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X-ray diffraction studies on photosystem I fragments from a blue-green alga, Anabaena variabilis, and spinach.

Photosystem I fragments were prepared from thylakoid membranes of a blue-green alga (Anabaena variabilis) and spinach by treatment with a detergent, Triton X-100. Equatorial X-ray diffraction patterns were recorded on films for oriented specimens of thylakoid membranes and photosystem I fragments. The thylakoid membranes and photosystem I fragments gave essentially the same equatorial diffraction patterns in both Anabaena variabilis and spinach, indicating that the major X-ray scatterers in these thylakoid membranes are the molecular assembly of photosystem I. The equatorial X-ray diffraction from the photosystem I fragments of Anabaena variabilis and spinach extends to the reciprocal space of 1/7 A-1. The diffraction pattern exhibits six to nine distinct maxima though they are diffuse, indicating that the arrangement of the constituent molecules in photosystem I has a definite geometrical regularity. The radial autocorrelation functions indicate that the maximal sizes of photosystem I in these thylakoid membranes are about 100 A, and the geometrical regularity does not correspond to a crystalline order. The X-ray diffraction patterns from photosystem I fragments from Anabaena variabilis and spinach are quite similar to each other, suggesting the possibility that the molecular structures of photosystem I in Anabaena variabilis and spinach have a fundamental similarity. These diffraction patterns, however, are different from that of the chromatophore obtained from a photosynthetic bacterium, Rhodospirillum rubrum.

Cyanobacteria↗

The Azolla-Anabaena azollae Relationship : XI. PHYCOBILIPROTEINS IN THE ACTION SPECTRUM FOR NITROGENASE-CATALYZED ACETYLENE REDUCTION.

Visible absorption spectra are presented for the Azolla caroliniana Willd.-Anabaena azollae Strass. association and the individual partners. Although absorption by the phycobiliproteins of the endophytic cyanobacterium clearly complements the absorption by the fern pigments, their contribution to the absorption spectrum of the association is effectively concealed by the preponderance of the Azolla pigments. Action spectra for nitrogenase-catalyzed C(2)H(2) reduction in both the Azolla-Anabaena association and the endophytic Anabaena demonstrate that quanta absorbed by the phycobiliproteins is as effective as that absorbed by chlorophyll a in driving this photosystem I-linked process. Under anaerobic conditions, the inhibition of photosystem II activity by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron did not selectively decrease the relative quantum yields in the region of phycobiliprotein absorption. At the well-below saturating light intensities used for the action spectra studies, the absolute rates of C(2)H(2) reduction were increased uniformly via respiratory-linked processes under aerobic conditions. The occurrence of phycobiliproteins in heterocysts of the endophytic Anabaena was demonstrated using fluorescence microscopy of intact filaments. Fluorescence micrographs of Anabaena cylindrica filaments are presented for comparison.

Journal Article↗

Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena.

Phycobiliprotein degradation can be initiated in cultures of the cyanobacterium Anabaena by removal of combined nitrogen from the medium. Certain strains of Anabaena differentiate cells specialized for aerobic nitrogen fixation (heterocysts) under such conditions. We describe here a procedure for the preparation of extracts from heterocysts or vegetative cells that contain an activity capable of degrading only the phycobiliproteins in a mixture of soluble Anabaena proteins in vitro. This activity increased under nitrogen starvation conditions or in ammonia-replete cultures treated with the glutamine synthetase inhibitor methionine sulfoximine. The increase in activity induced by nitrogen starvation was prevented by chloramphenicol or by carbon starvation. Under all these conditions, phycobiliprotein degradative activity assayed in vitro was correlated with the loss of phycobiliprotein absorbance in vivo. Finally, starvation of a met auxotroph of Anabaena for methionine (in the presence of ammonia) did not induce phycobiliprotein degradation in vivo or the increase in proteinase activity. Together with direct measurements of ppGpp, these results indicate that proteolysis in Anabaena is not controlled by compounds associated with the stringent response in Escherichia coli. Since the increase in proteinase activity appears to be regulated by the same variables that control heterocyst differentiation, the activity should provide a useful biochemical marker for the early events of differentiation.

Adenosine Triphosphate↗

Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences.

Use of the sacB gene (J. L. Ried and A. Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

Cyanobacteria↗

Adenosylcobalamin-dependent ribonucleotide reductase from the blue-green alga, Anabaena sp. Purification and partial characterization.

