G. P. Jagtap

Rapid molecular detection of Candidatus Liberibacter asiaticus, the bacterium associated with citrus huanglongbing (greening) disease of Mandarin using polymerase chain reaction

Huanglongbing (HLB or citrus greening or yellow shoot disease) is a devastating disease of citrus caused by nonculturable, fastidious phloem limited bacterium, Candidatus Liberibacter asiaticus and threatens the citrus industry in Asian countries. The putative causal agent of the disease is transmitted through insect vector or grafting with diseased budwood. The polymerase chain reaction (PCR) diagnosis is a more reliable and sensitive diagnostic tool for detecting greening bacterium than other conventional approaches like electron microscopy, DNA-DNA hybridization and immunofluorescence (IF) for detection of citrus greening. Results reveal that sodium sulphite method of DNA isolation provided higher yield and better quality DNA than other methods. To confirm the reliability of PCR, the greening bacterium was also detected in graft-inoculated plants, which showed typical greening symptoms. Results show amplification of 450 bp in PCR suggesting sampling in March is more suitable for PCR detection of greening bacterium. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a Candidatus Liberibacter asiaticus free nursery system. Keywords: Citrus greening, huanglongbing (HLB), molecular detection, polymerase chain reaction (PCR), phloem-limited bacterium. African Journal of Biotechnology Vol. 12(29), pp. 4623-4629

Polymerase chain reaction detection of Candidatus liberibacter asiatic associated with citrus huanglonbing

T investigation was conducted to assess the association of genetic diversity of parental lines with mean performance of hybrids, mid parent heterosis, better parent heterosis and sca effects. The material for this study consisted of five male-sterile lines, eight pollinator lines, 40 F1 crosses, which were generated through Line x Tester matings. Whole of the material was grown in a Randomized Complete Block Design with three replications. All the parental lines were screened to detect polymorphism in the form of RAPD markers. Genetic diversity among the parental lines was determined by RAPD markers and morphological characters. The genetic distances so obtained were correlated with F1 mean performance and heterosis. Positive correlation was obtained between molecular marker diversity and F1 mean performance, heterosis over better parent but the value of correlation coefficient was found to be non-significant. In contrary, negative correlation was obtained between taxonomic distance and F1 mean performance; better parent heterosis. Our results have indicated that genetic-distance measures based on RAPDs may be useful for the grouping of parents, but not for predicting heterotic combinations in pearl millet.Present studies in citrus orchards confirmed that Citrus Greening or Citrus huanglongbing (HLB), a heat sensitive citrus disease, is associated with the presence of “Candidatus liberibacter asiatic” in Maharashtra. It is associated with nonculturable phloem-restricted and gram-negative bacteria which is the members of the alpha subdivision of the proteobacteria. It is responsible for the decline of citrus orchards in Maharashtra and other citrus growing areas in the country. Molecular diagnosis of Candidatus liberibacter asiatic by using polymerase chain reaction which is used for detection of the disease when pathogen present is very low concentration in disease sample. Results revealed that among the three DNA isolation method viz., commercial kit method, sodium sulphite method and membrane bard nucleic acid technique, sodium sulphite method is cost effective for commercial use. The comparative cost of detection by various combination of reagent and sampling time was determined and cost effective technology was standardized and validated. The DNA extraction method by sodium sulphite, Klen Tag enzyme and 450 bp set of primers were identified to give best amplification in PCR system of diagnosis. The comparative cost of detection by various combination of reagent and sampling time was determined and cost effective technology was standardized and validated.T analysis of extracellular polymeric substances (EPS) from bacteria traditional methods requires chemical treatments and complicated separation procedures. Analysis of Low molecular weight compounds on Convention MALDI-MS is very difficult due to conventional organic matrixes like DHB or CHCA matrix background peaks are interfere with samples peaks at low molecular range .For avoiding those problems we developed a method, combining Hi-Speed centrifugation or physical extraction method with