Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase.

BACKGROUND: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. METHODS: C-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. RESULTS: Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. CONCLUSION: DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. GENERAL SIGNIFICANCE: Although, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.

DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans.

The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine-specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypomethylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry. As compared to wild type D. radiodurans cells, at least 54 proteins were differentially expressed in Δdam mutant. Among these, 39 metabolic enzymes were differentially expressed in Δdam mutant. The most prominent change was DNA adenine hypomethylation induced de-repression of pyruvate dehydrogenase complex, E1 component (aceE) gene resulting in 10 fold increase in the abundance of corresponding protein. The observed differential expression profile of metabolic enzymes included increased abundance of enzymes involved in fatty acid and amino acid degradation to replenish acetyl Co-A and TCA cycle intermediates and diversion of phosphoenolpyruvate and pyruvate into amino acid biosynthesis, a metabolic rewiring attempt by Δdam mutant to restore energy generation via glycolysis-TCA cycle axis. This is the first report of DNA adenine hypomethylation mediated rewiring of metabolic pathways in prokaryotes.

Synthesis and extracellular accumulation of silver nanoparticles by employing radiation-resistant Deinococcus radiodurans, their characterization, and determination of bioactivity.

There has been rapid progress in exploring microorganisms for green synthesis of nanoparticles since microbes show extraordinary diversity in terms of species richness and niche localization. Microorganisms are easy to culture using relatively inexpensive and simple nutrients under varied conditions of temperature, pressure, pH, etc. In this work, Deinococcus radiodurans that possesses the ability to withstand extremely high radiation and desiccation stress has been employed for the synthesis of silver nanoparticles (AgNPs). D. radiodurans was able to accumulate AgNPs in medium under various conditions, and process optimization was carried out with respect to time, temperature, pH, and concentration of silver salt. AgNPs were characterized using UV/vis spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy. The microbially synthesized AgNPs exhibited good antimicrobial activity against both Gram-negative and Gram-positive organisms and anti-biofouling activity. Their ability to inhibit growth and proliferation of cancer cell line was also examined, and it could be seen that AgNPs synthesized using D. radiodurans exhibited excellent anticancer activity.

Bio/chemoinformatics in India: an outlook.

With the advent of significant establishment and development of Internet facilities and computational infrastructure, an overview on bio/chemoinformatics is presented along with its multidisciplinary facts, promises and challenges. The Government of India has paved the way for more profound research in biological field with the use of computational facilities and schemes/projects to collaborate with scientists from different disciplines. Simultaneously, the growth of available biomedical data has provided fresh insight into the nature of redundant and compensatory data. Today, bioinformatics research in India is characterized by a powerful grid computing systems, great variety of biological questions addressed and the close collaborations between scientists and clinicians, with a full spectrum of focuses ranging from database building and methods development to biological discoveries. In fact, this outlook provides a resourceful platform highlighting the funding agencies, institutes and industries working in this direction, which would certainly be of great help to students seeking their career in bioinformatics. Thus, in short, this review highlights the current bio/chemoinformatics trend, educations, status, diverse applicability and demands for further development.

Comparative study of Plasmodium falciparum erythrocyte membrane protein 1-DBLα domain variants with respect to antigenic variations and docking interaction analysis with glycosaminoglycans.

The variant surface antigen PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) encoded by the polymorphic multi-copy var gene family plays an important role in parasite biology and the host-parasite interactions. Sequestration and antigenic variation is an essential component in the survival and pathogenesis of Plasmodium falciparum and contributes to chronic infection. The DBLα domain of PfEMP1 is a potential target for immuno-epidemiological studies and has been visualized as a vaccine candidate against severe malaria. Specific host receptors like heparin, heparan sulphate, blood group A and complement receptor 1 have been reported to bind the DBLα domain. Although heparin has been experimentally shown to disrupt the parasite-host interaction and effectively disrupt rosetting, the binding sites for the DBLα domain and the mechanism behind heparin-mediated rosette inhibition have not been elucidated. In this study, 3D structures and epitopes of the DBLα domain in 3D7 and in two Indian isolates have been predicted and compared. We have carried out docking studies on DBLα domains with human GAG receptors (heparin and heparan sulphate) to predict the strength of association between the protein-ligand interactions. The DBLα domain structures showed extensive diversity and polymorphism in their binding sites. The docking results indicate that heparin binds more effectively with high affinity as compared to heparan sulphate with some common interacting residues. These common residues can play an important role in rosetting and will aid in the designing of inhibitors specific to the interactions between DBLα and heparin or heparan sulphate would be important in malaria treatment. Thus it may lead to the development of novel interference strategies to block red blood cell invasion and provide protection against malaria.

Serratia odorifera mediated enhancement in susceptibility of Aedes aegypti for chikungunya virus.

BACKGROUND & OBJECTIVES: The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito. METHODS: Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10 th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis. RESULTS: The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV. INTERPRETATION & CONCLUSIONS: The results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane.

Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus.

