The atypical thioredoxin 'Alr2205', a newly identified partner of the typical 2-Cys-Peroxiredoxin, safeguards the cyanobacterium Anabaena from oxidative stress.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was found to be evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulfide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (the typical 2-Cys-Peroxiredoxin from Anabaena), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulfide bond between the two active site cysteines (Cys-38 and Cys-41). However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulfide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulfide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.

Loss of 2-Cys-Prx affects cellular ultrastructure, disturbs redox poise and impairs photosynthesis in cyanobacteria.

In a striking similarity to plant chloroplasts, the cyanobacterium Anabaena displays very low catalase activity, but expresses several peroxiredoxins (Prxs), including the typical 2-Cys-Prx (annotated as Alr4641), that detoxify H2 O2 . Due to the presence of multiple Prxs, the precise contribution of Alr4641 to the oxidative stress response of Anabaena is not well-defined. To unambiguously assess its in vivo function, the Alr4641 protein was knocked down using the CRISPRi approach in Anabaena PCC 7120. The knockdown strain (An-KD4641), which showed over 85% decrease in the content of Alr4641, was viable, but grew slower than the control strain (An-dCas9). An-KD4641 showed elevated levels of reactive oxygen species and the expression of several redox-responsive genes was analogous to that of An-dCas9 subjected to oxidative stress. The knockdown strain displayed reduced filament size, altered thylakoid ultrastructure, a marked drop in the ratio of phycocyanin to chlorophyll a and decreased photosynthetic parameters compared to An-dCas9. In comparison to the control strain, exposure to H2 O2 had a more severe effect on the photosynthetic parameters or survival of An-KD4641. Thus, in the absence of adequate catalase activity, 2-Cys-Prx appears to be the principal Prx responsible for maintaining redox homoeostasis in diverse photosynthetic systems ranging from chloroplasts to cyanobacteria.

Voyage of selenium from environment to life: Beneficial or toxic?

Selenium (Se), a naturally occurring metalloid, is an essential micronutrient for life as it is incorporated as selenocysteine in proteins. Although beneficial at low doses, Se is hazardous at high concentrations and poses a serious threat to various ecosystems. Due to this contrasting 'dual' nature, Se has garnered the attention of researchers wishing to unravel its puzzling properties. In this review, we describe the impact of selenium's journey from environment to diverse biological systems, with an emphasis on its chemical advantage. We describe the uneven distribution of Se and how this affects the bioavailability of this element, which, in turn, profoundly affects the habitat of a region. Once taken up, the subsequent incorporation of Se into proteins as selenocysteine and its antioxidant functions are detailed here. The causes of improved protein function due to the incorporation of redox-active Se atom (instead of S) are examined. Subsequently, the reasons for the deleterious effects of Se, which depend on its chemical form (organo-selenium or the inorganic forms) in different organisms are elaborated. Although Se is vital for the function of many antioxidant enzymes, how the pro-oxidant nature of Se can be potentially exploited in different therapies is highlighted. Furthermore, we succinctly explain how the presence of Se in biological systems offsets the toxic effects of heavy metal mercury. Finally, the different avenues of research that are fundamental to expand our understanding of selenium biology are suggested.

Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

Novel molecular insights into the anti-oxidative stress response and structure-function of a salt-inducible cyanobacterial Mn-catalase.

KatB, a salt-inducible Mn-catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological-biochemical function of KatB at the molecular level. Anabaena overexpressing the wild-type KatB protein (KatBWT) detoxified H2 O2 efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F-2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X-ray crystallography-based analysis showed that F-2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F-2, through its interaction with F-66 and W-43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn-catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.

Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase.

Cysteine desulfurases, which supply sulfur for iron-sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe-S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.

Down-Regulation of the Alternative Sigma Factor SigJ Confers a Photoprotective Phenotype to Anabaena PCC 7120.

