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Cyanobacterial DnaJ offers thermo-tolerance and effectively prevents aggregation of denatured protein in coordination with DnaK. The hypothetical protein All3048 of Anabaena sp. PCC7120 was found to be a 24 kDa DnaJ III protein with a putative J-domain at the extreme N-terminus. This paper decodes the role of All3048 in thermo-tolerance and as a co-chaperon of DnaK. Semi-quantitative and RT-PCR results showed up-accumulation of all3048 in heat, UV-B, cadmium, arsenic and salt. BL21/pET-28a-all3048, all3048(1-95) and all3048(31-128) reduced the heat stress-induced ROS generation by 40 %, 21 % and 24 % as compared to BL21/pET-28-a. Conformational properties of All3048 and its truncated variants were assessed using bis ANS, guanidine hydrochloride and acrylamide quenching. All3048(1-95), All3048 and All3048(31-128) increased DnaK ATPase activity by 8.6, 8.2, and 2.5 fold, respectively. The thermostability investigated using DSC and DSF methods affirmed the relative stability of All3048 and All3048 (31-128), whereas All3048 (1-95) was the least stable. All3048 is a novel cyanobacterial DnaJ III that imparts heat stress tolerance in E. coli; however, only the J-domain present at N-terminus was sufficient for stimulating DnaK's ATPase activity., Competing Interests: Declaration of competing interest No conflicts, informed consent, human or animal rights applicable., (Copyright © 2023 Elsevier B.V. All rights reserved.)

WD40 domain-containing proteins are one of the eukaryotes' most ancient and ubiquitous protein families. Little is known about the presence and function of these proteins in cyanobacteria in general and Anabaena in particular. In silico analysis confirmed the presence of WD40 repeats. Gene expression analysis indicated that the transcript levels of both the target proteins were up-regulated up to 4 fold in Cd and drought and 2-3 fold in heat, salt, and UV-B stress. Using a fluorescent oxidative stress indicator, we showed that the recombinant proteins were scavenging reactive oxygen species (ROS) (4-5 fold) more efficiently than empty vectors. Chromatin immunoprecipitation analysis (ChIP) and electrophoretic mobility shift assay (EMSA) revealed that the target proteins function as transcription factors after binding to the promoter sequences. The presence of kinase activity (2-4 fold) in the selected proteins indicated that these proteins could modulate the functions of other cellular proteins under stress conditions by inducing phosphorylation of specific amino acids. The chosen proteins also demonstrated interaction with Zn, Cd, and Cu (1.4-2.5 fold), which might stabilize the proteins' structure and biophysical functions under multiple abiotic stresses. The functionally characterized Alr0671 and All2352 proteins act as transcription factors and offer tolerance to agriculturally relevant abiotic stresses., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Nanotechnology is one of the most promising and advanced disciplines of science that deals with synthesis, characterization and applications of different types of Nanomaterials (NMs) viz. nanospheres, nanoparticles, nanotubes, nanorods, nanowires, nanocomposites, nanoalloys, carbon dots and quantum dots. These nanosized materials exhibit different physicochemical characteristics and act as a whole unit during its transport. The unique characteristics and vast applications of NMs in diverse fields viz. electronics, agriculture, biology and medicine have created huge demand of different type of NMs. Conventionally physical and chemical methods were adopted to manufacture NMs which are expensive and end up with hazardous by-products. Therefore, green synthesis exploiting biological resources viz. algae, bacteria, fungi and plants emerged as a better and promising alternative due to its cost effective and ecofriendly approach and referred as nanobiotechnology. Among various living organisms, cyanobacteria have proved one of the most favourable bioresources for NMs biosynthesis due to their survival in diverse econiches including metal and metalloid contaminated sites and capability to withstand high levels of metals. Biosynthesis of metallic NMs is accomplished through bioreduction of respective metal salts by various capping agents viz. alkaloids, pigments, polysaccharides, steroids, enzymes and peptides present in the biological systems. Advancement in the field of Nanobiotechnology has produced large number of diverse NMs from cyanobacteria which have been used as antimicrobial agents against Gram positive and negative human pathogens, anticancer agents, luminescent nanoprobes for imaging of cells, antifungal agents against plant pathogens, nanocatalyst and semiconductor quantum dots in industries and in bioremediation in toxic pollutant dyes. In the present communication, we have reviewed cyanobacteria mediated biosynthesis of NMs and their applications in various fields., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins., (© 2022. The Author(s).)

