Anabaena Cells Research Articles

A novel bacterium Saprospira sp. strain PdY3 forms bundles and lyses cyanobacteria

A helical filamentous cyanobactericidal bacterium was isolated from Dianchi Lake, a eutrophic freshwater lake in Kunming City of the Yunnan Province in China using a special solid medium. This species was designated strain PdY3. This bacterium was identified as a novel Saprospira sp. on the basis of its morphological characteristics and 16S rDNA sequence. Strain PdY3 showed apparent group behavior on the solid medium, forming orderly, bundle-like group structures. These bundles moved as groups. Individuals in a bundle responded to the bundle as a whole. PdY3 also showed group behavior and formed a three-dimensional reticular structure when co-cultured with Anabaena in liquid media. This helical bacterium lysed cyanobacteria through direct contact and its group behavior greatly accelerated the cyanobactericidal process. Our experiments showed that PdY3 caused lysis of 64% of Anabaena cells within 1 day and that its cyanobactericidal range was broad. These results underscore potential application of Saprospira on the control of blooms of cyanobacteria. PdY3 group behavior might allow a more efficient capture of bacterial prey.

Inactivation of algal blooms in eutrophic waterof drinking water supplies with the photocatalysisof TiO2 thin film on hollow glass beads

Photocatalytic inactivation of algae, Anabaena, Microcystis, and Melosira, was carried out with TiO2-coated Pyrex hollow glass beads under the illumination of UV light (370 nm wavelength). After being irradiated with UV light in the presence of the TiO2-coated Pyrex glass beads, Anabaena and Microcystis, known as typical cyanobacteria, lost their photosynthetic activity, and the string of Anabaena cells and the colonies of Microcystis cells were completely separated into individual spherical ones. In the case of Melosira, which is a typical diatom, however, somewhat lower photocatalytic inactivation efficiency was obtained, which was believed to be due to the presence of the inorganic siliceous wall surrounding the cells of Melosira. The TiO2-coated hollow glass beads could successfully be employed for the practical application in a eutrophicated river under sunlight. More than 50% of the chlorophyll-a concentration could be reduced by the action of TiO2 photocatalysis.

Exogenous expression of the wheat chloroplastic fructose-1,6-bisphosphatase gene enhances photosynthesis in the transgenic cyanobacterium, Anabaena PCC7120

The paper reports a study on the genetic regulation of photosynthesis by introducing the gene encoding wheat chloroplastic fructose-1,6-bisphosphatase (FBPase) into the cyanobacterium Anabaena PCC7120. The gene was RT-PCR amplified from wheat and modified by replacement of the 5′-terminal encoding sequence with optimal and A/T-rich codons to promote prokaryotic expression. The resultant FBPase gene was ligated downstream of the strong promoter, PpsbA of expression vector pRL-439, then inserted into of shuttle vector pDC-08. The resulting shuttle expression vector (pDC-fbp) was transferred into the filamentous, heterocystour cyanobacterium, Anabaena PCC7120, by the tri-parental conjugation transfer method. Protein expression of FBPase in the transgenic Anabaena was 126.5% higher than in wild type cells, and the enzyme activity of transgenic cells was 1.41-fold higher than that of wild type cells. Under atmospheric conditions of 360 μmol mol−1 CO2, Anabaena cells overexpressing the FBPase gene further showed increases in net photosynthesis (117.2%) and true photosynthesis (122.5%) as compared to wild type cells. In addition, transgenic Anabaena grew faster and contained more Chl a than did wild type cells. Together, these results indicate that introduction of the wheat chloroplastic FBPase gene into Anabaena increase photosynthesis and cell growth; furthermore, these trends were more evident under stress condition (higher CO2 concentration). This is the first report of enhanced photosynthesis in cyanobacteria expressing genes from higher plants.

Photochromicity of Anabaena Sensory Rhodopsin, an Atypical Microbial Receptor with a cis-Retinal Light-adapted Form

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin characterized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.

Regulation of CO2 on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.

