Browsing by Author "Yogesh Mishra"

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A comparative study of antioxidative defense system in the copper and temperature acclimated strains of Anabaena doliolum
(2008) Yogesh Mishra; Poonam Bhargava; Riti Thapar; Ashish Kumar Srivastava; Lal Chand Rai
This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu2+ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu2+ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu2+ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu2+ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu2+ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu2+ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu2+ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu2+ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu2+ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu2+ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu2+ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu2+ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM). © 2008 Springer Science+Business Media B.V.
Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7210
(Nature Publishing Group, 2017) Yogesh Mishra; Michael Hall; Roland Locmelis; Kwangho Nam; Christopher A. G. Söderberg; Patrik Storm; Neha Chaurasia; Lal Chand Rai; Stefan Jansson; Wolfgang P. Schröder; Uwe H. Sauer
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 (CP) and, if present, the resolving Cys (CR). 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. © 2017 The Author(s).
Acute cadmium toxicity and post-stress recovery: Insights into coordinated and integrated response/recovery strategies of Anabaena sp. PCC 7120
(Elsevier B.V., 2021) Akanksha Srivastava; Subhankar Biswas; Sandhya Yadav; Sanjiv Kumar; Vaibhav Srivastava; Yogesh Mishra
Cyanobacteria, the first photoautotrophs have remarkable adaptive capabilities against most abiotic stresses, including Cd. A model cyanobacterium, Anabaena sp. PCC 7120 has been commonly used to understand cyanobacterial plasticity under different environmental stresses. However, very few studies have focused on the acute Cd toxicity. In this context, Anabaena was subjected to 100 μM Cd for 48 h (acute Cd stress, ACdS) and then transferred into the fresh medium for post-stress recovery (PSR). We further investigated the dynamics of morpho-ultrastructure, physiology, cytosolic proteome, thylakoidal complexes, chelators, and transporters after ACdS, as well as during early (ER), mid (MR), and late (LR) phases of PSR. The findings revealed that ACdS induced intracellular Cd accumulation and ROS production, altered morpho-ultrastructure, reduced photosynthetic pigments, and affected the structural organization of PSII, which subsequently hindered photosynthetic efficiency. Anabaena responded to ACdS and recovered during PSR by reprogramming the expression pattern of proteins/genes involved in cellular defense and repair; CO2 access, Calvin-Benson cycle, glycolysis, and pentose phosphate pathway; protein biosynthesis, folding, and degradation; regulatory functions; PSI-based cyclic electron flow; Cd chelation; and efflux. These modulations occurred in an integrated and coordinated manner that facilitated Anabaena to detoxify Cd and repair ACdS-induced cellular damage. © 2021 Elsevier B.V.
AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses
(2009) Yogesh Mishra; Neha Chaurasia; Lal Chand Rai
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 °C), NaCl (6% w/v), carbofuron (0.025 mg ml-1), CdCl2 (4 mM), CuCl2 (1 mM), and UV-B (10 min) 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. © 2009 Elsevier Inc. All rights reserved.
Author Correction: Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7120 (Scientific Reports (2017) DOI: 10.1038/s41598-017-17044-3)
(Nature Publishing Group, 2018) Yogesh Mishra; Michael Hall; Roland Locmelis; Kwangho Nam; Christopher A. G. Söderberg; Patrik Storm; Neha Chaurasia; Lal Chand Rai; Stefan Jansson; Wolfgang P. Schröder; Uwe H. Sauer
The original version of this Article contained an error in the title of the paper, where "Anabaena sp. PCC 7120" was incorrectly given as "Anabaena sp. PCC 7210". This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file. © 2018 The Author(s).
