Iron induced metabolic changes in the diazotrophic cyanobacterium Anabaena PCC 7120

Iron induced changes in growth, N2-fixation, CO2 fixation and photosynthetic activity were studied in a diazotrophic cyanobacterium Anabaena PCC 7120. Iron at 50 microM concentration supported the maximum growth, heterocyst frequency, CO2 fixation, photosystem I (PS I), photosystem II (PS II) and nitrogenase activities in the organism. Higher concentration of iron inhibited these processes. Chl a and PS II activities were more sensitive to iron than the protein and PS I activity.     

Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima

An isolate of the cyanobacterium Anabaena from paddy fields was cultured and identified as Anabaena fertilissima based on morphometric features and 16S rRNA gene sequence matching. Cell extracts prepared using bead beater hydrolyzed casein. The caseinolytic protease with native molecular mass of 49 kDa was purified using ammonium sulfate fractionation, hydrophobic, affinity and ion-exchange chromatography, and gel filtration. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified protease was resolved in 17-kDa homologue of microcompartment protein and 27-kDa fragment of unknown protein. The enzyme in native state was digested with gelatin and fibrin in substrate gels producing bands corresponding to ca. 49 kDa. Moreover, a plasmin-specific substrate d-Val-Leu-Lys p-nitroanilide was also hydrolyzed with apparent K _m?=?0.18 mM and V _max?=?4.9?×?10^?7?M?s^?1; while Ca²⁺ stimulated, phenylmethanesulfonyl fluoride, leupeptin, and chelators completely abolished the amidolytic activity. The enzyme exhibited pH and temperature stability over a wide range. Upon incubation with fibrinogen, the Aα- and Bβ-chains preferentially cleaved, though the products thus resolved on SDS-PAGE moved at masses different from those of thrombin- and plasmin hydrolysates, and unlike thrombin, cross-linking of fibrinopeptides was not observed. In the plate assays, fibrinolysis was revealed at comparable strengths to that of plasmin, and the dissolute so obtained upon SDS-PAGE lacked bands corresponding to γ-dimer. Consequently, the degraded D-Dimer peptides appeared. The cyanobacterial protease displayed several unique properties not found in microbial and snake venom fibrinolytic enzymes.     

Relationship of combined nitrogen sources to salt tolerance in freshwater cyanobacterium Anabaena doliolum

Higher concentrations of NaCl inhibit the growth and reduce the specific growth rate of the freshwater cyanobacterium Anabaena doliolum. Among the nitrogen sources tried, nitrate protected the cyanobacterial cells most from salt toxicity. However, supplementing of medium with nitrate could increase the adaptability of the cells at sublethal doses but it would not permit growth at otherwise lethal doses of 300 mmol 1^-1 NaCl. Nitrate uptake was proportionally related to the NaCl level in the medium. The uptake of sodium was minimum when nitrate was simultaneously available to the cells, indicating the interaction of nitrate with the Na⁺ carrier. Na⁺ efflux was maximum in N², ammonia, urea and nitrate in decreasing order. This led to the conclusion that Na⁺ influx plays the critical role in salt tolerance, rather than its efflux.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Ecological rationale for H2 metabolism during aquatic blooms of the cyanobacterium Anabaena

Summary Nitrogenase-produced H₂ serves to remove excess intracellular O₂ during vigorous growth periods (blooms) of the nuisance cyanobacterium Anabaena. In two naturally-occurring species, A. oscillarioides and A. spiroides, nitrogen fixation (acetylene reduction) showed a high degree of resistance to O₂ inactivation. Under the influence of supersaturated O₂ concentrations, commonly encountered in lake blooms, elevated cellular ATP levels and enhanced uptake hydrogenase and nitrogenase activities were observed in actively growing filaments. Oxygen enhancement of nitrogenase activity appears mediated through localized uptake hydrogenase reactions. Hydrogen assimilated by hydrogenase is combined with O₂ in a Knallgas reaction, leading to the formation of H₂O and ATP via a respiratory chain. This combination of activities appears poised at O₂ removal and allows Anabaena to dominate O₂ supersaturated surface waters while maintaining optimal nitrogenase activity. Hence, instead of being a wasteful dissipation of reducing power, H₂ evolution via nitrogenase ultimately affords protection from O₂ while constituting a source of ATP through subsequent H₂ metabolism.     

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120

From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium ANABAENA PCC 7120 has been deduced. ANABAENA has nine DNA MTases. Four are associated with Type II restriction enzymes (AVAI, AVAII, AVAIII and the newly recognized inactive AVAIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AVAVI, DmtB/M.AVAVII, DmtC/M. AVAVIII and DmtD/M.AVAIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AVAV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.     

