Alok Kumar Shrivastava

Comparative proteomics unveils cross species variations in Anabaena under salt stress

UNLABELLED 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.

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.

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.

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.

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 (

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses.

HAsO_4^{- 2 }

Arsenic and cadmium are inhibitors of cyanobacterial dinitrogenase reductase (nifH1) gene

) 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.

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

This paper provides the first proteomic evidence of arsenic (As) tolerance and interactive regulatory network between primary and secondary metabolism in the medicinal plant, Artemisia annua. While chlorophyll fluorescence and photosynthetic rate depicted mild inhibition, there was a significant enhancement in PSI activity, whole chain, ATP, and NADPH contents in 100 μM As treatments compared to the control plants. However, a decrease in the above variables was recorded under 150 μM treatments. Proteomic decoding of the survival strategy of A. annua under As stress using 2-DE followed by MALDI-MS/MS revealed a total of 46 differentially expressed protein spots. In contrast to other plants where As inhibits photosynthesis, A. annua showed appreciable photosynthetic CO2 assimilation and allocation of carbon resources at 100 μM As concentration. While an increased accumulation of ATP synthase, ferredoxin-NADP(H) oxidoreductase, and FeS-rieske proteins supported the operation of cyclic electron transport, mdr ABC transporter protein and pcs gene might be involved in As detoxification. The most interesting observation was an increased accumulation of LEAFY like novel protein conceivably responsible for an early onset of flowering in A. annua under As stress. This study not only affirmed the role of energy metabolism proteins but also identified potential candidates responsible for As tolerance in plants.