Department of Biotechnology

Permanent URI for this collectionhttps://ir.nbu.ac.in/handle/123456789/947

Browse

Filters

2011 - 2019182020 - 20235

Advanced Search

Filter by

Settings

Search Results

Now showing 1 - 10 of 23
  • Thumbnail Image
    ItemOpen Access
    Revealing molecular genetics of boron tolerance/resistance in bacteria using in-vivo evolutionary engineering and high throughput tools
    (University of North Bengal, 2022) Sen, Subhajit; Chakraborty, Ranadhir
    Boron (B), the fifth element is of pivotal biological importance in maintaining viable plant growth without causing mortal harm to biotic community of the surrounding soil. Over use of boron based compounds might result in causing boron toxicity and alteration of tolerance of boron by tolerant bacteria, capable of growing in presence of boron in atmosphere up to a certain level. In the present study, boron tolerant bacterial strains were isolated from soil samples with prolonged and chronic overexposure towards boron compounds in order to derive the fine line between boron toxicity and tolerance among relevant bacteria population and to estimate any possible diversities within the tolerant species. The fifth element, boron (B), is crucial for maintaining healthy plant growth without seriously harming the biotic community of the surrounding soil. When boron is used excessively (attaining concentrations which can cause boron toxicity), tolerant bacteria that grew in the presence of boron up to a certain level may change or enhance their B-tolerance. To determine the precise boundary between boron toxicity and tolerance among the relevant bacterial population and to gauge any potential diversities within the tolerant species, boron-tolerant bacterial strains were isolated from soil samples with prolonged and chronic overexposure to boron compounds. Identification of diversity in boron tolerant bacteria would be done for the first time in North Bengal area with an avenue in further studying the possible mechanisms of tolerance development and probable methods for transfer of the genotypic factors or environmental causes alleviating tolerance levels. From the 16S rRNA based phylogenetic and biochemical analysis it was found that all the boron-tolerant bacterial strains isolated boron contaminated agricultural fields belongs to three different genera, Lysinibacillus, Enterococcus and Bacillus. A couple of boron tolerant strains, Lysinibacillus sp. strain OL1 and Enterococcus sp. strain OL5, found to be tolerate 230 mM and 210 mM boron maximally in form of boric acid respectively, were ultimately selected for in-depth study. They, OL1 and OL5, were also tolerant to several other heavy metals and metalloids, such as, arsenic, cadmium, copper, mercury, zinc and nickel. In comparison to other metal pollutants, boric acid [B(OH)3] (approximately 96%) and a minor quantity of borate anion [B(OH)-4] are the two forms of boron that are the most mobile in soil. Both boric acid and borate can interact reversibly with different types of biomolecules like adenosine monophosphate, riboflavin, pyrimidine nucleotides, pyridoxine, ascorbic acid, and various sugar molecules (apiose, ribose, etc.) that contain an adjacent cis-hydroxyl group in their structure. Following growth kinetics and by Abstract measuring the amount of intracellular boron, it was primarily established that they were able to tolerate boron by using an inducible active efflux mechanism. This efflux mechanism enabled to maintain relatively less amount of boron in the intracellular milieu compared to the external B concentration. In order to validate the hypothesis, a potent efflux inhibitor PAβN was used; it was found that in the presence of PAβN (100 μg/ml) the level of B-tolerance got significantly reduced to 150 mM and 160 mM boron respectively, indicating that PAβN-sensitive-efflux pumps were partially responsible for B-tolerance. Three different omics tools, genomics, transcriptomis and proteomics, were used to generate data. Omics data were used to explore molecular mechanisms of boron tolerance in Lysinibacillus sp. OL1. RNA-free genomic DNA of OL1 was used for the construction of pair- end library for sequencing on NextSeq 500 system using Illumina platform. On the other hand, genomic DNA library of the strain OL5 was prepared using the Ion Xpress Plus fragment library kit (Thermo Fisher Scientific, USA) for Ion S5 next-generation sequencing platform (Thermo Fisher Scientific, USA). After annotation using NCBI Prokaryotic Genome Annotation Pipeline, it was found that the genome of OL1/OL5 isolates consisted of several number of efflux associated genes, 54/17 genes associated with MFS transporter, 263/283 genes associated with ABC transporter, 4/1 genes associated with SMR transporter, 2/1 genes associated with RND transporter and 4/2 genes associated with MATE transporter. Both the genomes were also containing a large number of genes associated with heavy metals and metalloids resistance. Total 33 and 30 heavy metals and metalloids resistance associated genes were present in the genome of OL1 and OL5 respectively. Beside all these, both OL1/OL5 genomes were found to contain at least 1/12 genes associated with phosphonate metabolism, 9/8 genes associated with auxin biosynthesis, 3/2 genes associated with siderophore or iron-siderophore transporter substrate binding, 8/2 genes associated with antioxidant activities, 78/40 genes associated with motility and chemotaxis, 6/39 genes associated with exo-polysaccharide production, and 7/ 23 genes associated with antimicrobial compounds