The ribonucleotide reductase from Anabaena 7119 has been purified approximately 60- to 80-fold by conventional techniques and adsorption to the affinity medium, Matrix Gel Red A. The enzyme from Anabaena resembles the adenosylcobalamin-dependent reductase from Lactobacillus leichmannii, in that it is a small molecule (molecular weight 72,000) with no subunit structure as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Unlike its prototype, the Anabaena reductase is absolutely dependent on a divalent cation for activity, Ca2+ being the most effective. In addition, the Anabaena reductase shows a simple pattern of alloteric control by deoxyribonucleotides. CTP reduction is stimulated by dATP, GTP by dTTP, and ATP by dGTP. No reduction is observed in the absence of effectors, and none of the effectors inhibits enzyme activity. Thus, the Anabaena ribonucleotide reductase can be more easily studied by kinetic analysis than the Lactobacillus enzyme, and should provide additional information as to the mechanism of action of this enzyme in a photosynthetic organism.

Cations, Divalent↗

X-ray structure of the ferredoxin:NADP+ reductase from the cyanobacterium Anabaena PCC 7119 at 1.8 A resolution, and crystallographic studies of NADP+ binding at 2.25 A resolution.

The crystal structure of the ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 has been determined at 2.6 A resolution by multiple isomorphous replacement and refined using 15.0 A to 1.8 A data, collected at 4 degrees C, to an R-factor of 0.172. The model includes 303 residues, the flavin adenine dinucleotide cofactor (FAD), one sulfate ion located at the putative NADP+ binding site and 328 water molecule sites. The structure of Anabaena FNR, including FAD, a network of intrinsic water molecules and a large hydrophobic cavity in the C-terminal domain, resembles that of the spinach enzyme. The major differences concern the additional short alpha-helix (residues 172 to 177 in Anabaena FNR) and residues Arg 100 and Arg 233 which binds NADP+ instead of Lys 116 and Lys 244 in the spinach enzyme. Crystals of a complex of Anabaena FNR with NADP+ were obtained. The model of the complex has been refined using 15 A to 2.25 A X-ray data, collected at -170 degrees C, to an R-factor of 0.186. This model includes 295 residues, FAD, the full NADP+ (with an occupancy of 0.8) and 444 water molecules. The 2'-5' adenine moiety of NADP+ binds to the protein as 2'-phospho-5'-AMP to the spinach FNR. The nicotinamide moiety is turned towards the surface of the protein instead of stacking onto the FAD isoalloxazine ring as would be required for hydride transfer. The model of the complex agrees with previous biochemical studies as residues Arg 100 and Arg 233 are involved in NADP+ binding and residues Arg77, Lys 53 and Lys 294, located on the FAD side of the enzyme, remain free to interact with ferredoxin and flavodoxin, the physiological partners of ferredoxin: NADP reductase.

Amino Acid Sequence↗

Fatty acid composition of planktonic species of Anabaena (cyanobacteria) with coiled trichomes exhibited a significant taxonomic value.

Twenty-six axenic strains of planktonic Anabaena with coiled trichomes belonging to 13 species were investigated by analyzing the pattern and content of their fatty acid composition, and by comparing their fatty acid composition with their morphological properties. In general, the planktonic Anabaena with coiled trichomes contained 14:0, 16:0, 16:1(cis-), 18:0, 18:1, 18:2, and 18:3(alpha) as their major fatty acid component, and were classified as Type 2 according to the Kenyon-Murata System. The Type 2 was further divided into two subtypes: Type 2A with 16:2 and 16:3, and Type 2B without 16:2 and 16:3. Among these Anabaena strains with coiled form, A. oumiana (NIES-73 and Ana Kas1) and A. eucompacta (Ana Chiba) contained Type 2B fatty acid composition, and other strains contained Type 2A. Among the strains with the latter type, A. circinalis (Ana Da) and A. curva (Ana Ao) had low levels of 18:3(alpha). Most Anabaena strains with coiled trichomes showed a strong correlation between morphological characteristics and fatty acid composition.

Anabaena↗

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control.

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

Anabaena↗

Determinative value of a portion of the nifH sequence for the genera Nostoc and Anabaena (cyanobacteria).

The taxonomic positions of Nostoc and Anabaena strains are currently disputed. We selected three Nostoc and Anabaena strains, using the classic criteria of morphology and life cycle. DNA sequences of a part of the nifH gene were determined from these strains and aligned with homologous sequences from 10 other Nostoc/Anabaena strains in the public databases. Phylogenetic reconstructions were carried out to test the consistency of the taxonomic placement of these strains. The phylogenetic trees do not separate these strains into distinct groups. Our results are in agreement with other molecular-based phylogenies that also fail to differentiate the Nostoc-Anabaena groups. The data suggest that the currently recognized genera Nostoc and Anabaena may in fact belong within a single, broadly defined genus.

Anabaena↗

Sucrose synthase and RuBisCo expression is similarly regulated by the nitrogen source in the nitrogen-fixing cyanobacterium Anabaena sp.

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.

Anabaena↗

Sucrose-phosphate phosphatase from Anabaena sp. strain PCC 7120: isolation of the protein and gene revealed significant structural differences from the higher-plant enzyme.