Oxyanions of selenium, selenite (SeO 3 ) 2- and selenate (SeO 4 ) 2- are toxic to terrestrial and aquatic biota but few microorganisms including cyanobacteria are resistant to high levels of selenite. Cyanobacteria evade selenite toxicity through bioreduction and synthesis of selenium nanoparticles (SeNPs). In this study, extracellular biosynthesis of SeNPs (Se 0 ) using cyanobacterium, Anabaena sp. PCC 7120 on exposure to sodium selenite and characterization was done by using UV-visible spectroscopy, SEM-EDX, TEM and FTIR analyses which confirmed spherical shape with size range of 5-50 nm diameter. These biogenic SeNPs demonstrated significant antibacterial and anti-biofilm activity against bacterial pathogens. Furthermore, these SeNPs showed high antioxidant activity at minimum concentration of 50 µg/mL and significant anti-proliferative activity against HeLa cell line with IC 50 value of 5.5 µg/mL. The SeNPs also induced accumulation of cancer cells in the sub-G1 phase which was clearly observed in cellular and nuclear morphology. These biofabricated SeNPs also reduced and decolorized toxic methylene blue dye significantly through photocatalytic degradation. Therefore Anabaena sp. PCC 7120 may be employed as a green bioresource to synthesize SeNPs with potential applications in medicine and environmental bioremediation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oxidative stress is common consequence of abiotic stress in plants as well as cyanobacteria caused by generation of reactive oxygen species (ROS), an inevitable product of respiration and photosynthetic electron transport. ROS act as signalling molecule at low concentration however, when its production exceeds the endurance capacity of antioxidative defence system, the organisms suffer oxidative stress. A highly toxic metabolite, methylglyoxal (MG) is also produced in cyanobacteria in response to various abiotic stresses which consequently augment the ensuing oxidative damage. Taking recourse to the common lineage of eukaryotic plants and cyanobacteria, it would be worthwhile to explore the regulatory role of glyoxalase system and antioxidative defense mechanism in combating abiotic stress in cyanobacteria. This review provides comprehensive information on the complete glyoxalase system (GlyI, GlyII and GlyIII) in cyanobacteria. Furthermore, it elucidates the recent understanding regarding the production of ROS and MG, noteworthy link between intracellular MG and ROS and its detoxification via synchronization of antioxidants (enzymatic and non-enzymatic) and glyoxalase systems using glutathione (GSH) as common co-factor., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Background: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves the characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising the CBS-CP12 domain and deciphering its role in abiotic stress tolerance., Methods: Molecular cloning, heterologous expression and protein purification using affinity chromatography were performed to obtain native purified protein Alr0765. The energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check the redox sensitivity of the target protein. Transcript levels under different abiotic stresses, as well as spot assay, CFU count, ROS level and cellular H2O2 level, were used to show the potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein-ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio, respectively., Results: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS, followed by MASCOT search analysis, confirmed the identity of the protein and ESI/MS revealed that the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with an almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a λ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control, which affirms the iron acquisition property of the protein. Moreover, the generation of the redox potential of 146 mV by Alr0765 suggested its probable role in maintaining the redox status of the cell under environmental constraints. As per CFU count recombinant, E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3 and 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in the cellular ROS level and total cellular H2O2 concentration validated the stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765., Conclusion: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis which provides tolerance against multiple abiotic stresses., (© 2020 Bentham Science Publishers.)

Abiotic stresses enhance the cellular level of reactive oxygen species (ROS) which consequently leads to toxic methylglyoxal (MG) production. Glyoxalases (GlyI & GlyII) catalyze the conversion of toxic MG into non-toxic lactic acid but their properties and functions have been overlooked in cyanobacteria. This is the first attempt to conduct a genome-wide analysis of GlyI protein (PF00903) from Anabaena sp. PCC7120. Out of total nine GlyI domain possessing proteins, only three (Alr2321, Alr4469, All1022) harbour conserve His/Glu/His/Glu metal binding site at their homologous position and are deficient in conserved region specific for Zn 2+ dependent members. Their biochemical, structural and functional characterization revealed that only Alr2321 is a homodimeric Ni 2+ dependent active GlyI with catalytic efficiency 11.7 × 10 6  M -1  s -1 . It has also been found that Alr2321 is activated by various divalent metal ions and has maximum GlyI activity with Ni 2+ followed by Co 2+  > Mn 2+  > Cu 2+ and no activity with Zn 2+. Moreover, the expression of alr2321 was found to be maximally up-regulated under heat (19 fold) followed by cadmium, desiccation, arsenic, salinity and UV-B stresses. BL21/pGEX-5X2-alr2321 showed improved growth under various abiotic stresses as compared to BL21/pGEX-5X2 by increased scavenging of intracellular MG and ROS levels. Taken together, these results suggest noteworthy links between intracellular MG and ROS, its detoxification by Alr2321, a member of GlyI family of Anabaena sp. PCC7120, in relation to abiotic stress., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Abiotic stresses enhance cellular reactive oxygen species (ROS) level which results in toxic methylglyoxal (MG) production. Glyoxalases catalyze the conversion of toxic MG into non-toxic lactic acid whose properties and function are still unknown in cyanobacteria. This is the first attempt to characterize All0580 from Anabaena sp. PCC7120 as GlyII using in silico and wet lab approaches. Data of functional complementation of E. coli GlyII mutant (ΔgloB), enzyme kinetics and ESI-MS analysis suggested that All0580 harbors distinctive GlyII activity. The catalytic efficiency of All0580 (3 × 10 6  M -1  s -1 ) is higher than Arabidopsis GlyII. AAS analysis revealed the presence of a binuclear Zn/Fe centre in All0580 active site. The qRT-PCR of the target gene revealed maximum up-regulation in salinity followed by drought, arsenic, heat, and UV-B stresses. BL21/pET-21a-all0580 showed 1.5 to 10 fold increased growth and up to 4 fold decreased intracellular MG level as compared to BL21/pET-21a cells under various abiotic stresses and MG. A 39% drop in ROS generation by BL21/pET-21a-all0580 under MG stress suggested its potential to manage MG toxicity. Above attributes suggest that the hypothetical protein All0580 is a novel GlyII of cyanobacteria which heterologously confers tolerance to multiple abiotic stresses in E. coli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In- silico and functional genomics approaches have been used to determine cellular functions of two hypothetical proteins All1122 and Alr0750 of Anabaena sp. PCC 7120. Motif analysis and multiple sequence alignment predicted them as typical α/β ATP binding universal stress family protein-A (UspA) with G-(2×)-G-(9×)-G(S/T) as conserved motif. qRT-PCR data under UV-B, NaCl, heat, As, CdCl 2, mannitol and methyl viologen registered approximately 1.4 to 4.3 fold induction of all1122 and alr0750 thus confirming their multiple abiotic stress tolerance potential. The recombinant E. coli (BL21) cells harboring All1122 and Alr0750 showed 12-41% and 23-41% better growth respectively over wild type control under said abiotic stresses thus revalidating their stress coping ability. Functional complementation on heterologous expression in UspA mutant E. coli strain LN29MG1655 (ΔuspA::Kan) attested their UspA family membership. This study tempted us to suggest that recombinant Anabaena PCC 7120 over expressing all1122 and alr0750 might contribute to the nitrogen economy in paddy fields experiencing array of abiotic stresses including drought and nutrient limitation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (C P ) and, if present, the resolving Cys (C R ). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 Å resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.

