of the cells are slightly increased in the presence of lindane up to 5 ppm. Moreover, in those conditions, lindane did not produce significant changes in the synthesis, degradation or activity of FNR. The high capability of Anabaena to tolerate lindane makes this cyanobacterium a good candidate for phytoremediation of polluted areas. RECOMMENDATION AND OUTLOOK: The results of this study show that cultures of Anabaena PCC 7119 tolerate lindane up to 5 ppm, without significant changes in the photosynthetic vitality index of the cells. However, a slight increase in phycobiliprotein synthesis is observed, which is related to total protein content. This change might be due to degradation of proteins less stable than phycobiliproteins. An identification of the proteins with altered expression pattern in the presence of the pesticide remains the subject of further work and will provide valuable information for the preparation of strains which are highly tolerant to lindane.

Anabaena↗

Myxol and 4-ketomyxol 2'-fucosides, not rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and proposal for the biosynthetic pathway of carotenoids.

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.

Anabaena↗

Genomic analysis of protein kinases, protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120.

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.

Anabaena↗

Transcription control of ribulose bisphosphate carboxylase/oxygenase activase and adjacent genes in Anabaena species.

The gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activase (rca) was uniformly localized downstream from the genes encoding the large and small subunits of RubisCO (rbcL and rbcS) in three strains of Anabaena species. However, two open reading frames (ORF1 and ORF2), situated between rbcS and rca in Anabaena sp. strain CA, were not found in the intergenic region of Anabaena variabilis and Anabaena sp. strain PCC 7120. During autotrophic growth of Anabaena cells, rca and rbc transcripts accumulated in the light and diminished in the dark; light-dependent expression of these genes was not affected by the nitrogen source and the concentration of exogenous CO2 supplied to the cells. When grown on fructose, rca- and rbc-specific transcripts accumulated in A. variabilis regardless of whether the cells were illuminated. Transcript levels, however, were much lower in dark-grown heterotrophic cultures than in photoheterotrophic cultures. In photoheterotrophic cultures, the expression of the rca and rbc genes was similar to that in cultures grown with CO2 as the sole source of carbon. Although the rbcL-rbcS and rca genes are linked and are in the same transcriptional orientation in Anabaena strains, hybridization of rbc and rca to distinct transcripts suggested that these genes are not cotranscribed, consistent with the results of primer extension and secondary structure analysis of the nucleotide sequence. Transcription from ORF1 and ORF2 was not detected under the conditions examined, and the function of these putative genes remains unknown.

Amino Acid Sequence↗

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.

Carbon Isotopes↗

Bioconcentration and metabolism of DDT, fenitrothion and chlorpyrifos by the blue-green algae Anabaena sp. and Aulosira fertilissima.

Anabaena and Aulosira fertilissima showed a marked ability to accumulate DDT, fenitrothion and chlorpyrifos. Although the maximum accumulation of DDT was almost the same in both organisms, there were significant differences in their abilities to accumulate fenitrothion and chlorpyrifos. Patterns of uptake of DDT under different treatments were also similar in both Anabaena and Aulosira, but there were significant differences in the patterns of accumulation of fenitrothion between these two organisms. In Aulosira the maximum accumulation of fenitrothion was observed on the second day, whereas, in Anabaena, maximum accumulation was noticed on the first day. A completely different pattern of accumulation of chlorpyrifos was observed in Aulosira, which continued to accumulate chlorpyrifos throughout the experimental period. Bioconcentration of DDT in Anabaena and Aulosira ranged from 3 to 1568 ppm (microg g(-1)) and 6 to 1429 ppm, respectively. Bioconcentration of fenitrothion and chlorpyrifos in Anabaena varied from 53 to 3467 ppm and 7 to 6779 ppm, respectively. In Aulosira the bioconcentration varied from 100 to 6651 ppm and 53 to 3971 ppm for fenitrothion and chlorpyrifos, respectively. Anabaena and Aulosira metabolised DDT to DDD and DDE. Amounts of these DDT metabolites detected in the organisms were dependent on the concentration of treatment. DDD was the major, and DDE the minor, metabolite. These organisms were not able to metabolise the organophosphorus insecticides, fenitrothion and chlorpyrifos.