Nano-particle assisted laser desorption/ionization (HC-Np-ALDI-MS) mass spectrometry (MS) without any chemical treatment or extraction methods, separation and purification of EPS. The objective of this study is to demonstrate the use of Hi-Speed centrifugation coupled with Nanoparticle-assisted laser desorption/ionization (NALDI) mass spectrometry (MS) as a powerful technique for rapid, simple and direct analysis of low molecular weight EPS secreted by bacteria. For this we prepared manganese oxide nanoparticles and developed a nano-PALDI MS method to analyze EPS extract from different bacteria’s by using Hi-Speed centrifugation. Our studies showed that nano-PALDI MS was selective for analysis of low molecular weight EPS. This study is a preliminary study reporting for the first time the feasibility of nano-PALDI MS techniques for the analysis of bacterial EPS without prior complicated purification and separation methodologies. The MnO2 Nano-particles working as matrix during MALDI-MS analysis and help to ionize the EPS Samples. The mass spectra did not show any background interference in the low-m/z range. The results reporting the successful use of the manganese dioxide particles as matrices for analysis of bacterial EPS has been outlined.I this study, survey and collected rhizospheric soil samples was done from different regions of Indogangetic plains in U.P. (Gorakhpur, Lucknow, Kanpur, Varanasi, Meerut and Mau). Physiochemical properties of rhizospheric soil were determined for their pH (ranges 7.0-8.4), EC-value (ranges 1.3-1.9ds/m) and organic carbon in soil (ranges 0.500-0.750 to 0.750-1.00% by permagnate method). Isolation of rhizobacterial populations were made by the different inoculation techniques (soil plate and serial dilution) on various general and specific culture media (Nutient agar, Jenson agar, Pikovshaya agar, Burk,s medium, NFB medium, Malate Medium and YEMA medium). A total of 143 rhizobacterial isolates of from rhizospheric soil samples that were visually characterized for their different morphotypes. As the different morphotypes were tested for the HCN production, Siderophore production and Phosphate solubilization, in which some of the isolates showed significant results. Prominent plant growth promoting rhizobacteria showing qualitative characteristics, further their samples were prepared in methanol and ethyl acetate for the phenolic compounds analysis (Sinapic acid, Ferulic acid, Veratric acid, p-coumaric acid, Vanillic acid, Syringic acid, p-hydroxybenzoic acid, Cinnamic acid, Benzoic acid, o-coumaric acid, m-coumaric acid, Caffeic acid, Salicylic acid, Protocatechuic acid and Gentisic acid). Secondary metabolite profiling for the phenylpropanoids i.e phenolic compounds was done to characterize rhizobacteria for the biomolecules production using HPLC standards. Some isolates were identified as prominent phenolic compounds producers. Identification and quantification of these phenolic compounds in culture filtrates, cell pellets and or in soil by these bacteria through LC-MS conditions is underway. During plant microbe’s interaction using phenolic compounds i.e. biomolecules as plant growth promotion by the defence system activation, plant pathogens suppression by production of inhibitory compounds and or symbiosis by the production of quorum sensing compounds in the ecosystem. Therefore, identification and quantification of phenolic compounds producing rhizobacteria may helpful to develop consortia for some metabolic interaction in rhizosphere that may influence plant growth and productivity.F foods and beverages have been in the Indian food menu since ages. In Himachal Pradesh people have developed traditional food processing technology for preparing conventional fermented foods from locally available substrates largely governed by the ethnic preference, agro-climatic conditions, socio-cultural ethos and religion. Seera is a traditional fermented food prepared in Bilaspur, Kangra, Hamirpur, Mandi, Shimla and Kullu districts of Himachal Pradesh. It is a starch based food made by soaking, crushing and fermenting wheat grains used to prepare sweet dish/snack generally served people during religious fast. Samples during seera fermentation were analyzed for various microbiological and biochemical parameters. The microflora isolated from seera mainly comprised Saccharomyces cerevisiae, Cryptococcus laurentii and Torulosporadelbrueckii among yeasts and Lactobacillus amylovorus and Leuconostocsp. among bacteria. The biochemical analysis of seera revealed that it is an acidic food having a pH of 3.4 and titrable acidity of 0.44%. It has 10.4±0.20 % (w/w) protein, 89.0±0.43 mg/g of dry matter total proteins, 87.4±1.51 