The present study provides data on the insertion of an extra copy of phytochelatin synthase (alr0975) in Anabaena sp. PCC 7120. The recombinant strain (AnFPN-pcs) compared to wild type showed approximately 22.3% increase in growth rate under UV-B, NaCl, heat, CuCl 2 , carbofuran, and CdCl 2 . It also registered 2.25-fold enhanced nitrogenase activity and 5-fold higher phytochelatin production. A comparison of the protein profile of wild type with the recombinant strain revealed that recombinant strain accumulated proteins belonging to the following categories: (i) detoxification (nutrient stress induced DNA binding protein, Mn-SOD, Alr0946 (CalA)), (ii) protein folding and modification (molecular chaperone DnaK, FKBP-type peptidyl-prolyl cis-trans isomerase), (iii) nucleotide and amino acid biosynthesis (dihydroorotase and Ketol-acid reductoisomerase), (iv) photosynthesis and respiration (coproporphyrinogen III oxidase, phycocyanin alpha chain, ferredoxin-NADP + reductase), and (v) transport (sugar transport ATP-binding protein). Thus, it can be concluded that, above category proteins with their respective role in scavenging reactive oxygen species, proper folding of unfolded proteins, and protection of protein from degradation, sustained carbon fixation and energy pool and active transport of sugar together conceivably help the recombinant cyanobacterium (AnFPN-pcs) to cope with abiotic stress employed in the present study. Such recombinant strains have potential for future use as biofertilizer.

Soil quality is an important factor and maintained by inhabited microorganisms. Soil physicochemical characteristics determine indigenous microbial population and rice provides food security to major population of the world. Therefore, this study aimed to assess the impact of physicochemical variables on bacterial community composition and diversity in conventional paddy fields which could reflect a real picture of the bacterial communities operating in the paddy agro-ecosystem. To fulfill the objective; soil physicochemical characterization, bacterial community composition and diversity analysis was carried out using culture-independent PCR-DGGE method from twenty soils distributed across eight districts. Bacterial communities were grouped into three clusters based on UPGMA cluster analysis of DGGE banding pattern. The linkage of measured physicochemical variables with bacterial community composition was analyzed by canonical correspondence analysis (CCA). CCA ordination biplot results were similar to UPGMA cluster analysis. High levels of species-environment correlations (0.989 and 0.959) were observed and the largest proportion of species data variability was explained by total organic carbon (TOC), available nitrogen, total nitrogen and pH. Thus, results suggest that TOC and nitrogen are key regulators of bacterial community composition in the conventional paddy fields. Further, high diversity indices and evenness values demonstrated heterogeneity and co-abundance of the bacterial communities.

Aldo/keto reductases (AKRs) constitute a multitasking protein family that catalyzes diverse metabolic transformations including detoxification of stress generated reactive aldehydes. Yet this important protein family is poorly understood particularly in cyanobacteria, the ecologically most diverse and significant group of micro-organisms. Present study is an attempt to characterize all putative AKRs of Anabaena sp. PCC 7120. In silico analysis, it revealed the presence of at least four putative AKRs in Anabaena PCC7120 genome. All four proteins share less than 40% sequence identity with each other and also with the identified members of AKR superfamily and hence deserve to be assigned in new families. Dissimilarity in sequences is also reflected through their substrate specificity. While reduction of trans-2-nonenal, a LPO-derived reactive aldehyde was common across the four proteins, these proteins were found to be activated during heat, salt, Cd, As, and butachlor treatments, and their ectopic expression in Escherichia coli conferred tolerance to the above abiotic stresses. These findings affirm the role of AKRs in providing a broad tolerance to environmental stresses conceivably by detoxifying the stress-generated reactive aldehydes.

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 μmol min -1  mg protein -1 , and K cat /K m 9.438 × 10 4  μM - 1 min -1 ). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.

The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An+ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An+ahpC than in wild type. An+ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An+ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40-60% compared to a 33-40% enhanced growth in An+ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An+ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An+ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Alkylhydroperoxide reductase (AhpC), a 1-Cys peroxiredoxin is well known for maintaining the cellular homeostasis. Present study employs proteome approach to analyze and compare alterations in proteome of Anabaena PCC7120 in overexpressing (An+ahpC), deletion (An∆ahpC) and its wild type. 2-DE based analysis revealed that the major portion of identified protein belongs to energy metabolism, protein folding, modification and stress related proteins and carbohydrate metabolism. The two major traits discernible from An+ahpC were (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins. Increased accumulation of proteins of light reaction, dark reaction, pentose phosphate pathway and electron transfer agent FDX for nitrogenase in An+ahpC and their simultaneous downregulation in AnΔahpC demonstrates its role in augmenting photosynthesis and nitrogen fixation. Proteomic data was nicely corroborated with physiological, biochemical parameters displaying upregulation of nitrogenase (1.6 fold) PSI (1.08) and PSII (2.137) in An+ahpC. Furthermore, in silico analysis not only attested association of AhpC with peroxiredoxins but also with other players of antioxidative defense system viz. thioredoxin and thioredoxin reductase. Above mentioned findings are in agreement with 33-40% and 40-60% better growth performance of An+ahpC over wild type and An∆ahpC respectively under abiotic stresses, suggesting its role in maintenance of metabolic machinery under stress., Significance: Present work explores key role of AhpC in mitigating stress in Anabaena PCC7120 through combined proteomic, biochemical and in silico investigations. This study is the first attempt to analyze and compare alterations in proteome of Anabaena PCC7120 following addition (overexpressing strain An+ahpC) and deletion (mutant An∆ahpC) of AhpC against its wild type. The effort resulted in two major traits in An+ahpC as (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

DNA-binding proteins (Dps) induced during starvation play an important role in gene regulation and maintaining homeostasis in bacteria. The nitrogen-fixing cyanobacterium, Anabaena PCC7120, has four genes annotated as coding for Dps; however, the information on their physiological roles is limiting. One of the genes coding for Dps, 'all3940' was found to be induced under different abiotic stresses in Anabaena and upon overexpression enhanced the tolerance of Anabaena to a multitude of stresses, which included salinity, heat, heavy metals, pesticide, and nutrient starvation. On the other hand, mutation in the gene resulted in decreased growth of Anabaena. The modulation in the levels of All3940 in Anabaena, achieved either by overexpression of the protein or mutation of the gene, resulted in changes in the proteome, which correlated well with the physiological changes observed. Proteins required for varied physiological activities, such as photosynthesis, carbon-metabolism, oxidative stress alleviation, exhibited change in protein profile upon modulation of All3940 levels in Anabaena. This suggested a direct or an indirect effect of All3940 on the expression of the above stress-responsive proteins, thereby enhancing tolerance in Anabaena PCC7120. Thus, All3940, though categorized as a Dps, is possibly a general stress protein having a global role in regulating tolerance to multitude of stresses in Anabaena.