Biochemical and proteomic analysis reveals oxidative stress tolerance strategies of Scenedesmus abundans against allelochemicals released by Microcystis aeruginosa
(Elsevier B.V., 2019) Simranjeet Kaur; Akanksha Srivastava; Sanjiv Kumar; Vaibhav Srivastava; Amrik Singh Ahluwalia; Yogesh Mishra
We studied the possible survival strategies of a green alga, Scenedesmus abundans, against allelochemicals secreted by Microcystis aeruginosa. We exposed the monoculture of S. abundans to a cell free-filtrate (allelochemicals)of M. aeruginosa at the start of our experiment and measured the growth behaviour, morphological changes and oxidative stress markers. The results suggest that exposure to allelochemicals induced oxidative stress in S. abundans, which had significantly reduced the growth of green alga with certain morphological changes. However, after seven days, S. abundans found ways to reduce oxidative stress by recovering its morphology and growth close to that of control. To understand possible survival strategies of test alga, we measured biochemical as well as protein level changes in S. abundans. Biochemical response of the green alga clearly showed that as a response to allelochemicals, enzymatic and non-enzymatic antioxidants were induced. Proteomic analysis showed that exposure to allelochemicals induced accumulation of 13 proteins on the 2-DE gel of S. abundans, which falls in three functional categories, i.e., (i)energy metabolism (photosynthesis, carbon fixation and respiration), (ii)ROS scavenging enzymes and molecular chaperones, and (iii)amino acid and protein biosynthesis. After chronic oxidative stress, these proteins presumably retained glycolysis, pentose phosphate pathway and turnover rate of the Calvin-Benson cycle. Moreover, these proteins assisted in the adequate detoxification of ROS and played an important role in the damage removal and repair of oxidized proteins, lipids and nucleic acids. Therefore, our study anticipates that S. abundans embraces biochemical and proteomic reprogramming to thrives against allelochemicals released by M. aeruginosa. © 2019
Bryophytes as Modern Model Plants: An Overview of Their Development, Contributions, and Future Prospects
(Springer, 2023) Sandhya Yadav; Suvajit Basu; Akanksha Srivastava; Subhankar Biswas; Raju Mondal; Vishal Kumar Jha; Sushil Kumar Singh; Yogesh Mishra
Model organisms are commonly employed in research as convenient tools for studying diverse biological processes. Plant research relied on several non-model plants until the Arabidopsis thaliana was developed as powerful model for identifying genes and determining their functions. To study the genetics of unique processes in different species, few other model photosynthetic organisms have recently been established, including Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, Chlamydomonas reinhardatii, Oryza sativa, Zea mays, Triticum dicoccoides, Populus trichocarpa, and Picea abies. However, when it comes to answering different biological problems, each of the current model plants has its own set of advantages and disadvantages, and many questions about land plant adaptation strategies at the level of morpho-physiology, development, and stress mitigation could not be adequately answered using these models. Furthermore, the high occurrence of embryo lethal mutations rendered studying the molecular basis of 3-dimensional (3-D) growth and gametogenesis unfeasible. Since bryophytes have a low cellular complexity and a dominant haploid gametophytic phase, they could be useful models not only for avoiding the aforementioned drawbacks, but also for functional genomics research and understanding the chronology of land plant evolution. These distinguishing characteristics and the advancement of sequencing technology have led to the development of some bryophytes as modern model plants, including Physcomitrium patens, Marchantia polymorpha, Anthoceros agrestis. Here, we review at how bryophytes became model plants, and how they have been able to answer crucial plant biology-related concerns like stress tolerance and evolutionary developmental (evo-devo) biology that other model plants have not been able to. © 2023, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
Cd-induced cytosolic proteome changes in the cyanobacterium Anabaena sp. PCC7120 are mediated by LexA as one of the regulatory proteins
(Elsevier B.V., 2023) Akanksha Srivastava; Arvind Kumar; Subhankar Biswas; Vaibhav Srivastava; Hema Rajaram; Yogesh Mishra
LexA, a well-characterized transcriptional repressor of SOS genes in heterotrophic bacteria, has been shown to regulate diverse genes in cyanobacteria. An earlier study showed that LexA overexpression in a cyanobacterium, Anabaena sp. PCC7120 reduces its tolerance to Cd stress. This was later shown to be due to modulation of photosynthetic redox poising by LexA under Cd stress. However, due to the global regulatory nature of LexA and the prior prediction of AnLexA-box in a few heavy metal-responsive genes, we speculated that LexA has a broad role in Cd tolerance, with regulation over a variety of Cd stress-responsive genes in addition to photosynthetic genes. Thus, to further expand the knowledge on the regulatory role of LexA in Cd stress tolerance, a cytosolic proteome profiling of Anabaena constitutively overexpressing LexA upon Cd stress was performed. The proteomic study revealed 25 differentially accumulated proteins (DAPs) in response to the combined effect of LexA overexpression and Cd stress, and the other 11 DAPs exclusively in response to either LexA overexpression or Cd stress. The 36 identified proteins were related with a variety of functions, including photosynthesis, C-metabolism, antioxidants, protein turnover, post-transcriptional modifications, and a few unknown and hypothetical proteins. The regulation of LexA on corresponding genes, and six previously reported Cd efflux transporters, was further validated by the presence of AnLexA-boxes, transcript, and/or promoter analyses. In a nutshell, this study identifies the regulation of Anabaena LexA on several Cd stress-responsive genes of various functions, hence expanding the regulatory role of LexA under Cd stress. © 2023 Elsevier B.V.
Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli
(2008) Neha Chaurasia; Yogesh Mishra; Lal Chand Rai
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 °C), NaCl (6% w/v), carbofuron (0.025 mg ml-1), CdCl2 (4 mM), CuCl2 (1 mM), 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. © 2008 Elsevier Inc. All rights reserved.
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
(Elsevier, 2016) Alok K. Shrivastava; Sarita Pandey; Shivam Yadav; Yogesh Mishra; Prashant K. Singh; Ruchi Rai; Shilpi Singh; Snigdha Rai; L.C. Rai
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 (mutantAn∆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. © 2016 Elsevier B.V.
Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts
(BioMed Central Ltd., 2020) Sandhya Yadav; Akanksha Srivastava; Subhankar Biswas; Neha Chaurasia; Sushil Kumar Singh; Sanjiv Kumar; Vaibhav Srivastava; Yogesh Mishra
Objective: Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results: We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics. © 2020 The Author(s).
Copper pretreatment augments ultraviolet B toxicity in the cyanobacterium Anabaena doliolum: A proteomic analysis of cell death
(2008) Poonam Bhargava; Arvind Kumar; Yogesh Mishra; Lal Chand Rai
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, 14C 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. © CSIRO 2008.
Development of Bryophytes as a New Model System to Understand the Phenomenon of Terrestrialization with Environmental Changes
(Springer Nature, 2019) Sandhya Yadav; Subhankar Biswas; Akanksha Srivastava; Yogesh Mishra
Bryophytes are earliest diverging lineages of the extant land plants with around 25,000 species distributed all over the world. Bryophytes can be further classified into three main classes viz. Liverworts, Hornworts and Mosses that grow on a wide range of habitats. Bryophytes, with high ecological and economic values, occupy a very important position in the evolution of terrestrial plants. During the transition of aquatic to terrestrial habitat (terrestrialization), bryophytes got exposed to global climate changes as well as dehydrating atmosphere of terrestrial habitats that led to the desiccation of plant tissues. In order to tolerate the environmental alterations and to protect themselves from abiotic stresses, bryophytes must have enabled themselves to develop certain adaptive strategies. In order to understand these adaptive strategies at molecular level, attempts have been made to develop certain bryophytes as new model system such as Physcomitrella patens and Marchantia polymorpha. In the current chapter we will addresses how does these model systems have been used to address the uniqueness of bryophytes in terms of their capabilities behind the conquering the land i.e. terrestrialization. © 2019, Springer Nature Singapore Pte Ltd.
Differential induction of enzymes and antioxidants of the antioxidative defense system in Anabaena doliolum exposed to heat stress
(2005) Yogesh Mishra; Poonam Bhargava; Lal Chand Rai
Anabaena doliolum subjected to 43, 48, 53 and 58°C temperature for 1, 2, 3 and 4 h, showed temperature and time-dependent increase in H 2O2 production and MDA contents. All the measured enzymes of the antioxidative defense system (SOD, CAT, APX and GR) showed increase in their activities at 43°C after 1 h of treatment, but at higher temperature their activity declined. The content of antioxidants (ASC, GSH, and α-TOC) increased significantly with rise in temperature as well as duration of treatment. This study clearly demonstrates that when enzymatic defense system becomes inactive, the antioxidants (GSH, and α-TOC) are induced to protect the cyanobacterium from heat stress. One of the major roles of these antioxidants appears to be the protection of PSII as reflected by an effect on O2 evolution up to 53°C. © 2005 Elsevier Ltd. All rights reserved.
Effect of pretreatment of salt, copper and temperature on ultraviolet-B-induced antioxidants in diazotrophic cyanobacterium Anabaena doliolum
(2006) Ashish Kumar Srivastava; Poonam Bhargava; Yogesh Mishra; Bideh Shukla; Lal Chand Rai
Effect of salt, copper, and temperature pretreatments on the UV-B-induced oxidative damage, measured in terms of peroxide and MDA (lipid peroxidation) contents, was studied in the diazotrophic cyanobacterium Anabaena doliolum. To understand the survival strategy enzymatic (superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase) and non-enzymatic (glutathione, ascorbate, α-tocopherol and carotenoid) antioxidants were studied. Among the various pretreatments salt was found to decrease and copper and temperature pretreatments increased the deleterious effects of UV-B. This study is the first to demonstrate that physical stress (high temperature) enhanced the damaging effect of UV-B more profoundly than chemical stresses (salt and copper). © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.