mg/g dry matter starch and 11.9±0.53 mg/g dry matter reducing sugars. The activities of amylase and protease were 3.6±0.36 U/g and 1.02±0.05 U/g respectively. A significant increase in thiamine, riboflavin, nicotinic acid and cyanocobalamin was observed during fermentation of seera. The level of essential amino acids especially methionine, phenylalanine, threonine, lysine and leucine also increased during seera fermentation. As seera is biologically enriched with vitamins and amino acids during fermentation so it forms a good source of nutrition to the people who consume it.T aim of this study was to optimize the medium and cultivation conditions and to investigate the possibility of applying Aureobasidium pullulans BS1 for the study of anti-oxidant activity and metal biosorption efficiency with the simultaneous biosynthesis of Pullulan. The A. pullulans BS1 was screened from the phylloplane of Brassica oleracea reported in our previous study by enrichment process. The various parameters (pH, Temperature, Time period of Incubation and types of Carbon and Nitrogen sources) were optimized. The optimal medium for pullulan production by this strain BS1 was 7.0% (w/v) glucose, 1.8% (w/v) soybean cake hydrolysate, 0.45% (w/v) K2HPO4, 0.1% (w/v) NaCl, 0.02% (w/v) MgSO4•7H2O, 0.05% (w/v) (NH4)2SO4, pH 6.5. The optimal cultivation conditions for pullulan production by this strain in 300-ml shake flask containing 50 ml of medium were observed at 28°C and with 180 rpm. Under these conditions, 6.5% (w/v) pullulan was produced within a time period of 3 days. No melanin like pigments in the medium was observed during the fermentation process, indicating that strain BS1 was a non-pigmented yeast strain. The metal absorption efficiency and the antioxidant properties of the isolated strain were found with simultaneous production of pullulan in the fermentation broth.Plants are rich source of alkaloids, steroids, terpinoids, phenolics, sweetners, bittering agents, pigments and perfumes. These are used as source for large number of industrial products, including agricultural chemicals, pharmaceuticals and food additives. There is a continued commercial demand for these metabolites in food and pharmaceutical industries. Earlier the production of plant secondary compounds was achieved through the field cultivation of plants. It is difficult to cultivate some plants outside of their original ecosystems. The isolation of these compounds through the extraction of organs and seeds of whole plants is difficult and costly. Various methods in biotechnology, like free cell suspension culture, elicitation, immobilization and biotransformation has been developed to accumulate desired products. Currently, much interest is focused on the large-scale culture of plant cells as sources of commercially important secondary metabolites. The present review is focused on biotechnological strategies developed so far to enhance secondary metabolites production.U comparative genomics and computational methods, we have identified the structural & functional aspect of a putative t-RNA dihydrouridine synthase encoding gene of Saccharomyces cerevisae (MTCC-181) by homology modeling approach. Dihydrouridine synthases (DUSs, EC 1.3.1.91) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. A putative protein of 668 amino acids was analyzed. It contains 12-FMN-binding conserve domain on the active site, characteristics of DUS_like_FMN super-family (cd02801, E-value=3.21e-63) which catalyzes the reduction of the 5, 6-double bond of a uridine residue on tRNA. Four catalytic residues 386 (Cys), 428 (Arg), 457 (His) and 459 (Arg) were found on the DUS-like conserved domain (294-557), among which an active site cysteine is important for catalysis, likely through the protonation of uracil during tRNA reduction. The protein have multi-domains with high similarity to COG0042 (CDD, 284597); a tRNA-dihydrouridine synthase (E-value=1.07e-62) and TIM barrel superfamily (cl09108, 296-550, E-value=3.83-20) which share a structurally conserved phosphate binding motif and in general have an eight β/α closed barrel structure. The structure of an FMN-binding α/β-barrel also has significant similarity to dihydroorotate dehydrogenases and dihydropyrimidine dehydrogenases. Three dimensional (3D) structure was constructed by modeller 9v7 modeling software by using the 1VHN (4.4e-15 & similarity =76%) as a template. Binding of substrate (uracil-t-RNA) and acceptor molecules (NADPH and FMN) were confirmed by Autodock 4.2.Abstract S ewage water is one of the sources that are responsible for many epidemic diseases. The Bioremediation is green technique that reduces contaminates & nutrient from sewage water. The sewage