and lytic enzymes production, which makes these two isolates a potential candidate for plant growth promoting rhizobacterial group. From the genomic and physiological analysis it was found that that OL1 have more genetic diversity and it can tolerate boron in more superior way compared to OL5. So, on the basis of these findings OL1 was selected for further studies. Total RNA and proteins were extracted from OL1 log phase growing culture according to standard methodology for transcriptomics and proteomics analysis. Following isolation, the proteome and transcriptome libraries were built and sequenced using the LC/MS and Illumina platforms, respectively. After the data had been Abstract sequenced and analysed, it was discovered that several stress response genes, efflux associated genes, energy metabolism, protein synthesis, and protein synthesis all played important roles in maintaining the integrity of the cell in the presence of an increased amount of extracellular boron. This result showed that multiple cellular metabolisms were involved in reducing the negative effects of boron on bacterial cells rather than just one. Furthermore, the most effective boron-tolerant isolate, OL1, has been subjected to in-vivo evolutionary engineering technique. It was discovered that OL-EC, an in-vivo evolutionary engineered cell, was able to tolerate 52.17% more boron than the OL1 wild type strain. The modified OL1-EC was able to withstand larger amounts of boron than the wild type of OL1, thus we tried to examine what mutations occurred in the OL1 genome and performed SNP-based mutational analysis. As a result of this analysis, we discovered that mutations occurred in a number of genes, including those encoding the ABC transporter ATP-binding protein, CPBP family intramembrane metalloprotease, transcription termination factor Rho, HEAT repeat domain-containing protein, GntR family transcriptional regulator, MarR family transcriptional regulator, oxidoreductase, FadR family transcriptional regulator, and a number of hypothetical proteins. We can infer from the results above that these advantageous mutations helped OL1-EC tolerate more boron. Future thorough studies utilising various omics methods, such as transcriptomics and proteomics, might give more details regarding the boron tolerance phenomenon in bacteria. OL1 and OL5 were both found to be positive for IAA production, phosphate solubilization, lytic enzyme production, motility, and biofilm production; this indicates that these boron tolerant isolates can promote plant growth in boron rich bare land and restore the soil quality. The plant growth promotion properties of these two isolates were also characterized. Finally, a metagenomic technique was used to study the microbial diversity of soil supplemented with boron. For metagenomic analysis, the total metagenomic DNAs from soil samples that had been modified with boron and those that hadn't were collected. Metagenomic libraries were then created. Ion torrent was then used to sequence the libraries. OTUs were created from the sequenced data, and comparative analysis was done. It was revealed from the metagenomic data that the gram positive phylum, Firmicutes is prevalent along with other bacterial genera. Interestingly, all of the recovered boron-tolerant strains belonged to Firmicutes. Therefore, it's probable to infer that high level of boron stress lead to a selection pressure that encourages the growth of Firmicutes-related Gram-positive bacteria. Additionally, it was noted that the phyla Acidobacteria, Actinobacteria, and Proteobacteria Abstract are abundant following Firmicutes. The entire study was designed to support the hypothesis that there are a variety of microbes in the soil of North Bengal that can tolerate boron levels above normal and that these microbes have an impact on the biological interactions between boron, bacteria, and soil systems, which have an impact on agriculture, the environment, and people's health.
  • Thumbnail Image
    ItemOpen Access
    Studies on antioxidant, antimicrobial and antidiabetic activities of some ethnomedicinal plants collected from Darjeeling Himalayan region, West Bengal
    (University of North Bengal, 2023) Das Vaskar; Ghosh Shilpi
    Since the dawn of human civilization, plants and plant-derived natural remedies have been a vital part of traditional healing. With advances in development of synthetic drug, their usage has dominated over natural products. However, with several examples of synthetic drug side effects in recent decades, there has been a huge increase in the hunt for natural and safer pharmaceuticals. Plants derived phytochemicals or bioactive substances are safer and more cost-effective resource for drug development. The rate of development of numerous devastating oxidative stress-related disorders such as cancer, diabetes, atherosclerosis, arthritis, Alzheimer's disease, and other neurological disorders has grown as people's lifestyles have changed. Plant phytochemicals have been described as effective free radical scavengers or quenchers, making them a rich source of natural antioxidants. Herbal medications have been utilized for antibacterial, antiviral, antiinflammatory, anti-rheumatid, antiallergic and other purposes. The use of ethnomedicinal herbs in traditional medicine dates back over 2000 years; and many more beneficial therapeutic metabolites are expected to be discovered during later years. Despite being abundantly available and offering economic, ecological and therapeutic values, ethnic plants have received very little attention. The aim