Alternative sigma factors belonging to Group 3 are thought to play an important role in the adaptation of cyanobacteria to environmental challenges by altering expression of genes needed for coping with such stresses. In this study, the role of an alternative sigma factor, SigJ, was analyzed in the filamentous nitrogen-fixing cyanobacterium, Anabaena sp. PCC 7120 by knocking down the expression of the sigJ gene (alr0277) employing an antisense RNA-mediated approach. In the absence of any stress, the knock-down (KD0277) or the wild-type strain both grew similarly. Upon exposure to high-intensity light, KD0277 showed substantially reduced bleaching of its pigments, higher photosynthetic activity and consequently better survival than the wild type. KD0277 also showed an enhanced accumulation of two carotenoids, which were identified as myxoxanthophyll and keto-myxoxanthophyll. Further, KD0277 was more tolerant to ammonium-triggered photodamage than the wild type. Moreover, PSII was better protected against photodamage in KD0277 than in the wild type. Down-regulation of sigJ in Anabaena PCC 7120, however, reduced its ability to cope with desiccation. This study demonstrates that down-regulation of the sigJ gene in Anabaena PCC 7120 differentially affects its ability to tolerate two environmentally relevant stresses, i.e. high-intensity light and desiccation.

A Salt-Inducible Mn-Catalase (KatB) Protects Cyanobacterium from Oxidative Stress.

Catalases, enzymes that detoxify H2O2, are widely distributed in all phyla, including cyanobacteria. Unlike the heme-containing catalases, the physiological roles of Mn-catalases remain inadequately characterized. In the cyanobacterium Anabaena, pretreatment of cells with NaCl resulted in unusually enhanced tolerance to oxidative stress. On exposure to H2O2, the NaCl-treated Anabaena showed reduced formation of reactive oxygen species, peroxides, and oxidized proteins than the control cells (i.e. not treated with NaCl) exposed to H2O2. This protective effect correlated well with the substantial increase in production of KatB, a Mn-catalase. Addition of NaCl did not safeguard the katB mutant from H2O2, suggesting that KatB was indeed responsible for detoxifying the externally added H2O2. Moreover, Anabaena deficient in KatB was susceptible to oxidative effects of salinity stress. The katB gene was strongly induced in response to osmotic stress or desiccation. Promoter-gfp analysis showed katB to be expressed only in the vegetative cells but not in heterocysts. Biochemically, KatB was an efficient, robust catalase that remained active in the presence of high concentrations of NaCl. Our findings unravel the role of Mn-catalase in acclimatization to salt/oxidative stress and demonstrate that the oxidative stress resistance of an organism can be enhanced by a simple compound such as NaCl.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation.

UNLABELLED: Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE: The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells.

Redox-dependent chaperone/peroxidase function of 2-Cys-Prx from the cyanobacterium Anabaena PCC7120: role in oxidative stress tolerance.

BACKGROUND: Cyanobacteria, progenitors of plant chloroplasts, provide a suitable model system for plants to study adaptation towards different abiotic stresses. Genome of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena PCC7120 harbours a single gene (alr4641) encoding a typical 2-Cys-Peroxiredoxins (2-Cys-Prxs). 2-Cys-Prxs are thiol-based peroxidases that also function as molecular chaperones in plants and other systems. The Alr4641 protein from Anabaena PCC7120 shows high level biochemical similarities with the plant 2-Cys-Prx. The physiological role played by the Alr4641 protein in Anabaena was addressed in this study. RESULTS: In Anabaena PCC7120, alr4641 transcript /Alr4641 protein was induced in response to abiotic stresses and its promoter was active in the vegetative cells as well as heterocysts. The wild-type Alr4641 protein or Alr4641 lacking the peroxidatic cysteine (Alr4641C56S) or the resolving cysteine (Alr4641C178S) existed as higher oligomers in their native form. The wild-type or the mutant Alr4641 proteins showed similar chaperone activity, but only the wild-type protein exhibited peroxidase activity indicating that unlike peroxidase activity, chaperone activity was not dependent on cysteines. In contrast to other 2-Cys-Prxs, chaperone/peroxidase activity of Alr4641 was dependent on its redox state and not oligomerization status. Alr4641 could protect plasmid DNA from oxidative damage and physically associate with NADPH-dependent thioredoxin reductase (NTRC). Like 2-Cys-Prxs from plants (e.g. rice), Alr4641 could detoxify various peroxides using NTRC as reductant. On exposure to H2O2, recombinant Anabaena PCC7120 strain over-expressing Alr4641 (An4641+) showed reduced content of reactive oxygen species (ROS), intact photosynthetic functions and consequently better survival than the wild-type Anabaena PCC7120, indicating that Alr4641 can protect Anabaena from oxidative stress. CONCLUSIONS: The peroxidase/chaperone function of Alr4641, its inherent transcriptional/translational induction under different abiotic stresses and localization in both vegetative cells and heterocysts could be an adaptive strategy to battle various oxidative stresses that Anabaena encounters during its growth. Moreover, the recombinant Anabaena strain over expressing Alr4641 showed higher resistance to oxidative stress, suggesting its potential to serve as stress-tolerant biofertilizers in rice fields.