Present study deals with the identification of a novel aldo/keto reductase, AKR17A1 from Anabaena sp. PCC7120 and adds on as 17th family of AKR superfamily drawn from a wide variety of organisms. AKR17A1 shares many characteristics of a typical AKR such as- (i) conferring tolerance to multiple stresses like heat, UV-B, and cadmium, (ii) excellent activity towards known AKR substrates (isatin and 2-nitrobenzaldehyde), and (iii) obligate dependence on NADPH as a cofactor for enzyme activity. The most novel attribute of AKR17A1, first reported in this study, is its capability to metabolize butachlor, a persistent rice field herbicide that adversely affects agro-ecosystem and non-target organisms. The AKR17A1 catalyzed- degradation of butachlor resulted into formation of 1,2-benzene dicarboxylic acid and 2,6 bis (1,1, dimethylethyl) 4,-methyl phenol as the major products confirmed by GC-MS analysis.

Unlabelled: This paper focuses on the gel-based membrane proteomics from diazotrophic cyanobacterium Anabaena PCC7120 by modifying the protocol of Hall et al. [1]. The bioinformatic analysis revealed that 59 (29 integral, 30 peripheral) of the 67 proteins identified were membrane proteins. Of the 29 integral proteins, except Alr0834, the remaining 28 contained 1-12 transmembrane helices. Sixteen integral proteins harboring signal peptides (Sec/TAT/LipoP) suggest that protein targeting in Anabaena involves both sec-dependent and sec-independent pathways. While majority of photosynthesis and respiration proteins (21 of 24) were confined to broad pH gradient the hypothetical and unknown (12 of 13), and cell envelope proteins (3 of 3) preferred the narrow pH range. Of the 5 transporters and binding proteins, Na(+)/H(+)-exchanging protein and Alr2372 were present in broad, pstS1 and cmpD in narrow and cmpA was common to both pH ranges. The distribution of proteins across pH gradient, thus clearly indicates the functional and structural diversity in membrane proteome of Anabaena. It requires mention that protochlorophyllide oxido-reductase, Na(+)/H(+)-exchanging protein, All1355, Alr2055, Alr3514, Alr2903 and Alr2751 were new entries to the 2DE membrane protein profile of Anabaena. This study demonstrates suitability of the modified protocol for the study of membrane protein from filamentous cyanobacteria., Significance: Anabaena sp. PCC7120 is used as a model organism due to its agriculture significance as biofertilizer, close resemblance with higher plant chloroplast and availability of full genome sequence. Although cytosolic proteome has been explored a lot membrane proteins are still understudied as they are notoriously difficult to display using 2-D technology. Identification and characterization of these proteins is therefore required to elucidate and understand cellular mechanisms. The purpose of this study was to develop a protocol suitable for membrane protein extraction from Anabaena. Additionally, by homology comparison or domain assignment a possible function could be ascribed to novel uncharacterized proteins which will serve as a useful reference for further detailed studies of membrane system in filamentous cyanobacteria. Resolution of membrane proteins ranging from least (single transmembrane helix) to highly hydrophobic (several transmembrane helices) one on 2D gels recommends the gel based approach for identification of membrane proteomics from filamentous cyanobacteria. This article is part of a Special Issue entitled: Proteomics in India., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India., (Copyright © 2015. Published by Elsevier B.V.)

Comparative proteomics together with physiological variables revealed different responses among three species of diazotrophic cyanobacterium Anabaena exposed to UV-B stress at the same time points. Perceptible decline in PSII activity, ATP pool, nitrogenase activity and respiration rate was observed for all the three species; this being maximum in Anabaena doliolum, followed by Anabaena sp. PCC 7120 and minimum in Anabaena L31. Statistical analysis of the protein abundance divided majority of them as early accumulated in A. L31, late accumulated in A. sp. PCC 7120 and downregulated in A. doliolum. Tolerance of A. L31 may be ascribed to post-translational modification reflected through the highest number of protein isoforms in its proteome followed by A. PCC 7120 and A. doliolum. Furthermore, increase in abundance of cyanophycinase, glutamine synthetase and succinate semialdehyde dehydrogenase in A. L31 suggests operation of an alternate pathway for assimilation of nitrogen and carbon under UV-B stress. An early accumulation of four proteins viz., glutamate ammonia ligase (Alr2328), transketolase (Alr3344), inorganic pyrophosphatase (All3570), and trigger protein (Alr3681) involved respectively in amino acid metabolism, energy metabolism, biosynthesis of cofactor and trigger protein and chaperone like activity across three species, suggests them to be marker of UV-B stress in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The classification of order Nostocales (Cyanobacteria) and inter relationships of morphologically similar taxa is still debatable due to ever changing morphological features. No attempt has been made to improve the morphological taxonomy despite the fact that it is the morphology that represents the totality of genes. To test the validity of morphological taxonomy and fine tune the phylogenetic relationships within the order Nostocales a new weighted morphology approach was applied by using 76 isolates and their 16S rRNA gene sequences. Further, the study was extended with morphological data set of the remaining 232 taxa for which no molecular data are yet available. Trichome aggregation, heterocyst shape, and akinete shape are suggested as important and stable features for identification. At 30% weight assignment to the selected morphological characters, morphological taxonomy found 36% compatible with 16S tree. Adding weight to the morphological characters considerably improved the congruence between the morphology and 16S rRNA-based phylogenetic trees of the order Nostocales. When the weighting procedure was extended to all the Nostocalean members irrespective of molecular data availability, it was found that Nostoc sphaericum and Nostoc microscopicum closely assembled in a single clade. Closer arrangement of Aulosira and Nodularia represent the subfamily aulosirae (Bornet and Flahault Ann Sci Nat Bot 7:223-224, 1888) while taxonomic affiliation of Cylindrospermum with Nostoc, Anabaena, and Raphidiopsis representing the subfamily anabaenae (Bornet and Flahault Ann Sci Nat Bot 7:223-224, 1888) was resolved.

Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein.

The present study compares protein diversity within three Anabaena species (Anabaena doliolum, Anabaena sp.PCC 7120 and Anabaena L31). 2-DE based analysis of 256 protein spots in control and 1, 3, 5, and 7days of salt treatment resulted into 96 proteins arching across fourteen functional categories were assigned to biochemical pathways using KOBAS 2.0. While 52.34% of the evaluated protein spots were common across three species, the remaining 47.66% fraction mainly comprised of the hypothetical and unknown proteins. PSORTb, CDD, Motifscan and Pfam revealed function and subcellular localization for 27 of the 31 hypothetical and unknown proteins. The differences in high salt tolerance (LC50) of A. doliolum over A. L31 was reflected by (i) many fold accumulation (as spot volumes) of Alr3090, Alr0803, peptidyl prolyl cis-trans isomerase and modulator of DNA gyrase proteins, and (ii) a better photosynthesis and energy homeostasis as indicated through photosystem activity, respiration, ATP and NADPH contents. Some common noteworthy salt effects include (i) photosystem damage, (ii) DNA damage repair, (iii) upregulated protein synthesis, (iv) enhanced sulphur metabolism, and (v) upregulated pentose phosphate pathway. 34 of the identified protein spots are novel entries to the Anabaena salt proteome. This study reveals the existence of separate strategies even within species to combat stress., Biological Significance: This study for the first time enumerates protein diversity in three Anabaena species employing their presence/absence and relative abundance. Proteomics integrated with physiology and bioinformatics deciphers differential salt tolerance among the studied species and is the first of its kind to predict the function of hypothetical and unknown proteins. Salt-induced proteomic alterations clearly demonstrate significant metabolic shifts and existence of separate molecular phenome among the species investigated. This may be responsible for niche specificity limiting their application as biofertilizer. Of the 96 identified proteins, a large chunk are new entries to the Anabaena salt proteome while some protein genes may be used as potential candidates for engineering salt tolerant cyanobacteria., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Butachlor an extensively used rice field herbicide negatively affects the cyanobacterial proliferation, yet the molecular mechanism underlying its toxicity in diazotrophic cyanobacteria is largely unknown. The present study focuses on the comparative proteomics to decode the molecular basis of butachlor toxicity/tolerance in three Anabaena species e.g. Anabaena sp. PCC 7120, Anabaena doliolum and Anabaena L31. 75 differentially expressed proteins from each Anabaena sp. included those involved in photosynthesis, C, N and protein metabolism, redox homeostasis, and signal transduction. While early accumulated proteins related to photosynthesis (atpA, atpB), carbon metabolism (glpx, fba and prk), protein folding (groEL, PPIase), regulation (orrA) and other function (OR, akr) appeared crucial for tolerance of Anabaena L31, the late accumulated proteins in Anabaena 7120 presumably offer acclimation during prolonged exposure to butachlor. Contrary to the above, a multitude of down-accumulated proteins vis-a-vis metabolisms augment sensitivity of A. doliolum to butachlor. A cluster of high abundant proteins (atpA, groEL, OR, AGTase, Alr0803, Alr0806, Alr3090, Alr3199, All4050 and All4051) common across the three species may be taken as markers for butachlor tolerance and deserve exploitation for stress management and transgenic development., Biological Significance: Cyanobacteria offer an eco-friendly alternative to chemical fertilizers for increasing productivity, especially in rice cultivation. This study is the first to compare the proteome of three diazotrophic cyanobacteria subjected to butachlor, a pre-emergent herbicide extensively used in rice paddy. Changes in protein dynamics over time along with physiological and biochemical attributes clearly provide a comprehensive overview on differential tolerance of Anabaena species to butachlor. Molecular docking further added a new dimension in identification of potential protein candidates for butachlor stress management in cyanobacteria. This study strongly recommends combined application of Anabaena spp. A. L31 and A. PCC7120 as biofertilizer in paddy fields undergoing butachlor treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min⁻¹ mg protein⁻¹) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min⁻¹ mg protein⁻¹) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (∆arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.

This study examines response of Anabaena sp. PCC 7120 to salt and UV-B stress by combining physiological, biochemical, proteomics and bioinformatics approaches. Sixty five significantly altered protein spots corresponding to 51 protein genes identified using MALDI-TOF MS/MS were divided into nine functional categories. Based on relative abundance, these proteins were grouped into four major sets. Of these, 27 and 5 proteins were up- and downregulated, respectively, both under salt and UV-B while 8 and 11 proteins showed accumulation in salt and UV-B applied singly. Some responses common to salt and UV-B included (i) enhanced expression of FeSOD, alr3090 and accumulation of MDA indicating oxidative stress, (ii) accumulation of PDH, G6P isomerase, FBPaldolase, TK, GAPDH and PGK suggesting enhanced glycolysis, (iii) upregulation of 6-PGD, 6PGL and NADPH levels signifying operation of pentose phosphate pathway, (iv) upregulation of Dps, NDK and alr3199 indicating DNA damage, and (v) accumulation of proteins of ribosome assembly, transcriptional and translational processing. In contrast, enhanced expression of RUBISCO, increased glycolate oxidase activity and ammonium content under salt signify the difference. Salt was found to be more damaging than UV-B probably due to a cumulative effect of ionic, osmotic and oxidative damage. A group of proteins having common expression represent decreased toxicity of salt and UV-B when applied in combination.

