Pivotal Role of Iron in the Regulation of Cyanobacterial Electron Transport.

Iron-containing metalloproteins are the main cornerstones for efficient electron transport in biological systems. The abundance and diversity of iron-dependent proteins in cyanobacteria makes those organisms highly dependent of this micronutrient. To cope with iron imbalance, cyanobacteria have developed a survey of adaptation strategies that are strongly related to the regulation of photosynthesis, nitrogen metabolism and other central electron transfer pathways. Furthermore, either in its ferrous form or as a component of the haem group, iron plays a crucial role as regulatory signalling molecule that directly or indirectly modulates the composition and efficiency of cyanobacterial redox reactions. We present here the major mechanism used by cyanobacteria to couple iron homeostasis to the regulation of electron transport, making special emphasis in processes specific in those organisms.

A new pentaplex-nested PCR to detect five pathogenic bacteria in free living amoebae.

Changes in water use and anthropogenic activity have major impacts on the quality of natural aquatic ecosystems, water distribution and wastewater plants. One of the main problems is the presence of some pathogenic microorganisms that are resistant to disinfection procedures when they are hosted by free living amoeba and that in many cases are hardly detectable by culture-based procedures. In this work we report a sensitive, low-cost procedure consisting of a pentaplex-nested PCR that allows simultaneous detection of Legionella pneumophila, Mycobacterium spp., Pseudomonas spp., Vibrio cholerae and the microcystin-producing cyanobacteria Microcystis aeruginosa. The method has been used to detect the presence of these pathogenic bacteria in water and inside free living amoeba. Its validation in 72 samples obtained from different water sources from Aragon (Spain) evidences that Mycobacterium and Pseudomonas spp are prevailing as amoeba-resistant bacteria.

Optimization of intracellular microcystin-LR extraction for its analysis by protein phosphatase inhibition assay.

Microcystins are toxins produced by some strains of cyanobacteria. Several methods have been developed to allow the quantification of microcystins, which are mainly endotoxins. Among those methods, the protein phosphatase inhibition assay is a good candidate as a screening method because of its sensitivity, simplicity and specificity. In this work a method for intracellular microcystin extraction in field water samples and lab cyanobacterial cultures prior to their analysis by protein phosphatase inhibition assay has been optimized. Microcystin-LR and Microcystis aeruginosa PCC 7806 were used as reference microcystin and strain, respectively, in order to optimize the protocol. The protocol consists on filtering the sample through a nylon filter of 0.8 microm, filter extraction with methanol 80% 0.1% trifluoroacetic acid (TFA) 0.1% tween 20, extract centrifugation and supernatant dilution (1/20). The establishment of an extraction protocol was carried out determining the extraction volume, time of extraction and number of extractions. The advantages of the method developed in this work are basically its simplicity and avoiding the use of specific and expensive equipment.

Exploring the interaction of microcystin-LR with proteins and DNA.

The physiological role of microcystin-LR is still under discussion, and since binding of microcystin-LR to proteins different from their main cellular targets was described, we have performed experiments in order to explore this interaction. A non-specific interaction of microcystin-LR with a variety of soluble proteins in vitro is disrupted when using organic solvents such as methanol. The isoelectric point of proteins is not affected by their interaction with microcystin-LR, even though the presence of microcystin-LR alters the pool of peptides obtained by tryptic digestions. Under the conditions tested, microcystin-LR does not exhibit affinity for DNA. Although it is unlikely that the non-specific binding of microcystin-LR to proteins has a physiological meaning, one must be aware of the fact that determinations of the toxin extracted from any biological sample may be affected by the presence of proteins in the extracts. Consequently, we strongly recommend use organic solvents and to lyophilise the tissue samples to guarantee the accessibility of these organic solvents to microcystin-LR when performing experiments with tissue or cell extracts.

Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp. PCC 7120.

Fur (ferric uptake regulator) proteins are principally responsible for maintaining iron homeostasis in prokaryotes. Iron is usually a scarce resource. Its limitation reduces photosynthetic rates and cell growth in cyanobacteria in general and especially in cyanobacteria that are fixing dinitrogen, a process that requires the synthesis of numerous proteins with a high content of iron. This paper shows that in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120, levels of furA mRNA and FurA protein increase significantly in response to nitrogen deprivation, and that furA up-regulation takes place specifically in proheterocysts and mature heterocysts. Great differences in a Northern blot, probed with furA, of RNA from an ntcA mutant relative to wild-type Anabaena sp. were attributable to binding of NtcA, a global regulator of nitrogen metabolism, to the promoter of furA and to the promoter of the furA antisense transcript alr1690-alpha-furA.

Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn(2+) are positive effectors in the binding to isiB and furA promoters.