Characterization of a plant (rice) translin and its comparative analysis with human translin.

MAIN CONCLUSION: For the first time, a plant (rice) translin was characterized. The rice translin protein, which was octameric in native state, bound efficiently to single-stranded DNA and RNA. Translin, a DNA-/RNA-binding protein, is expressed in brain, testis and in certain malignancies. It is involved in chromosomal translocation, mRNA metabolism, transcriptional regulation and telomere protection. Studies from human, mice, drosophila and yeast have revealed that it forms an octameric ring, which is important for its function. In spite of the absence of neuronal functions and cancer processes, translin is present in plant systems, but information on plant translin is lacking. Here we report the characterization of a plant (rice) translin. Translin cDNA from O. sativa was cloned into an expression vector; protein was over-expressed in E. coli and subsequently purified to homogeneity. Circular dichroism and homology-based modeling showed that the rice translin protein was similar to the other translin proteins. Native PAGE and gel-filtration analyses showed rice translin to form an octamer and this octameric assembly was independent of disulphide bonds. Rice translin bound to single-stranded DNA sequences like human translin, but not to the double-stranded DNA. Rice translin bound more efficiently to linear DNA (with staggered ends) than open or closed circular DNA. Rice translin also bound to RNA, like its human counterpart. Rice translin displays all the characteristic properties of the translin group of proteins and does indeed qualify as a bonafide "translin" protein. To our knowledge, this is the first report wherein the translin protein from a plant source has been functionally characterized. Understanding the translin biology from plant systems will give the new insights into its functional role during plant development.

Unravelling the involvement of protein disorder in cyanobacterial stress responses.

This study explored the involvement of Intrinsically Disordered Proteins, (IDPs) in cyanobacterial stress response. IDPs possess distinct physicochemical properties, which allow them to execute diverse functions. Anabaena PCC 7120, the model photosynthetic, nitrogen-fixing cyanobacterium encodes 688 proteins (11 % of total proteome) with at least one intrinsically disordered region (IDR). Of these, 130 proteins that showed >30 % overall disorder were designated as IDPs. Physico-chemical analysis, showed these IDPs to adopt shapes ranging from 'globular' to 'tadpole-like'. Upon exposure to NaCl, 41 IDP-encoding genes were found to be differentially expressed. Surprisingly, most of these were induced, indicating the importance of IDP-accumulation in overcoming salt stress. Subsequently, six IDPs were identified to be induced by multiple stresses (salt, ammonium and selenite). Interestingly, the presence of these 6-multiple stress-induced IDPs was conserved in filamentous cyanobacteria. Utilizing the experimental proteomic data of Anabaena, these 6 IDPs were found to interact with many proteins involved in diverse pathways, underscoring their physiological importance as protein hubs. This study lays the framework for IDP-related research in Anabaena by (a) identifying, as well as physiochemically characterizing, all the disordered proteins and (b) uncovering a subset of IDPs that are likely to be critical in stress adaptation.