Mosquito midgut plays a crucial role in its vector susceptibility and pathogen interaction. Identification of the sustainable microflora of the midgut environment can therefore help in evaluating its contribution in mosquito-pathogen interaction and in turn vector competence. To understand the bacterial diversity in the midgut of Aedes aegypti mosquitoes, we conducted a screening study of the gut microbes of these mosquitoes which were either collected from fields or reared in the laboratory "culture-dependent" approach. This work demonstrated that the microbial flora of larvae and adult Ae. aegypti midgut is complex and is dominated by gram negative proteobacteria. Serratia odorifera was found to be stably associated in the midguts of field collected and laboratory reared larvae and adult females. The potential influence of this sustainable gut microbe on DENV-2 susceptibility of this vector was evaluated by co-feeding S. odorifera with DENV-2 to adult Ae. aegypti females (free of gut flora). The observations revealed that the viral susceptibility of these Aedes females enhanced significantly as compared to solely dengue-2 fed and another gut inhabitant, Microbacterium oxydans co-fed females. Based on the results of this study we proposed that the enhancement in the DENV-2 susceptibility of Ae. aegypti females was due to blocking of prohibitin molecule present on the midgut surface of these females by the polypeptide of gut inhabitant S. odorifera.

Characterization of the role of the RadS/RadR two-component system in the radiation resistance of Deinococcus radiodurans.

Deinococcus radiodurans shows extraordinary tolerance to DNA damage, and exhibits differential gene expression and protein recycling. A putative response regulator, the DRB0091 (RadR) ORF, was identified from a pool of DNA-binding proteins induced in response to gamma radiation in this bacterium. radR is located upstream of drB0090, which encodes a putative sensor histidine kinase (RadS) on the megaplasmid. Deletion of these genes both individually and together resulted in hypersensitivity to DNA-damaging agents and a delayed or altered double-strand break repair. A ΔradRradS double mutant and a ΔradR single mutant showed nearly identical responses to gamma radiation and UVC. Wild-type RadR and RadS complemented the corresponding mutant strains, but also exhibited significant cross-complementation, albeit at lower doses of gamma radiation. The radS transcript was not detected in the ΔradR mutant, suggesting the existence of a radRS operon. Recombinant RadS was autophosphorylated and could catalyse the transfer of γ phosphate from ATP to RadR in vitro. These results indicated the functional interaction of RadS and RadR, and suggested a role for the RadS/RadR two-component system in the radiation resistance of this bacterium.

Morphometric and allozyme variation in Culex tritaeniorhynchus mosquito populations from India.

Four populations of Culex tritaeniorhynchus (Giles) (Diptera: Culicidae), collected from Bellary, Cuddalore, Pune, and the Microbial Containment Complex laboratory culture in India were analyzed for morphological and allozyme variation. Multivariate analysis based on eight morphological characteristics and three morphometric indices was used to investigate the morphological variations among the four populations. Principal component analysis of the data suggested that siphon, saddle, and anal gills related variables were most important. Discriminant factor analysis of morphological data revealed that the four populations form significantly different clusters which can be differentiated from each other based on siphon, saddle, and pectin teeth related variables. Allozyme electrophoresis of the four populations revealed that the mean heterozygosity per locus value had high variation, ranging from 0.0879 to 1.794. Fst values between 0 and 0.519 suggested genetic differentiation within these populations. Fis values ranged from 0 to 1 with most of the values closer to 1. The allelic frequencies and Nei's genetic identity values showed that genetic differences between populations were small, but significant. Some of the morphological and allozyme variations in the Cx. tritaeniorhynchus populations could be partly attributed to the environmental conditions. The findings suggested that transition of morphological characters and allozyme variations in Cx. tritaeniorhynchus populations seem to be consequences of influence and selection by the environmental conditions. These results indicated that populations of Cx. tritaeniorhynchus in non-endemic areas of Japanese encephalitis (JE) virus infection have higher adaptability as compared to endemic areas of JE infection.

Dengue-2-virus-interacting polypeptides involved in mosquito cell infection.

For the design of effective antiviral strategies, understanding the fundamental steps of the virus life cycle, including virus-host interactions, is essential. We performed a virus overlay protein binding assay followed by proteomics for identification of proteins from membrane fractions of A7 (Aedes aegypti) cells, C6/36 (Aedes albopictus) cells and the midgut brush border membrane fraction of Ae. aegypti mosquito that bind to dengue-2 virus. Actin, ATP synthase ß subunit, HSc 70, orisis, prohibitin, tubulin ß chain, and vav-1 were identified as dengue-2-virus-binding proteins. Our results suggest that dengue-2 virus exploits an array of housekeeping proteins for its entry in mosquito cells.

Prediction of B cell and T cell epitopes of DBLalpha domain in Plasmodium falciparum malaria vaccine candidate var gene.

P. falciparum encodes PfEMP-1 which is important in pathogenicity and virulence. We have analyzed the structure of the Duffy binding like (DBLalpha) domain of var genes and predicted the antigenic sites and T and B cell epitopes which present a highly variable picture with major implications in immune interactions and vaccine design.

Rapid detection of Escherichia coli by using antibody-conjugated silver nanoshells.