Transport of leucine in the cyanobacterium Anabaena variabilis

The amino acid leucine was transported by the cyanobacterium Anabaena variabilis. The K _m for transport was 10.8 M; the V _max was 8.7 nmoles min^–1 mg^–1 chlorophyll a. Transport of leucine was energy dependent: uptake of leucine was inhibited in the dark, and by DCMU and cyanide. Transport was neither dependent on nor enhanced by Na⁺. Prior growth of cells with leucine did not repress transport of [¹⁴C]-leucine. Alanine, glycine, valine, and methionine were strong competitive inhibitors of leucine uptake; serine, threonine, isoleucine, norleucine, and d-alanine competitively inhibited to a lesser degree. Other amino acids or amino acid analogues, including d-leucine, -aminoisobutyrate, and d-serine did not inhibit the transport of leucine.Abbreviations Chl a chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TES N-tris(hydroxymethyl)-2-aminoethane-sulfonic acid - TCA trichloroacetic acid - Tris N-tris(hydroxymethyl)aminoethane     

Regulation of urea-uptake in the cyanobacterium Anabaena doliolum

Abstract The utilization of urea was studied in the cyanobacterium Anabaena doliolum . The uptake of urea was unaltered in the presence of ammonium. The cells receiving ATP exogenously showed an induced level of urea-uptake as compared with the control cells. Urease inhibitor acetohydroxamic acid and hydroxyurea as well as glutamate analogue, MSO, did not affect the uptake of urea. These results suggest: (1) urea and ammonia have different uptake sites, (2) urea-uptake is an energy dependent process, and (3) during short-term experiments, urea uptake is not linked with the enzyme urease or the ammonium assimilating enzyme glutamine synthetase.     

Geographical segregation of the neurotoxin-producing cyanobacterium Anabaena circinalis

Blooms of the cyanobacterium Anabaena circinalis are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisons (PSPs). Although there is a worldwide distribution of A. circinalis, there is a geographical segregation of neurotoxin production. American and European isolates of A. circinalis produce only anatoxin-a, while Australian isolates exclusively produce PSPs. The reason for this geographical segregation of neurotoxin production by A. circinalis is unknown. The phylogenetic structure of A. circinalis was determined by analyzing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP-producing A. circinalis formed two distinct 16S rRNA gene clusters. A molecular probe was designed, allowing the identification of A. circinalis from cultured and uncultured environmental samples. In addition, probes targeting the predominantly PSP-producing or non-PSP-producing clusters were designed for the characterization of A. circinalis isolates as potential PSP producers.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Occurrence and identification of UDP-N-acetylmuramyl-pentapeptide from the cyanobacterium Anabaena cylindrica

UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) is well known to be a key intermediate of bacterial peptidoglycan biosynthesis. We first detected the occurrence of UDP-MurNAc-pentapeptide in the cyanobacterium Anabaena cylindrica (NIES-19), and identified the structure of this pentapeptide by two-dimensional 1H-1H and 1H-13C NMR correlation experiments and mass spectra.     

NaCl-induced oxidative damage in the cyanobacterium Anabaena doliolum

Results obtained with graded concentrations of NaCl (20–200 mM) show decrease in the chlorophyll ‘a’ contents of Anabaena with increasing concentration of NaCl except at extremely low concentration of NaCl (5–20 mM). 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 O₂ 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. Received: 15 August 2001/Accepted: 20 September 2001     

Minutissamides E–L,antiproliferative cyclic lipodecapeptides from the cultured freshwater cyanobacterium cf. Anabaena sp.

The extract of UIC 10035, a strain obtained from a sample collected near the town of Homestead, South Florida, showed antiproliferative activity against MDA-MB-435 cells. Bioassay-guided fractionation led to the isolation of a series of cyclic lipodecapeptides, named minutissamides E–L (1–8). The planar structures were determined by analysis of HRESIMS, tandem MS, and 1D and 2D NMR data, and the stereoconfigurations were assigned by LC–MS analysis of the Marfey’s derivatives after acid hydrolysis. Minutissamides E–L (1–8) exhibited antiproliferative activity against MDA-MB-435 cells with IC₅₀ values ranging between 1 and 10 μM. The structures of minutissamides E–L (1–8) were closely related with those of the previously reported lipopeptides, puwainaphycins A–E and minutissamides A–D, characterized by the presence of a lipophilic β-amino acid and three non-standard amino acids NMeAsn, OMeThr and Dhb (α,β-dehydro-α-aminobutyric acid). The strain UIC 10035 was designated as cf. Anabaena sp. on the basis of morphological and 16S rRNA gene sequence analyses.     