Journal Article↗

Interaction of Endosulfan and malathion with blue-green algae Anabaena and Aulosira fertilissima.

The growth of Anabaena and Aulosira fertilissima was adversely affected by endosulfan even at 1 microg ml(-1). The inhibition was significantly above 50% at 20 microg ml(-1) throughout the incubation. Anabaena survived up to 500 microg ml(-1) of malathion, but was completely bleached in the presence of 50 microg ml(-1) of endosulfan. Aulosira was more sensitive to malathion than Anabaena and recovered to control levels only at 10 microg ml(-1). The morphology and hetercyst frequency were not altered in Anabaena. Aulosira cultures were dull brown in colour at 20 microg ml(-1) of endosulfan with the filaments clumping, instead of the usual mat formation. Both malathion and endosulfan considerably lowered (14)C uptake and nitrogenase activities in Aulosira. Nitrogen fixation was unaffected in Anabaena as the amounts of ethylene produced were equal to, or above, control levels. The impact of these insecticides on photosynthesis in Anabaena was only slight.

Journal Article↗

The Azolla, Anabaena azollae Relationship: I. Initial Characterization of the Association.

Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source.Morphological aspects of the symbiotic association show the confinement of the Anabaena azollae within the leaf cavity of the Azolla. Procedures were established for the isolation of pure preparations of Anabaena azollae and Azolla chloroplasts. It has not yet been possible to grow the isolated alga in independent culture.Photochemical activities of the isolated alga and fern chloroplasts were measured by spectrophotometric assays for photosystems I and II as well as by P700-content (photosystem I) and delayed light emission (photosystem II). In the algal fraction, both photosystems were repressed when compared to freeliving Anabaena cylindrica, but the relative ratio of photosystem I to photosystem II may be appreciably greater in Anabaena azollae. Azolla chloroplasts were generally comparable to spinach chloroplasts.A comparison of the chlorophyll a and b content of Azolla fronds with and without the symbiotic alga resulted in an estimate that in the symbiotic association, the Anabaena azollae accounts for from 7.5 to 15% of the total chlorophyll.

Journal Article↗

Regulation of expression of nitrate and dinitrogen assimilation by anabaena species.

Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more NO(3) into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species.

Journal Article↗

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.

Amino Acid Sequence↗

A Zoogloea sp. associated with blooms of Anabaena flos-aquae.

Bacteria were found attached to the heterocysts of Aphanizomenon flos-aquae and embedded within the mucilage of both anabaena flos-aquae and Microcystis aeruginosa in freshwater plankton. Electron microscopy of thin sections preceding the peak of an Anabaena flos-aquae bloom showed that the density of bacterial cells was 7.4 X 10(5) cells/ml in the planktonic macroenvironment and 2.6 X 10(11) cells/ml within the microenvironment of cyanobacterial mucilage. The bacteria occurred in aggregates and isolation required that these be dispersed by homogenizing at 50 000 rpm with glass beads. This procedure yielded a single bacterial isolate from blooms of Anabaena flos-aquae during 2 consecutive years. The isolate was flagellated, catalase- and oxidase-positive. Gram-negative, and rod-shaped to pleomorphic. Observation that the isolate required a pH greater than 8 for consistent growth, could not grow alone on liquid media but could grow alone on the corresponding solid media, could grow in liquid media only in the presence of Anabaena, formed tough mucilagenous colonies on solid media only in the presence of Anabaena extract, and rapidly assimilated but did not respire extracellular 14C-labelled organic matter produced by Anabaena suggested that the occurrence of the bacterium in cyanobacterial mucilage was not coincidental but reflected an obligatory bacterial requirement for the biological or physicochemical microenvironment of the mucilage. The bacterial isolate occurred in three growth forms. Either as a planktonic swarmer cell (which showed a positive chemotactic response to the cyanobacterium) embedded in cyanobacterial mucilage, or embedded in its own mucilage derived, in part, from a low molecular weight (below 1300) carbohydrate secreted by the cyanobacterium. These cultural, biochemical, and ecological characteristics suggest that the isolate is a new species in the genus Zoogloea and of potential importance in phytoplankton ecology.

Cyanobacteria↗