under the influence of Eichhornia & Pistia species for twenty days shows a significant change in the concentration of impurities. The change was observed by Physico-chemical, Nutrients, Microbial Factors & Solid analysis with different interval of days. The percentage of nutrients and other parameters absorbed by Eichhornia was better than Pistia. The level of impurities was reduced by the remarkable amount by activated charcoal & alum treatment. Key words Sewage, Bioremediation, Eichhornia, chemical analysis, charcoal. Sewage is a water borne waste derived from home, animal house, food-processing industries, hotels, restaurants, hospitals, service stations, etc. It is recommended that Biofertilizer is not a substitute, but a supplement to chemical fertilizers for maximizing not only the yield but also agriculture system 1stabilityC based on Zeolites brought revolution in oil refining and petrochemical production in recent years due to their size selectivity and gained much research interest all over the world. The most well known and widely used zeolite is ZSM-5 (pores: 0.51 x 0.55 nm, 0.53 x 0.56 nm). ZSM-5 has been used successfully in aromatic isomerisation, methanol upgradation and many other reactions in organic chemistry. On the other hand anhydrous aluminium chloride has been found to have a high efficiency in isomerisation reactions due to its strong Lewis acidic nature. But application of this catalyst is limited due to its high corrosiveness. In our present work we have used ZSM-13, which has not been normally used for catalyst preparation as main matrix due to its high monobranching selectivity. And we have tried to increase its (ZSM-13) catalytic performance by modifying the micropores present in it with aluminium chloride impregnation, reducing corrosive nature of aluminium chloride. Three samples of aluminium chloride impregnated ZSM-13 containing 0.015, 0.025, 0.05 mol of anhydrous aluminium chloride have been synthesized by batch impregnation method and thoroughly characterized by XRD, FTIR, SEM, TEM, AFM and BET surface area analysis. Both XRD and FTIR analysis show that the pores present in the ZSM-13 sample is finely impregnated with aluminium chloride. The SEM images of the samples show the surface morphology. Comparison shows that pure ZSM-13 has some morphological change on the surface after impregnation with aluminium chloride. The TEM image of pure ZSM-13 shows the micro pores present in it. The specific surface area of ZSM-13 has been increased after aluminium chloride incorporation. This is very significant since the isomerisation reaction is very much sensitive to specific surface area. All the three samples synthesized show very high conversion capacity of normal alkanes like n-hexane, n-octane compared to pure ZSM-13. This is due to the increase in activity of the active sites present in pure ZSM-13 after incorporation of aluminium chloride. This enhanced performance of aluminium chloride impregnated ZSM-13 also clearly indicates that pores present in ZSM-13 have not been blocked by anhydrous aluminium chloride. Leaching test of the catalysts shows that aluminium chloride is not leached out from any catalyst surface. Thus the aluminium chloride impregnated ZSM-13 sample is found to be very promising hydroisomerization catalyst and can be very safely used for practical purpose.N Phospho ribosyltransferase (NMPRTase) is an enzyme catalyses the biosynthesis of NAD+. It is generally expressed more in cancerous cell lines. The inhibitors targeted to NMPRTase reduce the cellular NAD+ concentration which results in apoptosis and cell death, hence can be used to treat cancer. These inhibitors could also target the NAD+ dependent enzymes like sirtuins indirectly, which were implicated in many diseases like diabetes, neurodegeneration and inflammation. Thus designing inhibitors to NMPRTase would be a good strategy to treat various disorders. In the present work, crystal structure of NMPRTase (2GVJ) with 2.1 Ao resolution, was employed for the energy based pharmacophore model generation for the crystal ligand (FK866) and virtual screening was carried out using Glide docking (Schrodinger). Commercial databases were used for virtual screening.Top30 potential hits were shortlisted by comparing pharmaophore fitness, docking score and hydrogen bonds with crystal ligand. These top hits are the potential lead compounds for treating cancer and other disorders.N pain occurs as a result of trauma or injury to a peripheral nerve due to damage or dysfunction of the nervous system under various disease conditions. Lysosomal Cysteine protease cathepsin S (CatS) plays a crucial role for the maintenance of neuropathic pain and spinal microglia