The Fur (ferric uptake regulator) protein is a global regulator in most prokaryotes that controls a large number of genes. Fur is a classical repressor that uses ferrous iron as co-repressor and binds to specific DNA sequences (iron boxes) as a dimer. Three different genes coding for Fur homologues have been identified in Anabaena sp. PCC 7120. FurA controls the transcription of flavodoxin, the product of the isiB gene, and is moderately autoregulated. In this work, the promoter of the furA gene was defined and the FurA protected regions in the furA and isiB promoters were identified, showing that the binding sites for Anabaena FurA contain A/T-rich sequences with a variable arrangement compared to the conventional 19-base pair Fur consensus. The influence of different factors on the interaction between FurA and the promoters was evaluated in vitro. The affinity of FurA for the DNA targets was significantly affected by the redox status of this regulator and the presence of Mn(2+). The optimal binding conditions were observed in the presence of both Mn(2+) and DTT. Those results suggest that, in addition to iron availability, FurA-DNA interaction is modulated by redox conditions.

Simultaneous occurrence of two different glyceraldehyde-3-phosphate dehydrogenases in heterocystous N(2)-fixing cyanobacteria.

Enzyme activity determinations and Western and Northern blot analyses have shown the presence of two catalytically different glyceraldehyde-3-phosphate dehydrogenases (GAPDH) in both vegetative cells and heterocysts of several N(2)-fixing Anabaena strains: (a) the gap2-encoded NAD(P)-dependent GAPDH2 (EC 1.2.1.59), the enzyme involved in the photosynthetic carbon assimilation pathway, which is present at higher levels in vegetative cells, and (b) the gap3-encoded NAD-dependent GAPDH3 (EC 1.2.1.12), presumably involved in carbohydrate anabolism and catabolism, which is the predominant GAPDH in heterocysts. In contrast, the gap1-encoded GAPDH1, which is the other NAD-dependent cyanobacterial GAPDH, is virtually absent in both cell types. These findings are discussed in the context of carbon metabolism of heterocystous N(2)-fixing cyanobacteria.

NADH oxidase activity of mitochondrial apoptosis-inducing factor.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFDelta1-120) exhibit NADH oxidase activity, whereas superoxide anion (O(2)(-)) is formed. AIFDelta1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFDelta1-120, which lacks FAD, has no NADH oxidase activity. However, native AIFDelta1-120, apoAIFDelta1-120, and the reconstituted (FAD-containing) holoAIFDelta1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFDelta1-120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFDelta1-120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter.

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.

Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca.

BACKGROUND: [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.

Expression of ferredoxin-NADP+ reductase in heterocysts from Anabaena sp.

The expression of ferredoxin-NADP+ reductase (FNR) from Anabaena sp. PCC 7119 in heterocysts and vegetative cells has been quantified. Specific reductase activity in heterocysts was approximately 10 times higher than in vegetative cells, corresponding to the increased FNR protein content. This was confirmed by immunoquantification of the FNR protein from whole filaments of Anabaena sp. PCC 7120 grown in media with and without combined nitrogen. Transcription of the petH gene was markedly enhanced in the absence of combined nitrogen. This suggests that the increased RNA level is mainly responsible for the up-regulation of FNR in heterocysts. As has been observed for nif genes, iron deficiency also increased transcription of petH. Characterization of the FNR purified from isolated heterocysts showed no detectable differences from the enzyme from vegetative cells. Although nitrogen stress was a key regulatory factor, localization of the petH gene in the genomic map of Anabaena PCC 7120 showed that this gene is not physically associated with the nif cluster.

The covalent linkage of a viologen to a flavoprotein reductase transforms it into an oxidase.

Chemical cross-linkage of the positively charged viologen N-methyl-N'-(aminopropyl)-4-4'-bipyridinium dibromide (APMV) to the enzyme ferredoxin-NADP+ reductase from the cyanobacterium Anabaena PCC 7119 has been performed using the carbodiimide 1-ethyl[3-(3-dimethylaminopropyl)]carbodiimide. 0.5-1 mol, depending on the preparation, is introduced for each mol enzyme. The residue involved in the covalent linkage with the viologen, Glu139, has been identified using HPLC separation of the modified proteolytic peptides and subsequent sequencing. Modification of the enzyme changes its catalytic specificity since it is able to react directly with oxygen; this is observed by a high NADPH oxidase activity, which is completely absent in the native enzyme. More important, this new enzymic activity is indicative of the intramolecular electron transfer between the natural redox cofactor FAD and the artificially introduced viologen. Electrons can also flow in the reverse direction, from the viologen to the FAD group, then to NADP+, when the reaction is performed using glassy-carbon electrodes to reduce the viologen. Cyclic voltammetry experiments have shown that there is a small catalytic current between the electrode and the enzyme which is not observed in the native enzyme.

Differential activities of heterocyst ferredoxin, vegetative cell ferredoxin, and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP(+) reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP(+), using thylakoids from vegetative cells.

Transcriptional and translational analysis of ferredoxin and flavodoxin under iron and nitrogen stress in Anabaena sp. strain PCC 7120.

In Anabaena sp. strain PCC 7120, vegetative cell ferredoxin synthesis under iron starvation was repressed 25-fold, whereas heterocyst ferredoxin synthesis decreased only 2.8-fold. Induction of flavodoxin under iron depletion was independent of the availability of combined nitrogen. Under iron stress but in the presence of combined nitrogen, fdxH and nifH genes were transcriptionally active; although excision of the 11-kb element seemed to be completed, nitrogenase activity and the fdxH gene product were not detectable.