Silver shells of 20 nm thickness have been deposited on silica particles of 200 nm diameter with narrow size distribution. Silver nanoshells dispersed in water exhibit a strong surface plasmon resonance band at 443 nm. This band was found to be very sensitive to rabbit immunoglobulin G antibodies, which were anchored on the nanoshells. These in turn could be utilized to detect the presence of small (approximately 5) to large numbers (approximately 10(9)) of Escherichia coli in water. The protocol presented here proves to be a specific, rapid, reliable, and inexpensive method to detect E. coli.

Distribution of aminopeptidase P like immunoreactivity in the olfactory system and brain of frog, Microhyla ornate.

The enzyme aminopeptidase P (AP-P) is encountered in diverse vertebrate and invertebrate phyla and is known to act on proteins and peptides by releasing their N-terminal amino acid when the penultimate amino acid is proline. The present study is the first attempt at visualizing distribution of this polypeptide in the brain of a vertebrate species. The distribution of this enzyme was studied immunocytochemically in the forebrain of frog Microhyla ornata using antisera directed against cytosolic aminopeptidase P (DAP-P) of Drosophila melanogaster. Receptor cells in the olfactory epithelium exhibited strong AP-P like immunoreaction (ir). Immunoreactive fibers arising from the olfactory epithelium as well as vomeronasal organ joined the olfactory nerve, entered into the olfactory bulb, or accessory olfactory bulb and terminated in distinct glomerular formations. Some immunoreactive fibers traveled caudally and terminated in discrete areas in the telencephalon or diencephalon. Strong AP-P-ir was also seen in the cells of pars intermedia and pars distalis of the pituitary. The pattern of immunoreactivity suggests a role for AP-P in the processing of olfactory information and in hypophysial regulation.

Biodegradation of allethrin, a pyrethroid insecticide, by an acidomonas sp.

Allethrin is a major mosquito repellent agent. To degrade allethrin present in used mats and the environment, a bacterium capable of utilizing allethrin was isolated. This isolate, an Acidomonas sp., grew in minimal medium with 16 mM: allethrin as sole source of carbon and degraded >70% of it in 72 h, with negligible residual metabolites in the medium. Culture filtrates collected after 48 h and 72 h showed presence of (i) cyclopropanecarboxylic acid, 2,2-dimethyl-3-(2-methyl-1-propenyl), (ii) 2-ethyl-1,3-dimethyl-cyclopent-2-ene-carboxylic acid (iii) chrysanthemic acid and (iv) allethrolone [2-cyclopenten-l-one, 4-hydroxy-3-methyl-2(-2-propenyl)] as the major metabolites with 2 minor metabolites. Allethrin is thus metabolized by a hydrolytic pathway followed by oxidation and dehydrogenation.

Deinococcus radiodurans strain R1 contains N6-methyladenine in its genome.

Methylation of DNA is known to be involved in DNA repair mechanisms in bacteria. Deinococcus radiodurans strain R1 on exposure to high radiation undergoes significant DNA damage, which is repaired without mutations. However, the presence of modified nucleotides has not been reported in its genome. We report here the detection of N6-methyladenine in the genome of D. radiodurans strain R1 using immunochemical techniques. This N6-methyladenine is not a part of GATC restriction-modification system. D. radiodurans cell extract also exhibited a DNA adenine methyltransferase activity which was reduced in the early post-irradiation recovery phase.

Potential application of protease isolated from Pseudomonas aeruginosa PD100

A protease isolated from Pseudomonas aeruginosa PD100 could act in the presence of SDS and Tween 80. This protease could be useful for degradation of protein in the presence of solvent, dehairing of cow skin and degradation of natural proteins. The immobilized protease showed 15-20% increases in temperature stability and the entrapped enzyme retained 83% of its initial activity after six cycles. With respect to properties of the enzyme and its capability for degradation of different protein sources, this protease finds potential application for waste treatment, used in detergents and leather industry.

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20.

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.

A novel mixed-ligand antimycobacterial dimeric copper complex of ciprofloxacin and phenanthroline.

A novel mixed-ligand Cu(II) complex of ciprofloxacin (cfH) and phenanthroline, is found to crystallize as a dimeric moiety containing monocationic and dicationic species. Two such dimeric moieties are found in the same unit cell leading to a dicationic cluster. The higher negative redox potential for this cluster dampens its antimycobacterial activity against M. smegmatis.

Structural chemistry and In vitro antitubercular activity of acetylpyridine benzoyl hydrazone and its copper complex against Mycobacterium smegmatis.

Acetylpyridine benzoyl hydrazone (APBH) 1 and its copper complex [[(APBH)CuCl](2)].(EtOH) 2 were structurally characterized by elemental analysis, magnetic measurements, spectroscopy, electrochemistry and single crystal X-ray diffraction studies. The ligand assumes Z-isomeric form and planar geometry in solid state, coordinating through pyridyl nitrogen, azomethine nitrogen and the carbonyl oxygen of the benzoyl group. The copper complex is dimeric and has a distorted octahedral geometry in which the two copper atoms are bridged by two chloride atoms. Antimycobacterial screening of ligand and its copper compound against Mycobacterium smegmatis shows clear enhancement in the antitubercular activity upon copper complexation.