In silico analysis followed by experimental validation leads us to propose that the predicted protein All0195 of Anabaena sp. PCC7120 showing enhanced expression under sodium arsenate (Na2HAsO4) stress belongs to the thioredoxin superfamily with structural similarity to bacterial arsenate reductase. The All0195 protein demonstrated C-X-TC-X-K, NTSG-X2-YR, and D-X2-L-X-KRP as functional motifs that show similarity to seven known bacterial arsenate reductase family protein homologs with Cys, Arg, and Pro as conserved residues. In view of physicochemical properties, such as aliphatic index, ratio of Glu + Lys to Gln + His, and secondary structure, it was evident that All0195 was also a thermostable protein. The predicted three-dimensional structure on molecular docking with arsenate oxyanion ([Formula: see text]) revealed its interaction with conserved Cys residue as also known for other bacterial arsenate reductase. In silico derived properties were experimentally attested by cloning and heterologous expression of all0195. Furthermore, this protein functionally complemented the arsenate reductase-deficient sodium arsenate-hypersensitive phenotype of Escherichia coli strainWC3110 (ΔarsC) and depicted arsenate reductase activity on purification. In view of the above properties, All0195 appears to be a new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120.

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (∆uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ∆uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS-PAGE and attested by immunoblotting. E. coli ∆uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl(2), H(2)O(2), UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl(2) demonstrated about 22.6-51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5 μM Cd(2+), 2 μM Co(2+), 0.5 μM Cu(2+), 500 μM Mn(2+), 1 μM Ni(2+), and 18 μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500 μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72 h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72 h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5 μM Cd(2+), while 2 μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.

Proteomics in conjunction with morphological, physiological and biochemical variables has been employed for the first time to unravel survival strategies of the diazotrophic cyanobacterium Anabaena sp. PCC7120 under Arsenic (As) stress. Significant reduction in growth, carbon fixation, nitrogenase activity and chlorophyll content after 1 day (1d) and recovery after 15 days (15d) of As exposure indicates the acclimation of the test organism against As stress. The formation of akinete like structures is a novel observation never reported before in Anabaena sp. PCC7120. Proteomic characterization using 2-DE showed average 537, 422 and 439 spots in control, 1 and 15d treatment respectively. MALDI-TOF and LC-MS of As-treated Anabaena revealed a total of 45 differentially expressed proteins, of which 13 were novel (hypothetical) ones. Down-regulation of phosphoglycerate kinase (PGK), fructose bisphosphate aldolase II (FBA II), fructose 1,6 bisphosphatase (FBPase), transketolase (TK), and ATP synthase on day 1 and their significant recovery on the 15th day presumably maintained the glycolysis, pentose phosphate pathway (PPP) and turnover rate of Calvin cycle, hence survival of the test organism. Up-regulation of catalase (CAT), peroxiredoxin (Prx), thioredoxin (Trx) and oxidoreductase appears to protect the cells from oxidative stress. Appreciable induction in phytochelatin content (2.4 fold), GST activity (2.3 fold), and transcripts of phytochelatin synthase (5.0 fold), arsenate reductase (8.5 fold) and arsenite efflux genes - asr1102 (5.0 fold), alr1097 (4.7 fold) reiterates their role in As sequestration and shielding of the organism from As toxicity. While up-regulated metabolic and antioxidative defense proteins, phytochelatin and GST work synchronously, the ars genes play a central role in detoxification and survival of Anabaena under As stress. The proposed hypothetical model explains the interaction of metabolic proteins associated with the survival of Anabaena sp. PCC7120 under As stress., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study examines the effects of 30 mg/kg butachlor on the cyanobacterial diversity of rice fields in Eastern Uttar Pradesh and Western Bihar in India. A total of 40 samples were grouped into three classes [(i) acidic, (ii) neutral, and (iii) alkaline soils], based on physicochemical and principle component analyses. Acidic soils mainly harbored Westillopsis, Trichormus, Anabaenopsis, and unicellular cyanobacteria; whereas Nostoc, Anabaena, Calothrix, Tolypothrix, and Aulosira were found in neutral and alkaline soils. Molecular characterization using 16S rRNA PCR and DGGE revealed the presence of 13 different phylotypes of cyanobacteria in these samples. Butachlor treatment of the soil samples led to the disappearance of 5 and the emergence of 2 additional phylotypes. A total of 40 DGGE bands showed significant reproducible changes upon treatment with butachlor. Phylogenetic analyses divided the phylotypes into five major clusters exhibiting interesting links with soil pH. Aulosira, Anabaena, Trichormus, and Anabaenopsis were sensitive to butachlor treatment, whereas uncultured cyanobacteria, a chroococcalean member, Westillopsis, Nostoc, Calothrix, Tolypothrix, Rivularia, Gloeotrichia, Fischerella, Leptolyngbya, and Cylindrospermum, appeared to be tolerant against butachlor at their native soil pH. Butachlor-induced inhibition of nitrogen fixation was found to be 65% (maximum) and 33% (minimum) in the soil samples of pH 9.23 and 5.20, respectively. In conclusion, low butachlor doses may prove beneficial in paddy fields having a neutral to alkaline soil pH.

Alkyl hydroperoxide reductase (AhpC) is a key component of a large family of thiol-specific antioxidant (TSA) proteins distributed among prokaryotes and eukaryotes. AhpC is involved in the detoxification of reactive oxygen species (ROS) and reactive sulfur species (RSS). Sequence analysis of AhpC from the cyanobacterium Anabaena sp. PCC 7120 shows that this protein belongs to the 1-Cys class of peroxiredoxins (Prxs). It has recently been reported that enhanced expression of this protein in Escherichia coli offers tolerance to multiple stresses such as heat, salt, copper, cadmium, pesticides and UV-B. However, the structural features and the mechanism behind this process remain unclear. To provide insights into its biochemical function, AhpC was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected to a maximum d-spacing of 2.5 Å using synchrotron radiation. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 80, b = 102, c = 109.6 Å. The structure of AhpC from Anabaena sp. PCC 7120 was determined by molecular-replacement methods using the human Prx enzyme hORF6 (PDB entry 1prx) as the template., (© 2011 International Union of Crystallography. All rights reserved.)