of this study was to screen and quantify different phytochemicals present in specific ethnomedicinal plants of Darjeeling district, West Bengal, Pin 734101, in light of the importance of natural products and their application as pharmaceuticals. The plant extracts were investigated for antioxidant, antimicrobial, and antidiabetic activities. Further, the cone extract of Thuja orientalis was fractionated by column chromatography for the isolation of active compound followed by characterization of the compound as Octacosanol. The antidiabetic potential of the compound was evaluated in streptozotocin (STZ) induced rat model. A systematic review of the literature has been assembled to provide the details of previous studies on the several bioactive substances found in plants and their antioxidant, antimicrobial and antidiabetic potential. For present investigation Thuja orientalis (THU), Tupistra nutans (TUP), Astilbe rivularis (AST), Calamus erectus (CAL), Zanthoxylum oxyphyllum (ZAN) and Artemisia vulgaris (ART) were collected from various locations of Darjeeling district. The plant materials were shed dried, powdered and extracted with various solvents. With the different solvents utilized, a difference in extraction yield was noticed. The yield percent was least with hot water and ethanol, while methanolic extracts produced the highest percentage of yield and therefore used for further studies. The plant methanolic extracts were examined qualitatively and quantitatively for the presence of phytochemicals. Among various secondary metabolites phenol, flavonoid, tannin, carbohydrate and reducing sugar were found to be present in all the extracts. The quantitative analysis showed THU cones having the highest quantity of phenol, flavonoid, tannin and total carbohydrate whereas ART leaf exhibited all these phytochemicals in least quantity. The plant extracts showed different degrees of antioxidant activities as measured by DPPH radical, H2O2, NO and ABTS scavenging assays. AST rhizome exhibited maximum antioxidant activity in assay based on DPPH, NO and ABTS assay, whereas THU cone showed highest activity with H2O2. The antibacterial potential of the plant extracts was tested against two gram positive (Bacillus amyloliquefaciens and Bacillus subtilis) and two gram negative (Flexibacter sp. and Aeromonas liquefaciens) bacteria. While comparing antibacterial effect of various plant extracts, the THU cone exhibited highest antibacterial activity against Bacillus amyloliquefaciens, Bacillus subtilis and Aeromonas liquefaciens whereas AST rhizome showed highest activity against Flexibacter sp. The plant extracts were effective in limiting the growth of tested fungi, namely, Aspergillus niger, Rhizopus stolonifer, and Fusarium oxysporum, as measured by radial growth bioassay. The extracts of THU cone and THU leaf effectively suppressed radial growth of all of the fungi examined, but TUP flower and CAL fruit extracts only partially inhibited the radial growth. Depending upon phytochemical constituents, antioxidant and antimicrobial activity, THU cone was found to exhibit the highest possible avenue for profiting from their use in pharmaceuticals. Hence THU cone was used for isolation and purification of bioactive molecule followed by its characterization by UV, IR and NMR spectroscopy and LC-ESI-MS analysis. The active compound in THU cone was characterized as Octacosanol with molecular formula C28H56O and molecular weight of 408. Octacosanol was found to inhibit the in vitro α-amylase activity, and its inhibitory effect was about two fold lesser than that of the positive control acarbose. In an in vivo antidiabetic assay on streptozotocin-induced diabetic rats, Octacosanol was found to be effective in restoring various blood parameters that had been altered by the induction of diabetes. Variable parameters evaluated was change in body weights, fasting blood sugar level, cholesterol, triglycerides and HDL cholesterol content, SGPT and SGOT activities and serum urea and creatinine level. Higher concentration of compound revealed higher reduction in the tested blood parameters than the lower concentration used in the study. Treatment with Octacosanol significantly improved and restored the histological structure of both liver and pancreas in STZ-induced diabetes rat. The molecule Octacosanol has potential application in pharmaceuticals due to its hypoglycemic, antimicrobial and antioxidant effects
  • Thumbnail Image
    ItemOpen Access
  • Thumbnail Image
    ItemOpen Access
    Search for molecular diversity of metallo-B-lactamase genes in eubacterial isolates of Karala and Mahananda rivers of West Bengal
    (University of North Bengal, 2021) Ranjan, Vivek Kumar; Chakraborty, Ranadhir
  • Thumbnail Image
    ItemOpen Access
  • Thumbnail Image
    ItemOpen Access
    Cloning and characterization of genes involved in biosynthesis of ginsenosides from Panax sokpayensis Shiva K. Sharma & Pandit
    (University of North Bengal, 2018) Gurung, Bhusan; Saha, Dr. Dipanwita, and Bhardwaj, Dr. P.K.
  • Thumbnail Image
    ItemOpen Access
    Serological and molecular detection of foliar fungal pathogens of Persea bombycina Kost and activation of defense response using bioinoculants
    (University of North Bengal, 2016) Acharya, Amrita,; Ghosh Dr. Shilpi Chakraborty, B.N.
  • Thumbnail Image
    ItemOpen Access
  • Thumbnail Image
    ItemOpen Access
  • Thumbnail Image
    ItemOpen Access
    Immunological responses and internal microbes of eisenia fetida
    (University of North Bengal, 2018) Saha, Tilak,; Chakraborty, Ranadhir,