Novel silver nanoparticle-antibiotic combinations as promising antibacterial and anti-biofilm candidates against multiple-antibiotic resistant ESKAPE microorganisms.

HYPOTHESIS: The emergence of Multiple Antibiotic Resistance (MAR) in ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens is a global challenge to public health. The inherent antimicrobial nature of silver nanoparticles (AgNPs) makes them promising antimicrobial candidates against antibiotic-resistant pathogens. This study explores the combination of AgNPs with antibiotics (SACs) to create new antimicrobial agents effective against MAR ESKAPE microorganisms. METHODS: AgNPs were synthesized using Streptococcus pneumoniae ATCC 49619 and characterized for structure and surface properties. The SACs were tested against ESKAPE microorganisms using growth kinetics and time-kill curve methods. The effect of SACs on bacterial biofilms and the disruption of cell membranes was determined. The in-vitro cytotoxicity effect of the AgNPs was also studied. FINDINGS: The synthesized AgNPs (spherical, 7.37±4.55 nm diameter) were antimicrobial against MAR ESKAPE microorganisms. The SACs showed synergy with multiple conventional antibiotics, reducing their antibacterial concentrations up to 32-fold. Growth kinetics and time-kill studies confirmed the growth retardation effect and bactericidal activity of SACs. Mechanistic studies suggested that these biofilm-eradicating SACs probably resulted in the loss of bacterial cell membrane integrity, leading to leakage of the cytoplasmic content. The AgNPs were highly cytotoxic against skin melanoma cells but non-cytotoxic to normal Vero cells.

Modulation of oxidative stress machinery determines the contrasting ability of cyanobacteria to adapt to Se(VI) or Se(IV).

Excess of selenium (Se) in aquatic ecosystems has necessitated thorough investigations into the effects/consequences of this metalloid on the autochthonous organisms exposed to it. The molecular details of Se-mediated adaptive response remain unknown in cyanobacteria. This study aims to uncover the molecular mechanisms driving the divergent physiological responses of cyanobacteria on exposure to selenate [Se(VI)] or selenite [Se(IV)], the two major water-soluble oxyanions of Se. The cyanobacterium, Anabaena PCC 7120, withstood 0.4 mM of Se(VI), whereas even 0.1 mM of Se(IV) was detrimental, affecting photosynthesis and enhancing endogenous ROS. Surprisingly, Anabaena pre-treated with Se(VI), but not Se(IV), showed increased tolerance to oxidative stress mediated by H2O2/methyl viologen. RNA-Seq analysis showed Se(VI) to elevate transcription of genes encoding anti-oxidant proteins and Fe-S cluster biogenesis, whereas the photosynthesis-associated genes, which were mainly downregulated by Se(IV), remained unaffected. Specifically, the content of typical 2-Cys-Prx (Alr4641), a redox-maintaining protein in Anabaena, was elevated with Se(VI). In comparison to the wild-type, the Anabaena strain over-expressing the Alr4641 protein (An4641+) showed enhanced tolerance to Se(VI) stress, whereas the corresponding knockdown-strain (KD4641) was sensitive to this stressor. Incidentally, among these strains, only An4641+ was better protected from the ROS-mediated damage caused by high dose of Se(VI). These results suggest that altering the content of the antioxidant protein 2-Cys-Prx, could be a potential strategy for modulating resistance to selenate. Thus, involvement of oxidative stress machinery appears to be the major determinant, responsible for the contrasting physiological differences observed in response to selenate/selenite in cyanobacteria.

Oxidative stress management in the filamentous, heterocystous, diazotrophic cyanobacterium, Anabaena PCC7120.