This study provides first-hand proteomic data on the survival strategy of Anabaena sp. PCC 7120 when subjected to long-term iron-starvation conditions. 2D-gel electrophoresis followed by MALDI-TOF/MS analysis of iron-deficient Anabaena revealed significant and reproducible alterations in ten proteins, of which six are associated with photosynthesis and respiration, three with the antioxidative defense system, and the last, hypothetical protein all1861, conceivably connected with iron homeostasis. Iron-starved Anabaena registered a reduction in growth, photosynthetic pigments, PSI, PSII, whole-chain electron transport, carbon and nitrogen fixation, and ATP and NADPH content. The kinetics of hypothetical protein all1861 expression, with no change in expression until day 3, maximum expression on the 7th day, and a decline in expression from the 15th day onward, coupled with in silico analysis, suggested its role in iron sequestration and homeostasis. Interestingly, the up-regulated FBP-aldolase, Mn/Fe-SOD, and all1861 all appear to assist the survival of Anabeana subjected to iron-starvation conditions. Furthermore, the N2-fixation capabilities of the iron-starved Anabaena encourage us to recommend its application as a biofertilizer, particularly in iron-limited paddy soils.

This study presents first hand data on the cloning and heterologous expression of Anabaena PCC 7120 all3940 (a dps family gene) in combating nutrients limitation and multiple abiotic stresses. The Escherichia coli transformed with pGEX-5X-2-all3940 construct when subjected to iron, carbon, nitrogen, phosphorus limitation and carbofuron, copper, UV-B, heat, salt and cadmium stress registered significant increase in growth over the cells transformed with empty vector under iron (0%), carbon (0.05%), nitrogen (3.7 mM) and phosphorus (2mM) limitation and carbofuron (0.025 mg ml(-1)), CuCl(2) (1 mM), UV-B (10 min), heat (47 degrees C), NaCl (6% w/v) and CdCl(2) (4mM) stress. Enhanced expression of all3940 gene measured by semi-quantitative RT-PCR at different time points under above mentioned treatments clearly demonstrates its role in tolerance against aforesaid abiotic stresses. This study opens the gate for developing transgenic cyanobacteria capable of growing successfully under above mentioned stresses., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The present study examines butachlor-induced inhibition of growth, photosynthetic pigments such as chlorophyll a, phycocyanin, allophycocyanin, phycoerythrin, photosystems I and II, whole chain electron transport, oxygen evolution, carbon fixation, ATP content, total thiol and glutathione contents of Aulosira fertilissima. For ascertaining if above mentioned changes are due to disturbance in plasma membrane integrity or proteins, fatty acid profiling and proteomics were done. Gas chromatographic (GC) analysis of fatty acid methyl esters (FAME) depicted a decrease in alpha-linolenic acid (C18:3) which appears responsible for plasma membrane instability. Enhanced lipid peroxidation and electrolyte leakage further attested the butachlor-induced cell damage. Butachlor-treated Aulosira exhibited significant and reproducible alternations in eight proteins as assessed by 2DE and LC-MS analysis of which phycocyanin alpha-chain, allophycocyanin beta-chain, C-phycocyanin alpha-subunit, ATP synthase beta-chain and FBP aldolase were associated with photosynthesis and respiration, peroxiredoxin with antioxidative defense system and GroES and NusB with protein folding and transcription termination respectively. However, a prolonged (15 d) butachlor treatment of Aulosira downregulated all the proteins except NusB. Reverse transcription PCR of the protein genes affirmed that aforesaid proteins were the gene products not artifacts. Downregulated GroES and over expressed NusB are critical proteins for cell death.

This study offers first hand information on the arsenite (As(III)) and arsenate (As(V))-induced oxidative stress and changes in antioxidative defense system of Anabaena doliolum. A requirement of 58 mM As(V) as compared to only 11 mM As(III) to cause 50% reduction in growth rate suggests that As(III) is more toxic than As(V) in the test cyanobacterium. In contrast to above, oxidative damage measured in terms of lipid peroxidation, electrolyte leakage and peroxide content were significantly higher after As(V) than As(III) treatment as compared to control. Similarly all the studied enzymatic parameters of antioxidative defense system except glutathione reductase (GR) and non-enzymatic parameters except glutathione reduced (GSH) showed greater induction against As(V) than As(III). Interestingly, higher increase in non-enzymatic counterpart than enzymatic in both the stresses suggests that detoxification is mainly managed by former than the later. This confirms the belief of pronounced stimulation of the antioxidative defense system by As(V) than As(III). In conclusion, the cyanobacterium may survive better in As(V) than As(III) contaminated fields because of its low toxicity and pronounced induction of antioxidative defense system., (Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study offers proteomic elucidation of heat pretreatment-induced alleviation of UV-B toxicity in Anabaena doliolum. Heat-pretreated cells exposed to UV-B showed improved activity of PSI, PSII, whole chain, (14)C fixation, ATP and NADPH contents compared to UV-B alone. Proteomic analysis using two-dimensional gel electrophoresis (2-DE), MALDI-TOF MS/MS and reverse transcription polymerase chain reaction (RT-PCR) of UV-B and heat pretreatment followed by UV-B-treated cells exhibited significant and reproducible alterations in nine proteins homologous to phycocyanin-alpha-chain (PC-alpha-chain), phycoerythrocyanin-alpha-chain (PEC-alpha-chain), hypothetical protein alr0882, phycobilisome core component (PBS-CC), iron superoxide dismutase (Fe-SOD), fructose-1,6-bisphosphate aldolase (FBA), nucleoside diphosphate kinase (NDPK), phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) large chain. Except the PEC-alpha-chain, hypothetical protein alr0882 and PBS-CC, all other proteins showed upregulation at low doses of UV-B (U2) and significant downregulation at higher doses of UV-B (U5). The disruption of redox status, signaling, pentose phosphate pathway and Calvin cycle appears to be due to the downregulation of Fe-SOD, NDPK, FBA, PRK and RuBisCo thereby leading to the death of Anabaena. In contrast to this, the upregulation of all the above proteins in heat-pretreated cells, harboring different heat shock proteins (HSPs) like 60, 26 and 16.6, followed by UV-B treatment than only the UV-B-treated ones suggests a protective role of HSPs in mitigating UV-B toxicity.