Preparation of TiO 2-coated hollow glass beads and their application to the control of algal growth in eutrophic water

Photocatalytic inactivation of algae, Anabaena, Microcystis and Melosira, was carried out with the TiO 2-coated pyrex hollow glass beads under the illumination of UV-A light. After being irradiated with UV-A light in the presence of the TiO 2-coated pyrex glass beads, Anabaena and Microcystis, known as typical cyanobacteria, lost their photosynthetic activity, and the string of Anabaena cells and the colonies of Microcystis cells were completely separated into individual spherical one. In the case of Melosira, which is a typical diatom, however, somewhat lower photocatalytic inactivation efficiency was obtained, which was believed to be due to the presence of the inorganic siliceous wall surrounding the cells of Melosira. The TiO 2-coated hollow glass beads could successfully be employed for the practical application at the eutrophicated river under sunlight. More than 50% of the chlorophyll- a concentration could be reduced by the action of TiO 2 photocatalysis.

BIOLOGICAL AVAILABILITY OF IRON TO THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe3+ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe3+] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe3+ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity

It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days’ experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity.

Fructose-containing oligosaccharides: novel compatible solutes in Anabaena cells exposed to salt stress

In response to salinity cyanobacteria synthesize osmoprotective compounds. It has been reported that Anabaena sp. accumulates sucrose (Suc) as a salt adaptation strategy. The aim of this study is to show that Anabaena sp. cells not only increase intracellular Suc concentration after addition of NaCl but also synthesizes fructose-containing-oligosaccharides (Fru-oligosaccharides). Sucrose–phosphate synthase (SPS) activity and polypeptide amount also increased in short-term treated cells. Suc and Fru-oligosaccharide accumulation, and SPS expression were reversed to control values when cells were returned to basal-medium growth conditions. In contrast to what is known in regard to plant fructan biosynthesis, Suc level did not determine the initiation of Fru-oligosaccharide synthesis in Anabaena. We conclude that the accumulation of Fru-oligosaccharides must be also considered as an adaptation mechanism to salt stress in Anabaena, and they should be included among the compatible solutes synthesized in response to salt stress in stenohaline cyanobacterium strains.

Lead accumulation in the aquatic fern Azolla filiculoides

In this study, we characterized lead (Pb 2+) accumulation and storage by the aquatic fern Azolla filiculoides. Lead precipitates were detected in the vacuoles of mesophyll cells of Azolla plants cultured for 6 d in rich growth medium containing 20 mg l –1 Pb 2+. Energy dispersive spectroscopy (EDS) analysis of the relative element content of leaves collected from these plants revealed a 100% increase in the levels of P, S, Na and Ca and a 40% decrease in Mg and Cl compared to the untreated plants. Both Azolla whole plants and isolated apoplasts were incubated for 6 d in 20 mg l –1 Pb 2+. Lead content in the whole plant composed 0.37%, 2.3% and 1.8% of the dry weight after 2, 4 and 6 d of growth, respectively, while the isolated Azolla apoplast contained 0.125%, 1.22% and 1.4% Pb 2+, respectively. Lead content in Azolla whole plant increase by 200%, 100% and 22% after 2, 4 and 6 d of growth, respectively, when compared to Azolla apoplast. Dark, electron dense deposits of lead were observed in light and transmission electron microscope in leaf cells treated with lead. All the observed lead deposits were localized in vacuoles while larger lead deposits were found in mature leaves than in young leaves. No lead deposits were found in cells of the cyanobiont Anabaena when the plants were exposed to similar conditions. Activity and content of V-H +-ATPase were studied in Azolla plants grown in the presence of 20, 40 and 80 mg l –1 of lead for a period of 4 d. Activity of V-H +-ATPase was increased by 190%, 210% and 220%, respectively, but the content of V-H +-ATPase was reduced by all lead concentrations.

Only iron-limited cells of the cyanobacteriumAnabaena flos-aquaeinhibit growth of the green algaChlamydomonas reinhardtii

