Madhu B. Tyagi

Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

Inactivation of cyanobacterial nitrogenase after exposure to ultraviolet-B radiation.

Exposure of the N2-fixing cyanobacterium Anabaena BT2 to ultraviolet-B radiation (2.5 W m−2) for 30 min resulted in complete loss of nitrogenase activity but 100% cell killing occurred only after a 90-min exposure. Inactivation of nitrogenase activity was not specific to Anabaena BT2; other species also showed a similar effect. The time required for 100% killing and inactivation of nitrogenase activity differed in various species, and this difference may be ascribed to the presence of different levels of UV-B protection mechanisms in individual species. Inhibition of nitrogenase activity was immediate, since exposure of cultures to UV-B for as little as 5 min elicited some inhibition of activity. The activity of UV-B-inhibited nitrogenase did not recover upon transfer of exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment of inhibitors of protein synthesis and PS-II activity, it was demonstrated that restoration of nitrogenase activity in a UV-B-treated culture occurred by fresh synthesis of nitrogenase polypeptide. Our findings suggest that estimation of nitrogenase activity in diazotrophic species may be used as a marker enzyme for assessing the impact of UV-B radiation.

UVB Shielding Role of FeCI3 and Certain Cyanobacterial Pigments

The shielding role of ferric iron (FeCl3) and certain cyanobacterial pigments (a brown‐colored pigment from Scytonema hofmanii culture filtrate and a pink extract from Nostoc spongiaeforme) against UVB‐induced damage in the filamentous, nitrogen‐fixing cyanobacterium Nostoc muscorum has been demonstrated. Addition of these colored compounds to agarose gels (1–3 mm thick) resulted in a considerable decrease in UVB transmittance through the gels. The lowest UVB transmittance (15%) occurred through a 3 mm gel containing 0.01% FeCl3 followed by S. hofmanii culture filtrate (40%) and N. spongiaeforme extract (50%). These substances appear to act as very efficient UVB‐absorbing screens. Percent survival and 14CO2 uptake of N. muscorum increased significantly if UVB exposure was given on gels containing FeCl3 or other UVB‐shielding substances. The highest protection of N. muscorum was recorded with FeCl3, followed by S. hofmanii culture filtrate and N. spongiaeforme extract. Such UV‐shielding substances if present in required concentration range may enhance the survival of cyanobacteria exposed to high levels of UVB.

Synthesis of ZnO nanoparticles using the cell extract of the cyanobacterium, Anabaena strain L31 and its conjugation with UV-B absorbing compound shinorine.

In the present work, we describe a cheap, unexplored and simple procedure for the synthesis of zinc oxide nanoparticles (ZnONPs) using the cell extract of the cyanobacterium, Anabaena strain L31. An attempt was also made to conjugate synthesized ZnONPs with a UV-absorbing water soluble compound shinorine. UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infra-red (FTIR) spectroscopy, transmission electron microscopy (TEM) and TEM-selected area electron diffraction (SAED) analyses were made to elucidate the formation and characterization of ZnONPs and ZnONPs-shinorine conjugate. The synthesized ZnONPs were characterized by a sharp peak at 370 nm in UV-vis spectrum. TEM images showed the formation of spherical shaped nanoparticles with an average size of 80 nm. Results of selective area electron diffraction (SAED) pattern showed a set of rings which suggested uniform shape with hexagonal structure of ZnONPs. XRD spectra confirmed the crystalline structure of particles. Conjugation of ZnONPs with shinorine was successfully achieved at pH 7.0 and 10mM concentration of shinorine. The conjugate showed a zeta potential value of -3.75 mV as compared to +30.25 mV of ZnONPs. The change in zeta potential value of ZnONPs-shinorine conjugate was attributed to the changes in the surface functionalities after conjugation. The generation of in vivo reactive oxygen species (ROS) by Anabaena strain L31 with treatment of ZnONPs-shinorine conjugate showed approximately 75% less ROS generation as compared to ZnONPs. Properties exhibited by the ZnONPs-shinorine conjugate suggest that it may be used as a potential agent in developing environmental-friendly sunscreen filters of biological origin.

Hepatotoxicity of Microcystis aeruginosa Strains Growing as Blooms in Certain Eutrophic Ponds

Critical assessment of five eutrophicated ponds of Varanasi city (India) revealed the presence of heavy blooms of cyanobacteria consisting mainly of Microcystis aeruginosa. Crude aqueous extracts of blooms as well as laboratory grown M. aeruginosa isolated from three ponds, namely Lakshmikund, Durgakund and Adityanagar showed toxicity in mouse bioassay test. Crude aqueous extracts from these samples caused death of test mice within 1h of administration (i.p.) with a LD50 of 60 mg/kg body weight and the treated animals showed clinical signs of hepatotoxicity. However such an effect was not associated with the blooms from Laatbhairov and Surajkund ponds suggesting that not all strains of M. aeruginosa are toxic. Based on spectral properties (λmax 230 nm), and comparison with standard microcystin-LR, the toxin is tentatively identified as microcystin-LR. The purified toxin caused death of test mice within 40 min of its administration with a LD50 of 100 µg/ kg body weight and induced gross morphological and functional changes in liver. A 1.55 fold increase in liver weight accompanied by deep red coloration most probably due to hemorrhage and blood pooling suggested the hepatotoxic properties of the toxin. Hepatotoxicity was also evident from the drastic increase (up to 2.5 fold) in activity of serum enzymes such as glutamate pyruvate transaminase/alanine aminotransferase (GPT/ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (APase) following toxin treatment. 14 C-labelling experiments demonstrated maximum accumulation (∼15%) of 14 C- toxin after 20 min. of toxin administration. Appreciable level of toxin was also detected in water of four ponds. In conclusion these results clearly demonstrate that microcystinproducing blooms of M. aeruginosa are common in eutrophicated ponds of Varanasi city but not all ponds harbour toxic blooms.