The present study describes the first isolation and characterization of a prokaryotic protein and gene for sucrose-phosphate phosphatase (SPP), the enzyme that catalyzes the terminal step in sucrose synthesis. For gene isolation, a 2,015-bp DNA fragment containing an open reading frame with about 31% amino acid identity to Synechocystis SPS was amplified from Anabaena sp. PCC 7120 DNA. Surprisingly, expression of the putative gene in Escherichia coli demonstrated that it encoded an SPP protein. The expressed protein cross-reacted with antibodies against the native form of Anabaena SPP and its biochemical properties were identical to those of the enzyme purified from the cyanobacterial cells. Comparisons of the Anabaena SPP with the higher-plant enzyme revealed important differences in the C-terminal region, molecular mass, subunit composition and immunoreactivity. Nevertheless, two conserved motifs, including four invariant aspartate residues similar to those found in members of the phosphohydrolase superfamily, were identified in the Anabaena SPP deduced amino acid sequence.

Amino Acid Sequence↗

A small multigene family encodes the rod-core linker polypeptides of Anabaena sp. PCC7120 phycobilisomes.

The cpc operon of Anabaena sp. PCC7120 is shown to encode ten genes: 5'-cpcB-cpcA-cpcC-cpcD-cpcE-cpcF- cpcG1-cpcG2-cpcG3-cpcG4-3'. The 3' portion of this operon includes four tandemly repeated genes encoding phycocyanin (PC)-associated, rod-core linker polypeptides of the phycobilisomes (PBS). The products of these four genes are most similar at their N termini, and overall are 50-61% identical and 68-76% similar to one another. The four CpcG proteins of Anabaena sp. PCC7120 are 41-47% identical and 62-65% similar to the single CpcG rod-core linker protein in Synechococcus sp. PCC7002. The N-terminal domains of the polypeptides are also more distantly related to the conserved domains of other types of rod-linker polypeptides associated with PC, phycoerythrin, and allophycocyanin (AP). Three of these rod-core linker proteins (CpcG1, CpcG2, and CpcG4) were demonstrated to occur in isolated PBS by N-terminal amino acid sequence analyses. These results indicate that previously proposed models for the PBS of Anabaena sp. are incorrect. It is suggested that the PBS of Anabaena sp. have eight peripheral rods, each of which interacts with the AP of the core via a specific rod-core linker (CpcG) polypeptide.

Amino Acid Sequence↗

Morphological, physiochemical and molecular characterization of Anabaena strains.

A set of 30 Anabaena strains, isolated from diverse geographical regions of India, were characterized using morphological and physiochemical attributes as well as molecular marker profiles. Significant differences were observed among the Anabaena strains with regard to the shape and size of trichomes and individual cells within a filament, besides qualitative and quantitative aspects of phycobiliprotein accumulation and activities of enzymes involved in nitrogen metabolism. Analyses of molecular polymorphisms in a selected set of 13 Anabaena strains, using primers based on repetitive sequences in the genome, led to unambiguous differentiation of the strains as well as understanding of their genetic relationships. Informative morphological, physio-chemical and molecular characters have been identified that could aid in differentiation and utilization of Anabaena strains as bioinoculants or as sources of pigments.

Anabaena↗

Outdoor cultivation of a nitrogen-fixing marine cyanobacterium, Anabaena sp. ATCC 33047.

Optimization of conditions for outdoor production of the nitrogen-fixing cyanobacterium Anabaena sp. ATCC 33047 has been pursued. In open ponds operated under semi-continuous regime biomass productivity values achieved ranged from 9 g (dry weight) m(-2) per day, in winter, to over 20 g m(-2) per day, in summer, provided that key operation parameters, including cell density, were optimized. Under these conditions the efficiency of solar energy conversion by the cells was fairly constant throughout the year, with photosynthetic efficiency values higher than 2%. The cyanobacterial biomass was rich in high-value phycobiliproteins, namely allophycocyanin and phycocyanin, for which open cultures of marine Anabaena represent a most interesting production system. The performance of Anabaena cultures operated under continuous regime in a closed tubular reactor has also been assessed outdoors, in winter. Biomass productivity values similar to those obtained in the ponds have been recorded for the closed system. Additionally, under these conditions, the cells excreted to the medium large amounts of a previously characterized exopolysaccharide, at production rates as high as 35 g m(-2) per day (1.4 g l(-1) per day). Properly operated closed cultures of this strain of Anabaena appear most suitable for outdoor mass production of valuable extracellular polysaccharides.

Anabaena↗

Catalysis of RNA cleavage by a ribozyme derived from the group I intron of Anabaena pre-tRNA(Leu).