Evolution of Genetically Modified (GM) Crops and The Scared World
(Elsevier, 2020) Subhankar Biswas; Akanksha Srivastava; Sandhya Yadav; Yogesh Mishra
Since antiquity, it was a key desire of human beings to develop new crops with higher nutritional values and good flavors. The classical plant breeding approaches has given many improved varieties of crops to the world but it has its own limitations, such as (1) genetic exchange can be done in the same species (2) while crossing, many traits are transferred along with the trait/s of interest that might have undesirable effects on yield potential. To overcome these limitations there was an emergence of new technology that is genetic engineering which has developed transgenic plants which are popularly known as genetically modified (GM) plants. First genetic engineering mediated transgenic tobacco plant was developed in 1987. After that, there was a boom in the development of GM crops such as “flavr-savr” tomato, Bt-cotton, corn, etc. In the journey of the past 3 decades, GM crops faced several vicissitudes but never stopped its improvement. The case-by-case study has demonstrated that the technique was never the problem but the consequences may be. In this chapter, we have briefly highlighted the necessity, current status and global impact of genetically modified plants. Apart from these, we have also addressed the question that why a large part of the human population is still scared of GM foods?. © 2021 Elsevier Inc. All rights reserved.
Excess copper induces anoxygenic photosynthesis in Anabaena doliolum: A homology based proteomic assessment of its survival strategy
(2008) Poonam Bhargava; Yogesh Mishra; Ashish Kumar Srivastava; Om Prakash Narayan; Lal Chand Rai
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 O2-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 O2-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 α-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 α-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. © 2007 Springer Science+Business Media B.V.
Exploring the membrane proteome of the diazotropic cyanobacterium Anabaena PCC7120 through gel-based proteomics and in silico approaches
(Elsevier, 2015) Sonia Sen; Chhavi Agrawal; Yogesh Mishra; Shweta Rai; Antra Chatterjee; Shivam Yadav; Shilpi Singh; L.C. Rai
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. © 2015 Elsevier B.V.
Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 7120
(2011) Yogesh Mishra; Michael Hall; Neha Chaurasia; Lal Chand Rai; Stefan Jansson; Wolfgang P. Schröder; Uwe H. Sauer
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 P212121, 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.
Gamma (γ)-radiation stress response of the cyanobacterium Anabaena sp. PCC7120: Regulatory role of LexA and photophysiological changes
(Elsevier Ireland Ltd, 2023) Akanksha Srivastava; Arvind Kumar; Subhankar Biswas; Rajender Kumar; Vaibhav Srivastava; Hema Rajaram; Yogesh Mishra
High radioresistance of the cyanobacterium, Anabaena sp. PCC7120 has been attributed to efficient DNA repair, protein recycling, and oxidative stress management. However, the regulatory network involved in these batteries of responses remains unexplored. In the present study, the role of a global regulator, LexA in modulating gamma (γ)-radiation stress response of Anabaena was investigated. Comparison of the cytosolic proteome profiles upon γ-radiation in recombinant Anabaena strains, AnpAM (vector-control) and AnlexA+ (LexA-overexpressing), revealed 41 differentially accumulated proteins, corresponding to 29 distinct proteins. LexA was found to be involved in the regulation of 27 of the corresponding genes based on the presence of AnLexA-Box, EMSA, and/or qRT-PCR studies. The majority of the regulated genes were found to be involved in C-assimilation either through photosynthesis or C-catabolism and oxidative stress alleviation. Photosynthesis, measured in terms of PSII photophysiological parameters and thylakoid membrane proteome was found to be affected by γ-radiation in both AnpAM and AnlexA+ cells, with LexA affecting them even under control growth conditions. Thus, LexA functioned as one of the transcriptional regulators involved in modulating γ-radiation stress response in Anabaena. This study could pave the way for a deeper understanding of the regulation of γ-radiation-responsive genes in cyanobacteria at large. © 2022 Elsevier B.V.