Induction of nitrate assimilation in the cyanobacterium Anabaena variabilis

Filaments of Anabaena variabilis Kütz strain ATCC 29413 grown in the absence of nitrate contain nitrate reductase that is active in permeabilized filaments, but not in intact, living filaments until they have been incubated for about 40 min in the presence of nitrate. The delayed acquisition of the ability to reduce nitrate is insensitive to chloramphenicol. Thus, switching on of enzyme activity in the presence of nitrate does not involve protein synthesis and nitrate reductase activity is not regulated by the amount of enzyme present.     

Characterization of akinete differentiation in the cyanobacterium Anabaena azollae

Differentiation of akinetes was investigated in the filamentous cyanobacterium Anabaena azollae Stras. In this organism all pre-existing vegetative cells are capable of developing into akinetes. Standard sporulation medium (SSM) was used to synchronously induce the formation of akinetes, while cultures in Allen and Arnon (AA/8) medium were used as controls.This paper describes the changes in photosynthetic pigments and total soluble proteins in these cultures over a 25-day period encompassing akinete differentiation. Heterocyst frequencies and nitrogenase activity were also monitored during the same period in both media. SDS-PAGE results indicated that specific proteins were synthesized in a manner correlated with akinete differentiation. The results demonstrate that in cultures undergoing akinete development, some of the photosynthetic pigments are maintained, nitrogen-fixation and heterocyst differentiation are suppressed, and the cells synthesize a variety of specific proteins.     

Photoinhibition and its wavelength dependence in the cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis the dependence of photoinhibition on fluence rate, duration and wavelength of irradiation were studied by measurements of oxygen production and fluorescence emission spectra. The analysis of the photosynthetic activity revealed that photoinhibition affects exclusively photosystem II (PS II), whereas photosystem I (PS I) remained largely unimpaired. Furthermore, PS II fluorescence emission decreased much faster in bleached than in unbleached controls.Studying the wavelength dependence of photoinhibition it was found that only radiation between 520 and 680 nm causes photoinhibition. This is about the same range of wavelengths which causes photobleaching. Fluorescence emission spectra of samples exposed to high fluence rates of 582 and 662 nm, respectively, essentially agree with those samples exposed to high fluence rates of white light, whereas the fluorescence emission spectra of samples exposed to blue light resemble those exposed to dim white light.NaN₃, a substance which prevents photobleaching, inhibits the photosynthetic O₂ production of Anabaena and, hence, enhances the photoinhibitory effect.     

Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120

Abstract The strengths of several promoters were assessed in the cyanobacterium Anabaena PCC 7120 by fusing them to luxAB , encoding bacterial luciferase. Two promoters, P _tac and P _psbA , with sequences nearly identical to consensus Escherichia coli σ ⁷⁰ promoters, gave as high or higher expression than the strong Anabaena promoter, P _rbc . P _npt , the natural promoter driving expression of the kanamycin-resistance determinant from Tn5, was poorly expressed in Anabaena . The Lac repressor partially repressed expression from P _tac , permitting regulated expression in Anabaena after induction with isopropyl thiogalactoside to a level 4–5-fold higher than without inducer.     

Physiological and structural responses of the cyanobacterium Anabaena cylindrica to aluminium

When adding aluminium (3.7–370 μ M ) as AlCl₃–6H₂0 to cultures of the nitrogen-fixing cyanobacterium Anabaena cylindrica , strain 1403/2a (CCAP), the following responses were observed: The effects of aluminium were dependent on pH. being most drastic at pH 6.0. At this pH the growth of A. cylindrica was significantly reduced by 3.7 μ M aluminium and completely inhibited by 370 μ M . The content of chlorophyll a and phycocyanin decreased after treatment with aluminium. Also, aluminium lowered the rates of both CO₂-fixation and N₂-fixation with total inhibition of both processes by 370 μ M . At the lower concentrations used the nitrogenase activity started to recover after about 100 h. The aluminium content in the cells increased with increasing concentration and with time. At 190 μ M the aluminium concentration in the cells represented 2.4 and 3.3% of the dry weight after 6 and 24 h, respectively. Clogging of filaments and lysis of vegetative cells were apparent at higher aluminium concentrations while the frequency of heterocysts increased in all concentrations used. The most pronounced ultrastructural changes included accumulation of cyanophycin granules and degradation of the thylakoids. The ultrastructure of the heterocysts was however not affected. It is concluded that major reasons for the toxicity are interactions with membranes and phosphate deficiency.