In the cyanobacterium Anabaena PCC7120, the precursor to tRNA(Leu) contains a 249-nucleotide group I intron that undergoes efficient self-splicing in vitro. By deleting the 5' and 3' splice sites, this intron has now been converted to an RNA enzyme that uses a guanosine nucleophile to cleave substrate RNAs (S) with multiple turnover. This Anabaena ribozyme has a second-order rate constant for RNA cleavage (kcat/Km)S that is 250-500-fold smaller than that of the Tetrahymena ribozyme, and a multiple-turnover rate constant at saturating S [kcat(mt)] that is approximately 400-fold larger. Several lines of evidence, including kinetic analysis of cleavage of phosphorothioate- and deoxynucleotide-substituted substrates and pH dependence, support the conclusion that both (kcat/Km)S and kcat(mt) are limited by the actual chemical cleavage step. In contrast, for the Tetrahymena ribozyme, it has been shown that neither of these rate constants reflects the chemical step. These kinetic differences are expected from the shorter guide sequence-substrate pairing of the Anabaena ribozyme; for example, weaker binding of RNA speeds product release during multiple turnover and thereby overcomes the rate-limiting product release observed for the Tetrahymena ribozyme. Thus, the large kinetic differences represent superficial rather than fundamental differences between these ribozymes. Furthermore, the strength of the guanosine-binding interaction, the stereospecificity for Rp-phosphorothioate at the cleavage site, and the 10(3)-fold slower cleavage with a deoxyribonucleoside leaving group are properties conserved between the Anabaena and Tetrahymena ribozymes. Finally, log(kcat/Km)S increases linearly with pH in the acid range where chemistry is rate-limiting and becomes pH-independent above pH 7, perhaps because a conformational step becomes rate-limiting; again, these are characteristics shared with the Tetrahymena ribozyme. We conclude that two group I ribozymes, although differing in the identity of many of their active site nucleotides, nevertheless provide functionally similar active sites for sequence-specific RNA cleavage.

Anabaena↗

Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors.

A region of the genome of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 containing the ntcB gene was identified. This region is located upstream from the nir operon involved in nitrate assimilation in this cyanobacterium. An Anabaena ntcB mutant was able to use ammonium and dinitrogen as sources of nitrogen for growth but was unable to assimilate nitrate. Enzymes of the nitrate reduction system were not synthesized in the ntcB mutant under derepression conditions. The transcription start-point of the Anabaena nir operon, which has been shown to be subjected to ammonium-stimulated repression and whose expression requires the global nitrogen regulator NtcA, was only weakly used in the ntcB mutant. The expression of the ntcB gene in strain PCC 7120 was also subjected to repression by ammonium and was found to take place from an NtcA-activated promoter located 31 bp upstream from the start of the ntcB gene. NtcB binds to the nir promoter region in vitro and protects a region localized just upstream from the NtcA-binding site in footprinting assays. These results showed that NtcB, a LysR-family protein, is required in addition to NtcA, a CAP-family protein, for the expression of genes encoding proteins specifically involved in nitrate assimilation in Anabaena sp. PCC 7120.

Anabaena↗

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation.

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca(2+)-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca(2+) reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca(2+)concentration {[Ca(2+)](i)}, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca(2+)](i) was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca(2+)](i) could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca(2+) ions play very important roles in hetR induction and heterocyst differentiation.

Anabaena↗

Growth of the cyanobacterium Anabaena on molecular nitrogen: NifJ is required when iron is limited.

The nifJ gene of Klebsiella pneumoniae encodes an oxidoreductase required for the transfer of electrons from pyruvate to flavodoxin, which reduces nitrogenase. The nifJ gene of Anabaena 7120, isolated from a cosmid bank, was found to contain an open reading frame encoding a 1197-aa protein. The deduced amino acid sequence shows 50% identity to the Klebsiella homolog. The nifJ gene in Anabaena 7120 was inactivated by chromosomal interruption. The resulting mutant was unable to grow on medium depleted of both iron and combined nitrogen but grew normally, fixing nitrogen, when iron was present. NifJ transcripts of 2.7 and 4.3 kb are induced by iron depletion irrespective of nitrogen status. One particular stretch of the Anabaena 7120 nifJ gene encodes 12 aa with no complementary matches in the Klebsiella protein. This insert contains five tandem repeats of the heptamer CCCCAGT. These heptamers, as well as heptamers and octamers of other related sequences, have been located in a number of cyanobacterial genomes but are usually not found within the coding region of a gene. The site of the Anabaena 7120 heptamers in the nifJ genes of other filamentous cyanobacteria contains a surprising diversity of repeated sequences, both octamers and heptamers. The corresponding protein inserts range in length from 1 to 21 aa, relative to Klebsiella NifJ.

Amino Acid Sequence↗