Reactive oxygen species (ROS) are inevitably generated as by-products of respiratory/photosynthetic electron transport in oxygenic photoautotrophs. Unless effectively scavenged, these ROS can damage all cellular components. The filamentous, heterocystous, nitrogen-fixing strains of the cyanobacterium, Anabaena, serve as naturally abundant contributors of nitrogen biofertilizers in tropical rice paddy fields. Anabaena strains are known to tolerate several abiotic stresses, such as heat, UV, gamma radiation, desiccation, etc., that are known to generate ROS. ROS are detoxified by specific antioxidant enzymes like superoxide dismutases (SOD), catalases and peroxiredoxins. The genome of Anabaena PCC7120 encodes two SODs, two catalases and seven peroxiredoxins, indicating the presence of an elaborate antioxidant enzymatic machinery to defend its cellular components from ROS. This article summarizes recent findings and depicts important perspectives in oxidative stress management in Anabaena PCC7120.

Purification, crystallization and preliminary crystallographic analysis of KatB, a manganese catalase from Anabaena PCC 7120.

Catalases are enzymes that play an important role in the detoxification of hydrogen peroxide (H2O2) in aerobic organisms. Among catalases, haem-containing catalases are ubiquitously distributed and their enzymatic mechanism is very well understood. On the other hand, manganese catalases that contain a bimanganese core in the active site have been less well characterized and their mode of action is not fully understood. The genome of Anabaena PCC 7120 does not show the presence of a haem catalase-like gene; instead, two ORFs encoding manganese catalases (Mn-catalases) are present. Here, the crystallization and preliminary X-ray crystallographic analysis of KatB, one of the two Mn-catalases from Anabaena, are reported. KatB was crystallized using the hanging-drop vapour-diffusion method with PEG 400 as a precipitant and calcium acetate as an additive. Diffraction data were collected in-house on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 2.2 Šresolution at 100 K. The tetragonal crystal belonged to space group P4(1)2(1)2 (or enantiomer), with unit-cell parameters a = b = 101.87, c = 138.86 Å. Preliminary X-ray diffraction analysis using the Matthews coefficient and self-rotation function suggests the presence of a trimer in the asymmetric unit.

A novel glutaredoxin domain-containing peroxiredoxin 'All1541' protects the N2-fixing cyanobacterium Anabaena PCC 7120 from oxidative stress.

Prxs (peroxiredoxins) are ubiquitous thiol-based peroxidases that detoxify toxic peroxides. The Anabaena PCC 7120 genome harbours seven genes/ORFs (open reading frames) which have homology with Prxs. One of these (all1541) was identified to encode a novel Grx (glutaredoxin) domain-containing Prx by bioinformatic analysis. A recombinant N-terminal histidine-tagged All1541 protein was overexpressed in Escherichia coli and purified. Analysis with the protein alkylating agent AMS (4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate) showed All1541 to form an intra-molecular disulfide bond. The All1541 protein used glutathione (GSH) more efficiently than Trx (thioredoxin) to detoxify H(2)O(2). Deletion of the Grx domain from All1541 resulted in loss of GSH-dependent peroxidase activity. Employing site-directed mutagenesis, the cysteine residues at positions 50 and 75 were identified as peroxidatic and resolving cysteine residues respectively, whereas both the cysteine residues within the Grx domain (positions 181 and 184) were shown to be essential for GSH-dependent peroxidase activity. On the basis of these data, a reaction mechanism has been proposed for All1541. In vitro All1541 protein protected plasmid DNA from oxidative damage. In Anabaena PCC 7120, all1541 was transcriptionally activated under oxidative stress. Recombinant Anabaena PCC 7120 strain overexpressing All1541 protein showed superior oxidative stress tolerance to H(2)O(2) as compared with the wild-type strain. The results suggest that the glutathione-dependent peroxidase All1541 plays an important role in protecting Anabaena from oxidative stress.

Mn-catalase (Alr0998) protects the photosynthetic, nitrogen-fixing cyanobacterium Anabaena PCC7120 from oxidative stress.