Cocultivation of iron-limited cells of the cyanobacterium Anabaena flos-aquae (Lyng.) Brèb. and the green alga Chlamydomonas reinhardtii Dangeard resulted in growth of Anabaena but not Chlamydomonas, even in the presence of excess exogenous iron. This effect was also observed during the cultivation of Chlamydomonas in a medium in which iron-limited Anabaena cells had been growing, but were removed prior to culture of Chlamydomonas. Conversely, iron-limited Chlamydomonas cells grew very well in medium from iron (nutrient)-sufficient, phosphate-limited, and nitrogen-limited Anabaena cultures. Iron-limited Anabaena cultures produced siderophores, while the other types of Anabaena cultures did not. Treatment of Anabaena iron-limited medium with activated charcoal completely removed the inhibitory effect on Chlamydomonas growth, and boiling the medium removed most of the inhibitory effect. Both the charcoal and the boiling treatments also removed siderophores from the medium. Partially purified Anabaena siderophore preparations were also inhibitory to Chlamydomonas growth. The inhibitory effect of iron-limited Anabaena medium could be partially overcome by addition of excess micronutrients (especially cobalt copper) but not by addition of iron. We suggest that Anabaena-derived siderophores, present only in iron-limited Anabaena medium, inhibit the growth of Chlamydomonas cells via a previously uncharacterized toxicity. This effect is different from previously described experiments in which cyanobacterial siderophores suppressed green algal growth via competition for limiting amounts of iron.Key words: Anabaena, Chlamydomonas, cocultivation, iron limitation, micronutrients; siderophores.

Cyanobacterial alkaline/neutral invertases. Origin of sucrose hydrolysis in the plant cytosol?

The aim of this work was to investigate the occurrence of invertase (Inv) in cyanobacteria. We describe the first isolation and characterization of prokaryotic alkaline/neutral Inv (A/N-Inv) genes. Two genes (invA and invB) were identified in Anabaena sp. PCC 7120, which share about 50-56% identity with plant A/N-Inv and encode proteins of about 53-55 kDa. The identification of these proteins was confirmed by biochemical and immunological studies with recombinant proteins and with the enzymes isolated from Anabaena cells. Expression analysis supported the important role of A/N-Inv in nitrogen-fixing growth conditions. Nevertheless, A/N-Inv activities were shown in all filamentous and unicellular cyanobacteria investigated, regardless of their capacity to fix dinitrogen. Searches in complete sequenced genomes showed that A/N-Inv homologues are restricted to cyanobacterial species and plants. In particular, filamentous nitrogen-fixing strains display two A/N-Inv genes and unicellular strains have only one. Phylogenetic analysis leads us to suggest that modern plant A/N-Inv might have originated from an orthologous ancestral gene after the endosymbiotic origin of chloroplasts.

NaCl-induced oxidative damage in the cyanobacterium Anabaena doliolum.

Results obtained with graded concentrations of NaCl (20-200 m M) show decrease in the chlorophyll 'a' contents of Anabaena with increasing concentration of NaCl except at extremely low concentration of NaCl (5-20 m M). The rate of Hill activity and oxygen evolution are found to be stimulated by lower concentrations of NaCl, but not at higher concentrations of NaCl. Results have demonstrated that the O(2) evolution process is relatively more sensitive to NaCl stress than the Hill activity. Further, the results show NaCl induced an increase in the rate of RNO bleaching and loss of total thiol (-SH) contents. Taken together, these results suggest a NaCl-induced general oxidative stress. Results on the effect of oxygen radical quenchers reveal a predominant role of singlet oxygen in the NaCl-induced general oxidative stress as evident from a higher quenching effect of sodium azide than formate and histidine on the rate of RNO bleaching in Anabaena cells. However, the rate of lipid peroxidation and SOD activity show a declining pattern in response to increasing concentrations of NaCl. There is the possibility of a NaCl-induced decrease in the rate of lipid peroxidation when the SOD activity is also lower. But the NaCl-induced decline in the SOD activity does suggest that symptoms of general oxidative stress at elevated levels of NaCl are apparently owing to collapse of intracellular defense of the cells against the toxic oxygen radicals, not because of the higher rate of photosynthetic activity.

Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development.

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was, however, not dependent on HetR.

Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state.

Fur (ferric uptake regulator) protein is a DNA-binding protein which regulates iron-responsive genes. Recombinant Fur from the nitrogen-fixing cyanobacterium Anabaena PCC 7119 has been purified and characterized, and polyclonal antibodies obtained. The experimental data show that Fur from Anabaena dimerizes in solution with the involvement of disulphide bridges. Cross-linking experiments and MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) MS also show several oligomerization states of Fur, and the equilibrium of these forms depends on protein concentration and ionic strength. In intact recombinant Fur, four cysteine residues out of five were inert towards DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)], and their modification required sodium borohydride. Metal analysis and electrospray ionization MS revealed that neither zinc nor other metals are present in this Fur protein. Purified recombinant Fur bound to its own promoter in gel-shift assays. Fur was shown to be a constitutive protein in Anabaena cells, with no significant difference in its expression in cells grown under iron-sufficient compared with iron-deficient conditions.