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 degrees C), NaCl (6% w/v), carbofuron (0.025mgml(-1)), CdCl(2) (4mM), CuCl(2) (1mM), and UV-B (10min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.

Background: Salinity is known to affect almost half of the world's irrigated lands, especially rice fields. Furthermore, cyanobacteria, one of the critical inhabitants of rice fields have been characterized at molecular level from many different geographical locations. This study, for the first time, has examined the molecular diversity of cyanobacteria inhabiting Indian rice fields which experience various levels of salinity., Results: Ten physicochemical parameters were analyzed for samples collected from twenty experimental sites. Electrical conductivity data were used to classify the soils and to investigate relationship between soil salinity and cyanobacterial diversity. The cyanobacterial communities were analyzed using semi-nested 16S rRNA gene PCR and denaturing gradient gel electrophoresis. Out of 51 DGGE bands selected for sequencing only 31 which showed difference in sequences were subjected to further analysis. BLAST analysis revealed highest similarity for twenty nine of the sequences with cyanobacteria, and the other two to plant plastids. Clusters obtained based on morphological and molecular attributes of cyanobacteria were correlated to soil salinity. Among six different clades, clades 1, 2, 4 and 6 contained cyanobacteria inhabiting normal or low saline (having EC < 4.0 ds m(-1)) to (high) saline soils (having EC > 4.0 ds m(-1)), however, clade 5 represented the cyanobacteria inhabiting only saline soils. Whilst, clade 3 contained cyanobacteria from normal soils. The presence of DGGE band corresponding to Aulosira strains were present in large number of soil indicating its wide distribution over a range of salinities, as were Nostoc, Anabaena, and Hapalosiphon although to a lesser extent in the sites studied., Conclusion: Low salinity favored the presence of heterocystous cyanobacteria, while very high salinity mainly supported the growth of non-heterocystous genera. High nitrogen content in the low salt soils is proposed to be a result of reduced ammonia volatilization compared to the high salt soils. Although many environmental factors could potentially determine the microbial community present in these multidimensional ecosystems, changes in the diversity of cyanobacteria in rice fields was correlated to salinity.

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn(2+) over 14 days showed that ZnCl(2) levels above 22 microM significantly reduced cell viability. After 3 days in 22 microM ZnCl(2), ca. 12% of the Zn(2+) was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 microM relative to 0 microM treatment. Radiolabeled (65)Zn showed Zn(2+) uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn(2+) uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 microM ZnCl(2) within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.

Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 degrees C), NaCl (6% w/v), carbofuron (0.025 mg ml(-1)), CdCl2 (4 mM), CuCl2 (1mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.

This study provides first-hand proteomic characterisation of Cu-pretreatment-induced augmentation of ultraviolet B toxicity in the cyanobacterium Anabaena doliolum Bharadwaja. Of the three treatments (i.e. Cu, UV-B and Cu + UV-B) tested, the UV-B treatment of Cu-pretreated Anabaena produced a greater inhibition of oxygen evolution, 14 C fixation, ATP and NADPH contents than UV-B alone. Proteomic analysis using two-dimensional gel electrophoresis (2DE), MALDI-TOF MS/MS and reverse transcription polymerase chain reaction (RT-PCR) of Cu, UV-B, and Cu + UV-B treated Anabaena exhibited significant and reproducible alterations in 12 proteins. Of these, manganese superoxide dismutase (Mn-SOD), iron superoxide dismutase (Fe-SOD) and peroxiredoxin (PER) are antioxidative enzymes; ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), phosphoribulokinase (PRK), flavodoxin (Flv), plastocyanin (PLC), phosphoglycerate kinase (PGK), phycocyanin (PC) and phycoerythrocyanin α-chain (PC α-chain) are linked with photosynthesis and respiration; and DnaK and nucleoside diphosphate kinase (NDPK) are associated with cellular processes and light signalling, respectively. However, when subjected to a high dose of UV-B, Cu-pretreated Anabaena depicted a severe down-regulation of DnaK, NDPK and Flv, probably because of inevitable oxidative stress. Thus, the augmentation of UV-B toxicity by Cu can be attributed to the down-regulation of DnaK, NDPK and Flv.

This study is the first to demonstrate operation of anoxygenic photosynthesis in copper acclimated Anabaena doliolum and to offer proteomic comparison with the control cells. The Cu-treated control strain showed a negative correlation in growth and intracellular Cu, partial inhibition of O(2)-evolution, PS II, PS I, whole chain, chlorophyll absorption, and nitrogenase activity. However, the acclimated strain growing in 250-fold excess Cu exhibited near normal growth, ATP content, PS I activity, carbon fixation, and almost complete inhibition of O(2)-evolution, PS II and chlorophyll absorption, but increased nitrogenase activity as compared to control. Proteomic decoding of the survival strategy of Cu-treated control and the acclimated strain using two-dimensional gel electrophoresis and MALDI-TOF MS analysis of proteins displaying significant and reproducible changes demonstrated involvement of transketolase, phycoerythrocyanin alpha-chain, iron superoxide dismutase (Fe-SOD), hypothetical protein alr 0803, manganese superoxide dismutase (Mn-SOD), phosphoribulokinase, and plastocyanin (PLC). Expression pattern of these proteins was attested at the transcriptional level using RT-PCR. Time course analysis of proteins of Cu-treated control strain revealed almost no change in PLC level, and a minor accumulation of transketolase, phycoerythrocyanin alpha-chain and both isoforms of SOD after 7 and recovery after 10 days. Acclimated strain under excess Cu, however, exhibited significant accumulation of both isoforms of SOD, plastocyanin, phosphoribulokinase and transketolase, which seem to counteract oxidative damage, serve as an alternate electron carrier from cytochrome b6/f complex to photosystem I and meet the NADPH and ATP requirements, respectively, under anoxygenic photosynthesis. In view of the kinetics of the hypothetical protein alr0803 (no change in expression level for 7, maximum after 10 and decline after 15 days) its involvement in metal homeostasis is suggested.