Role of the non-haem, manganese catalase (Mn-catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn-catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat(+). The Alr0998 protein could be immunodetected in AnKat(+) cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat(+) cells but not in the wild-type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn-catalase. In response to oxidative stress, the AnKat(+) showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress-inducible 2-Cys-Prx protein. Treatment of wild-type Anabaena PCC7120 with H(2)O(2) caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO(2) fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat(+) strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn-catalase.

Unique functional insights into the antioxidant response of the cyanobacterial Mn-catalase (KatB).

KatB, a hexameric Mn-catalase, plays a vital role in overcoming oxidative and salinity stress in the ecologically important, N2-fixing cyanobacterium, Anabaena. The 5 N-terminal residues of KatB, which show a high degree of conservation in cyanobacteria, form an antiparallel ß-strand at the subunit interface of the KatB hexamer. In this study, the contribution of these N-terminal non-active site residues, towards the maintenance of the structure, biochemical properties, and redox balance was evaluated. Each N-terminal amino acid residue from the 2nd to the 7th position of KatB was individually mutated to Ala (to express KatBF2A/KatBF3A/KatBH4A/KatBK5E/KatBK6A/KatBE7A) or this entire 6 amino acid stretch was deleted (to yield KatBTrunc). All the above-mentioned KatB variants, along with the wild-type KatB protein (KatBWT), were overproduced in E. coli and purified. In comparison to KatBWT, the KatBF2A/KatBH4A/KatBTrunc proteins were less compact, more prone to chemical/thermal denaturation, and were unexpectedly inactive. KatBF3A/KatBK5E/KatBK6A showed biophysical/biochemical properties that were in between that of KatBWT and KatBF2A/KatBH4A/KatBTrunc. Surprisingly, KatBE7A was more thermostable with higher activity than KatBWT. On exposure to H2O2, E. coli expressing KatBWT/KatBE7A showed considerably reduced formation of ROS and increased survival than the other KatB variants. Utilizing the KatB structure, the molecular basis responsible for the altered stability/activity of the KatB mutants was delineated. This study demonstrates the physiological importance of the N-terminal ß-strand of Mn-catalases in combating H2O2 stress and shows that the non-active site residues can be used for rational protein engineering to develop Mn-catalases with improved characteristics.

Functional and mechanistic insights into the differential effect of the toxicant 'Se(IV)' in the cyanobacterium Anabaena PCC 7120.

Selenium, an essential trace element for animals, poses a threat to all forms of life above a threshold concentration. The ubiquitously present cyanobacteria, a major photosynthetic biotic component of aquatic and other ecosystems, are excellent systems to study the effects of environmental toxicants. The molecular changes that led to beneficial or detrimental effects in response to different doses of selenium oxyanion Se(IV) were analyzed in the filamentous cyanobacterium Anabaena PCC 7120. This organism showed no inhibition in growth up to 15 mg/L sodium selenite, but above this dose i.e. 20-100 mg/L of Se(IV), both growth and photosynthesis were substantially inhibited. Along with the increased accumulation of non-protein thiols, a consistent reduction in levels of ROS was observed at 10 mg/mL dose of Se(IV). High dose of Se(IV) (above 20 mg/L) enhanced endogenous reactive oxygen species (ROS)/lipid peroxidation, and decreased photosynthetic capability. Treatment with 100 mg/L Se(IV) downregulated transcription of several photosynthesis pathways-related genes such as those encoding photosystem I and II proteins, phycobilisome rod-core linker protein, phycocyanobilin, phycoerythrocyanin-associated proteins etc. Interestingly, at a dose range of 10-15 mg/L Se(IV), Anabaena showed an increase in PSII photosynthetic yield and electron transport rate (at PSII), suggesting improved photosynthesis. Se was incorporated into the Anabaena cells, and Se-enriched thylakoid membranes showed higher redox conductivity than the thylakoid membranes from untreated cells. Overall, the data supports that modulation of photosynthetic machinery is one of the crucial mechanisms responsible for the dose-dependent contrasting effect of Se(IV) observed in Anabaena.