An ABC-type, high-affinity urea permease identified in cyanobacteria.

Urea is an important nitrogen source for many microorganisms, but urea active transporters have not been characterized at a molecular level in any bacterium. Cells of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 exhibited the capacity to take up [14C]-urea from low-concentration (<1 microM) urea solutions. The Ks of Anabaena cells for urea was about 0.11 microM, and the observed uptake activity involved the transport and metabolism of urea. In contrast to urease, which was constitutively ex-pressed, expression of the high-affinity urea uptake activity was subjected to nitrogen control. In an Anabaena ureG (urease-) mutant, a concentrative, active transport of urea could be demonstrated. We found that a mutant of open reading frame (ORF) sll0374 from the Synechocystis genomic sequence lacked urea transport activity. This ORF encoded a conserved component of an ABC-type transporter, but it is not clustered together with any other possible transporter-encoding gene. An Anabaena homologue of sll0374, urtE, was isolated and found to be part of a cluster of genes, urtABCDE, putatively encoding all the elements of an ABC-type permease. Although the longest transcript that we could detect only covered urtABC, the impairment of urea transport by inactivation of urtA, urtB or urtE suggested that the whole gene cluster is expressed producing the urea permease. Expression was induced under nitrogen-limiting conditions, and a complex promoter regulated by the cyanobacterial global nitrogen control transcription factor NtcA was found upstream from urtA. Our work adds urea to the known substrates of the versatile class of ABC-type transporters and suggests the involvement of a transporter of this superfamily in urea scavenging by some bacteria in natural environments.

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.

Differential Effects of Thiobencarb Toxicity on Growth and Photosynthesis of Anabaena variabilis with Changes in Phosphate Level

A thiobencarb dose of 3 mg L−1 reduced the protein content of Anabaena variabilis, whereas it elevated the carbohydrate content. Measurements of Anabaena growth, photosynthetic activity, and respiration rate revealed that the high dose of phosphate (0.53 mM) exerted no additional toxic effect to thiobencarb toxicity. Recovery of Anabaena cells from the inhibitory effect of thiobencarb occurred immediately after its reculture in herbicide free-medium. Maximum uptake of thiobencarb was associated with high biomass yield.

A Single Arginyl Residue in Plastocyanin and in Cytochrome c6 from the Cyanobacterium Anabaenasp. PCC 7119 Is Required for Efficient Reduction of Photosystem I

Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c(6) from Anabaena (Molina-Heredia, F. P., Diaz-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and Synechocystis (De la Cerda, B., Diaz-Quintana, A., Navarro, J. A. , Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.

Differential regulation of nitrogenase activity by ionic and osmotic stresses and permeable sugars in the Cyanobacterium Anabaena sp. strain L-31

Nitrogenase (acetylene reduction) activity in Anabaena sp. strain L-31 is significantly enhanced by the addition of sucrose, but is inhibited upon addition of sodium chloride. The positive effect of sucrose is not a general osmotic stress effect since non-permeable osmolytes (mannitol or polyethylene glycol (PEG)) do not influence nitrogenase activity. Unlike enteric bacteria, Anabaena cells take up and metabolise sucrose and incorporate products of its catabolism into proteins. Cultures inhibited in photosynthesis retain the ability to take up sucrose but do not show acetylene reduction activity, even when supplemented with sucrose. Addition of an inhibitor of transcription (rifampicin) or of a repressor of nitrogenase biosynthesis (ammonium chloride) abolishes the positive effect of sucrose on acetylene reduction activity. Cultures grown with permeable sugars (glucose, fructose and sucrose) show significantly higher levels of dinitrogenase reductase (Fe-protein of nitrogenase complex) while those grown with NaCl lack the protein. Fe-protein content is not affected by non-permeable solutes. Thus, exogenous sucrose elevates dinitrogenase reductase synthesis but does not appear to support the requirement of reductant for nitrogenase activity. The data substantiate our previous finding that the ionic and osmotic stresses differentially regulate cyanobacterial nitrogenase activity and explain the relatively superior osmotolerance of diazotrophic cyanobacterial strains, as compared with their sensitivity to salinity stress.