Poonam Bhargava

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture, mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.

Cadmium mitigates ultraviolet-B stress in Anabaena doliolum: Enzymatic and non-enzymatic antioxidants

Impact of ultraviolet-B (UV-B) and Cd, applied individually and in combination, measured in terms of oxygen-evolution, chlorophyll (Chl) and protein contents, lipid peroxidation, and enzymatic and non-enzymatic antioxidants of Anabaena doliolum, revealed a greater oxidative damage induced by UV-B than by Cd. While superoxide dismutase (SOD) showed a greater stimulation by UV-B than Cd, the activities of catalase (CAT) and glutathione reductase (GR) declined after UV-B treatment. Cd treatment, however, enhanced the activities of ascorbate peroxidase (APX) and GR. CAT activity increased at low but decreased at high dose of Cd. Increase in carotenoid (Car) content in UV-B treated cells suggested a shielding effect of Car against UV-B stress. A 15-and 10-fold rise in α-tocopherol (α-TOC) content at high dose of Cd and/or UV-B offered testimony to the antioxidant role of α-TOC.

Molecular characterization and the effect of salinity on cyanobacterial diversity in the rice fields of Eastern Uttar Pradesh, India

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

Differential response of antioxidative defense system of Anabaena doliolum under arsenite and arsenate stress.

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

Metagenomes from the Saline Desert of Kutch

ABSTRACT We provide the first report on the metagenomic approach for unveiling the microbial diversity in the saline desert of Kutch. High-throughput metagenomic sequencing of environmental DNA isolated from soil collected from seven locations in Kutch was performed on an Ion Torrent platform.

Proteomic evaluation of the non-survival of Anabaena doliolum (Cyanophyta) at elevated temperatures

This paper presents proteomic evidence for the non-survival of Anabaena doliolum at elevated temperatures (43°C, 48°C, 53°C and 58°C), when photosystem II (PSII), carbon fixation, ATP and NADPH contents were significantly decreased. A total of 215, 200, 261, 229 and 99 spots were clearly visible in two-dimensional gel electrophoresis (2DE) gels of the cyanobacterium from the control and those subjected to 1 h treatment at the above temperatures, respectively. Proteomic analysis using 2DE, MALDI-TOF MS/MS and reverse transcription polymerase chain reaction of Anabaena exposed to the above temperatures displayed significant and reproducible alterations in 13 proteins homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1, 6-bisphosphate aldolase (FBA), fructose-1, 6-bisphosphatase (FBPase), keto-acid reductoisomerase, phycocyanin-α-chain, peroxiredoxin, ATP synthase-β-chain, RNA binding protein, nucleoside diphosphate kinase (NDPK), GroES, phycoerythrocyanin-α-chain, AhpC/TSA family and phycobilisome rod-core linker (PBS-RCL) protein. Except Prx, GroES and ATP synthase-β-chain, other metabolic and oxidative stress proteins were down-regulated at temperatures over 48°C. The down-regulation of FBA, GAPDH, and FBPase beyond 48°C possibly disrupted glycolysis, the pentose phosphate pathway and the Calvin cycle, thereby leading to the death of Anabaena above 48°C. Notwithstanding the above, an appreciable down-regulation of NDPK (involved in protein phosphorylation) and AhpC/TSA (specific scavenger of reactive sulphur species), may lead to loss of signalling and accumulation of reactive sulphur species, respectively, which contribute further to temperature stress. Appreciable induction of heat-shock proteins (HSPs), required for maintenance of metabolic proteins during thermal stress, as well as metabolic proteins up to 48°C and their down-regulation at 53°C and 58°C suggested a close interlinking of these proteins. However, down-regulation of HSP60, HSP26 and HSP 16.6 appears to be responsible for cell death.

Metagenomic Approach for Understanding Microbial Population from Petroleum Muck

ABSTRACT Petroleum products play a major role in fueling the economy of the world but the pollution they create has become a critical issue. Understanding the diversity present in pipeline muck will help with the exploration of new microbial strains with better hydrocarbon degrading capacities for bioremediation of polluted sites. This study provides an analysis of petroleum muck using next generation sequencing.

Copper pretreatment augments ultraviolet B toxicity in the cyanobacterium Anabaena doliolum: a proteomic analysis of cell death

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.

A single gene all3940 (Dps) overexpression in Anabaena sp. PCC 7120 confers multiple abiotic stress tolerance via proteomic alterations

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

Molecular survey of basidiomycetes and divergence time estimation: An Indian perspective

This study outlines the biodiversity of mushrooms of India. It reveals the molecular biodiversity and divergence time estimation of basidiomycetes from Gujarat, India. A total of 267 mushrooms were collected from 10 locations across the state. 225 ITS sequences were generated belonging to 105 species, 59 genera and 29 families. Phylogenetic analysis of Agaricaceae reveals monophyletic clade of Podaxis differentiating it from Coprinus. Further, the ancient nature of Podaxis supports the hypothesis that gasteroid forms evolved from secotioid forms. Members of Polyporaceae appeared polyphyletic. Further, our results of a close phylogenetic relationship between Trametes and Lenziteslead us to propose that the genera Trametes may by enlarged to include Lenzites. The tricholomatoid clade shows a clear demarcation for Entolomataceae. However, Lyophyllaceae and Tricholomataceae could not be distinguished clearly. Distribution studies of the mushrooms showed omnipresence of Ganoderma and Schizophyllum. Further, divergence time estimation shows that Dacrymycetes evolved in the Neoproterozoic Era and Hymenochaetales diverged from Agaricomycetes during the Silurian period.