Rhizosphere of rice plants harbor bacteria with multiple plant growth promoting features

114 diazotrophic bacteria from the rice rhizosphere of five districts of Eastern Uttar Pradesh (India) were isolated and screened for plant growth promoting (PGP) activities employing standard microbiological and biochemical techniques. All these isolates showed nitrogenase activity in the range of 0.23 to 1.72 μmol C 2 H 4 mg -1 protein h -1 . Further analysis showed that 84 (73.68%) isolates were Indole-3-acetic acid (IAA) producer; the value of IAA production ranged from 10.1 to 353.0 μg IAA mg -1 protein. IAA production occurred solely in the medium supplemented with tryptophan. P solubilization activity was observed in 28 (24.56%) isolates, the activity being in the range of 38.50 to 321.0 P released μg mg -1 protein. 45 (39.46%) isolates were capable of producing siderophore, the range of production being 4.50 to 223.26 μg mg -1 protein. Analysis of molecular diversity was made by amplified ribosomal DNA restriction analysis (ARDRA) and denaturating gradient gel electrophoresis (DGGE), which exhibited distinct differences among all the isolates. Of the 114 isolates, twenty one (21) isolates showed multiple plant growth promoting traits and were potent in terms of PGP activities. These isolates were identified on the basis of 16S rDNA sequencing and belonged to the genera Pantoea, Bacillus, Microbacterium, Pseudomonas, Sphingomonas, Ancylobacter, Enterobacter, Advenella, γ-proteobacterium strain VA3S1, Rhizobium and Agrobacterium . Findings of this study suggest that certain isolates may be exploited for developing a potential source of biofertilizer. Key words: Plant growth promoting rhizobacteria, N 2 fixation, amplified ribosomal DNA restriction analysis, indole-3-acetic acid, siderophore, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis

Induction and differential expression of certain novel proteins in Anabaena L31 under UV-B radiation stress

For examining how UV-B radiation alters the proteome of the N2-fixing cyanobacterium, Anabaena L31, we extracted proteins from cultures irradiated with UV-B + white light and controls (white light irradiated) and analyzed the proteins using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Twenty one proteins, including two hypothetical proteins (HPs) were identified and placed in eight functional categories. However several of the proteins were housekeeping proteins involved in key metabolic processes such as carbon, amino acid biosynthesis and energy metabolism, certain proteins seem to have a role in stress (antioxidative enzymes), translation, cellular processes and reductases. Two novel HPs (all3797 and all4050) were characterized in detail. These two were over-expressed after UV-B irradiation and characterized as FAS 1 (all3797) and PRC barrel-like (all4050) proteins. Bioinformatics analysis revealed that the genes of both the HPs have promoter regions as well as transcription binding sites in their upstream region (UTR). Promoters present in all3797 genes suggest their crucial role against UV-B and certain other abiotic stresses. To our knowledge these novel proteins have not been previously reported in any Anabaena strains subjected to UV-B stress. Although we have focused our study on a limited number of proteins, results obtained shed light on the highly complicated but poorly studied aspect of UV-B radiation-mediated changes in the proteome and expression of proteins in cyanobacteria.

Microbial Community Composition and Functions Through Metagenomics

Metagenomic approaches have provided a better understanding of microbial diversity and function across the terrestrial biome. Initial studies on soil metagenomics involved construction of libraries and sequencing of cloned genes to know the product encoded, but now a days direct sequence-based information plays an important role in functional profiling of environmental DNA. The rich information obtained from soil metagenome provides new insight into the taxonomic and functional diversity of soil microorganism. Some of the techniques of molecular biology research such as clone-based gene sequence analysis, molecular fingerprinting, next-generation sequencing, and many others have proved very useful in analyzing unknown environmental DNA sample and opened a flux gate of exciting research finding. Additionally, development of new environmental DNA isolation method as well as improved cloning systems has accelerated the pace of research. More importantly, metagenomic tools have resulted in discovery of several novel genes coding for protease, lipase, amylase, alcohol oxidoreductase, antibiotic resistance, etc., from ecological niches including meadows, crop fields, and others. With metagenomic approaches, new dimension in the characterization of complex microbial community has been attained. Surely, metagenomic approaches can be used to build a predictive understanding of how microbial diversity and function vary across terrestrial biome.