Shilpi Singh

Comparative proteomics reveals association of early accumulated proteins in conferring butachlor tolerance in three N2-fixing Anabaena spp.

UNLABELLED 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.

Effect of age on the isozymes of lactic dehydrogenase of the heart and the brain of rat

Abstract Lactic dehydrogenase (LDH) has five isozymes, H4, H3M1, H2M2, H1M3 and M4, which differ from each other in their kinetic properties ( Dawson et al., 1964 ). These workers have shown that the turn over number of the chicken M4-LDH for the forward reaction, pyruvate → lactate, is higher than that of H4-LDH and the isozyme composition of the LDH of the skeletal muscle of the rabbit changes during its development. Brody (1965) has shown that the LDH pattern of the skeletal muscle of adult rat changes after denervation of the muscle. This shows that the isozymes of LDH are subject to alterations with the change in the environment of the tissue. We show here that the isozyme patterns of LDH of the brain and the heart of rat change in a manner that may decrease the capacity of these tissues to tolerate anaerobic condition with increasing age.

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.

Effect of standardized extract of Bacopa monnieri (CDRI-08) on testicular functions in adult male mice.

ETHNOPHARMACOLOGICAL RELEVANCE Bacopa monnieri (BM) has been used in India since the time of Rig-Veda for augmentation of learning, memory, brain health etc. AIM OF THE STUDY The memory augmenting effect of BM is well documented. CDRI-08 is a standardized extract of Bacopa monnieri, but its effect on the male reproductive health has not been investigated. Therefore, the aim of the present study was to examine the effect of CDRI-08 administration on the male reproductive organs with special emphasis on testis in adult mice. MATERIALS AND METHODS CDRI-08, containing at least 55% bacosides (the major constituent of BM), was investigated for its effect on testicular functions in adult Parkes (P) mice. A suspension of CDRI-08 was orally administered in doses of 40 and 80mgkg-1 body weight day-1 for 28 days and various male reproductive end points were evaluated. RESULTS Compared to control, CDRI-08 treatment caused a significant increase (p<0.05) in spermatogenic cell density (germinal epithelial height: control, 55.03±4.22 vs 40mg, 67.15±2.65 and 80mg, 69.93±3.76; and tubular diameter: control, 206.55±2.62 vs 80mg, 253.23±12.19), PCNA index (control, 59.85±2.09 vs 40mg, 82.17±1.56 and 80mg, 84.05±3.51) and in steroidogenic indices in the testis, and in sperm viability (control, 0.67±0.010 vs 80mg, 0.80±0.04) in cauda epididymidis of the treated mice. On the other hand, however, the same treatment caused a significant decrease (p<0.05) in abnormal sperm morphology (control, 21.72±1.06 vs 40mg, 10.63±1.50 and 80mg, 15.86±0.87) in cauda epididymidis, and in lipid peroxidation level in testis of the treated mice compared to controls. CONCLUSION The results suggest that treatment with CDRI-08 extract improves sperm quality, and spermatogenic cell density and steroidogenic indices in the testis of P mice.

In silico characterization and transcriptomic analysis of nif family genes from Anabaena sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.

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

The enzyme nitrogenase complex is a key component conferring nitrogen fixation in all known diazotrophs. This study for the first time examines the impact of As, Na, Cd, Cu and butachlor on component II (dinitrogenase reductase, nifH1) of nitrogenase from diazotrophic cyanobacterium Anabaena sp. PCC7120 using in silico and wet lab approaches. The nifH1 of Anabaena is a glycine-rich stable protein having DNA-binding properties and shows close similarity with free living compared with symbiotic diazotrophs. Phylogenetic tree revealed an adverse effect of the selected stresses on close homologs across the diazotroph community. The protein interaction network demonstrated the presence of nirA, glnA, glnB, alr4255 and alr2485 proteins besides nif proteins, suggesting their involvement in nitrogen fixation along with nifH1. Homology modelling and docking under As, Na, Cd, Cu and butachlor revealed an interaction between stressors and nifH1 protein which was further validated by a transcript of the gene through quantitative real-time PCR (qRT-PCR). Presence of binding sites for As, Na, Cd and Cu on oxyR promoter attested their adverse affects on nifH1. Maximum down-regulation of nifH1 in Cd and As followed by salt, copper and butachlor revealed that arsenic and cadmium were most potential inhibitors of nitrogenase of diazotrophic community, which might negatively affect crop yield.

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.

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.

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.