Isolation and characterization of two different flavodoxins from the eukaryote Chlorella fusca.

Two different molecular forms of flavodoxin from the green alga Chlorella fusca have been purified to homogeneity and their properties compared. The molecular masses are 22 kDa (flavodoxin I) and 20 kDa (flavodoxin II). Western blots of axenic crude extract show the two bands. Both are single polypeptide chains and their N-terminal sequences differ but are very similar. Each form contains 1 mol of FMN/mol of apoprotein, exhibits a typical flavodoxin u.v.-visible absorption spectrum and does not contain covalently bound phosphate. The oxidation-reduction properties of the FMN in the flavodoxins differ considerably. Redox potentials of flavodoxin I at pH8 are -240 mV for the oxidized/semiquinone couple and -350 mV for the semiquinone/hydroquinone couple. Flavodoxin II gives more electronegative values: -278 mV and -458 mV respectively. Flavodoxin II fulfils better the redox requirements for photosynthetic electron transport and, as expected, it is more efficient at mediating NADP+ photoreduction in the photosynthetic electron flow. A new h.p.l.c. method for flavodoxin purification is described, which is useful for the isolation of very similar anionic proteins.

Electron paramagnetic resonance as a tool for monitoring overexpression in Escherichia coli of fully functional flavodoxin.

Overexpression of foreign proteins in Escherichia coli is usually tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Western blot analysis. However, when metalloproteins of flavoenzymes are expressed, the correct assembly of the polypeptide chain to the cofactor cannot be verified using these methods. We have used EPR spectroscopy and Mancini tests to monitor the expression of holoflavodoxin from the cyanobacteria Anabaena in E. coli. Flavodoxin from Anabaena sp PCC 7119 was cloned in the plasmid pTrc 99b after the trc promotor, which is activated in the presence of isopropyl-beta-D-thiogalactoside. Two hours after induction, most of the recombinant apoflavodoxin is already synthesized. However, only 65% of this protein is assembled to flavin mononucleotide (FMN). Harvesting of the cells to obtain all the flavodoxin in the holo form must be carried out 10 h after induction. Addition of FMN to the culture media increases the synthesis of holoflavodoxin by about 17%. The presence of flavin adenine dinucleotide or riboflavin in the culture inhibits the accumulation of semiquinone flavodoxin in the cells.

Riboflavin 3'- and 5'-sulfate, two novel flavins accumulating in the roots of iron-deficient sugar beet (Beta vulgaris).

Roots from iron-deficient sugar beet grown in the presence of calcium carbonate exhibit a yellow color and autofluorescence typical of flavin-like compounds, whereas roots of control, iron-sufficient plants exhibited no yellow color and extremely low autofluorescence. The two major flavins whose accumulation is induced by iron deficiency have been shown to be different from riboflavin, FMN, and FAD by reversed-phase high performance liquid chromatography. These flavins, accounting for 82 and 15% of the total flavin concentration in deficient roots, have been shown unequivocally to be riboflavin 3'-sulfate and riboflavin 5'-sulfate, respectively, by electrospray-mass spectrometry, inductively coupled plasma emission spectroscopy, infrared spectrometry, and 1H nuclear magnetic resonance. These flavin sulfates have not been found previously in biological systems. The localization of riboflavin sulfates in deficient roots is similar, but not identical, to that of high iron reductase activity. The concentration of riboflavin sulfates has been estimated from root extracts to be at least 1 mM. We hypothesize, based on the similar localization of flavin and that of iron reduction, that the accumulation of riboflavin sulfates induced by iron deficiency may be an integral part of the turbo iron-reducing system in sugar beet roots.

Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase.

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains.

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same Km values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.

Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacteria Anabaena variabilis.

The isolation and characterization of ferredoxin-NADP+ -oxidoreductase from Anabaena variabilis, a nitrogen-fixing, filamentous cyanobacterium, is described. Purified enzyme was obtained in four steps with a 55% yield and 300-fold purification utilizing chromatographic separations on DEAE-cellulose and Cibacron Blue-Sepharose columns. The enzyme is quite similar but not identical to the spinach enzyme as judged by isoelectric focusing, molecular weight determination, and amino acid composition. N-terminal sequence analysis allowed identification of 28 of the first 33 residues. Alignment with the corresponding sequences from spinach and Spirulina FNR preparations was possible. A higher degree of homology was found with the Spirulina enzyme than with the spinach enzyme. Small differences with the spinach enzyme were also shown by absorption and circular dichroism spectral measurements. Oxidation-reduction potential measurements of the bound FAD coenzyme show an Em = -320 mV at pH 7 for the two-electron process. Complex formation between the reductase and ferredoxin from the same organism was observed by difference absorption spectroscopy with a Kd = 4 microM. Similar Kd and difference absorption properties were observed on complex formation with spinach ferredoxin.