Functional characterization of two WD40 family proteins, Alr0671 and All2352, from Anabaena PCC 7120 and deciphering their role in abiotic stress management.

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.

Alr0671 and All2352 are novel WD40 proteins of Anabaena capable of regulating biochemical functions and abiotic stress tolerance by acting as a transcription factor and mediating DNA-protein interaction.

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches.

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.

Identification and functional characterization of four novel aldo/keto reductases in Anabaena sp. PCC 7120 by integrating wet lab with in silico approaches.

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.

Alr2954 of Anabaena sp. PCC 7120 with ADP-ribose pyrophosphatase activity bestows abiotic stress tolerance in Escherichia coli.

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 × 104 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.

A novel alkyl hydroperoxidase (AhpD) of Anabaena PCC7120 confers abiotic stress tolerance in Escherichia coli.

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.

A new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120.

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.

Salt and UV-B induced changes in Anabaena PCC 7120: physiological, proteomic and bioinformatic perspectives.

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.

All3048, a DnaJ III homolog of Anabaena sp. PCC7120 mediates heat shock response in E. coli and its N-terminus J-domain stimulates DnaK ATPase activity.

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.

Physiological metal uptake by Nostoc punctiforme.

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.

Cyclophilin anaCyp40 regulates photosystem assembly and phycobilisome association in a cyanobacterium.

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.

Regulation of antioxidant defense and glyoxalase systems in cyanobacteria.

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.

Bioinformatic and expression analyses of genes mediating zinc homeostasis in Nostoc punctiforme.

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.

Alr2321, a multiple stress inducible glyoxalase I of Anabaena sp. PCC7120 detoxifies methylglyoxal and reactive species oxygen.

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 Zn2+ dependent members. Their biochemical, structural and functional characterization revealed that only Alr2321 is a homodimeric Ni2+ dependent active GlyI with catalytic efficiency 11.7 × 106 M-1 s-1. It has also been found that Alr2321 is activated by various divalent metal ions and has maximum GlyI activity with Ni2+ followed by Co2+ > Mn2+ > Cu2+ and no activity with Zn2+. 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.

Molecular and biochemical characterization of All0580 as a methylglyoxal detoxifying glyoxalase II of Anabaena sp. PCC7120 that confers abiotic stress tolerance in E. coli.

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 × 106 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.

Molecular characterization of two novel proteins All1122 and Alr0750 of Anabaena PCC 7120 conferring tolerance to multiple abiotic stresses in Escherichia coli.

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/ß 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, CdCl2, 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.

Overexpression of phytochelatin synthase (pcs) enhances abiotic stress tolerance by altering the proteome of transformed Anabaena sp. PCC 7120.

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, CuCl2, carbofuran, and CdCl2. 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.

Comparative proteomics of wild type, An+ahpC and An∆ahpC strains of Anabaena sp. PCC7120 demonstrates AhpC mediated augmentation of photosynthesis, N2-fixation and modulation of regulatory network of antioxidative proteins.

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.

UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species.

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.

Exploring the membrane proteome of the diazotropic cyanobacterium Anabaena PCC7120 through gel-based proteomics and in silico approaches.

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.

Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

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.