activation. Thus, effective CatS inhibitors may be of significant therapeutic importance. In this study, a 3D pharmacophore mapping studies were undertaken for different series of synthetic derivatives. A five point pharmacophore with three hydrogen bond acceptors (A) one hydrogen bond donor (D), and one hydrophobic feature (H) as pharmacophoric features were developed. The pharmacophore hypothesis was validated with enrichment calculation best pharmacophore further yielded a statistically significant 3D-QSAR model, with a correlation coefficient of R2 = 0.992 for training set compounds. The model generated showed excellent predictive power, with a correlation coefficient of Q2 = 0.7. The model was then employed as 3D search query to screen against public and private compound libraries (Asinex, BITS database) in-order to identify a new scaffold. Best hit compounds were selected from virtual screening for the enzyme inhibitory In-vitro activity against CatS enzyme. Inhibitors IC50 value below 50μM were considered as potential selective non-peptidic and non-covalent inhibitors for CatS. Backbone structural scaffold features and the contour maps delivered from the built 3D QSAR models could serve as building blocks in designing novel drug molecules for CatS.C is an opportunistic fungus, known to cause systemic and life threatening infections in immunosuppressed patients. In our attempt to dealt with this ominous fungal pathogen we have successfully fabricated ultrafine poly (D,L-lactide-coglycolide), PLGA, nanofibrous membrane loaded with different concentrations of quercetin dihydrate, natural flavones with antioxidant and antimycolytic activity, using acetone-dichloromethane as solvent system. The bulk fabrication of these nanofibrous membrane was performed via electrospinning technique. The morphology of resulting nanofibrous membrane with or without quercetin dihydrate was examined using field emission scanning electron microscopy (FE-SEM). The nanofibrous membrane was also subjected to detailed analysis by Fourier transform infrared spectrometry (FTIR). The antimycolytic effect of these quercetin dihydrate loaded nanofibrous membrane on to the viability of C. albicans cells was established using modified XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay in 6 well polystyrene microtiter plates. Scanning electron Microscopic (SEM) visualization of this C. albicans treated quercetin dihydrate loaded nanofibrous membrane showed drastic reduction in the intense network of yeast, hyphae and pseudohyphae forms. These highly loaded nanofibrous membrane can be efficiently used as an antimycolytic agent as well as can be exploited as cost effective wound dressings and protective bandages.T the science of naming and classifying organisms, is the foundation of biology. Identification and documentation of all forms of life on earth and proper nomenclature of species provide the framework for organizing biological information (Kumar and Jain, 2011). Existing morphology-based diagnostic approaches used by traditional taxonomists are often cumbersome and based on ontogeny. Barcoding organizations note that taxonomists have identified only 15 per cent of all living species over the past 250 years (Ebach and Holdrege, 2005).M oilseeds (eg copra, palm kernels and groundnuts) need proper processing in mills before oil extraction to increase the yield of oil. The efficient and economical utilization of feed stocks is highly essential in oil producing industries. Coconut has the highest productivity and is less susceptible to abnormal climatic condition. The production of coconut oil and its byproducts, raw and fried cake, is an important source of income for women in coastal areas of India. Hence, identification of optimal pretreatment conditions of coconut nut kernel is very important for high yield of coconut oil. The optimum processing conditions can be found by incorporating reliable and efficient statistical design methodologies such as central composite design (CCD), and ANN.September 13-15, 2012 R oleaginous fungi have emerged as an alternative feedstock for biodiesel production in the form of microbial lipids (single-cell-oils, SCOs). Filamentous fungi are known to degrade and utilize a wide range of lignocellulosic waste materials. Therefore, it is economically promising to develop a biodiesel production process using oleaginous fungi grown on such renewable carbon sources. The main intent of the present work was to evaluate the growth and lipid accumulation by a newly isolated Aspergillus terreus IBB M1 on Eucalyptus bark waste (EBW) and corn cobs, this fungal strain was earlier shown to produce SCO (54 % w/w ) on glucose, in our laboratory (Khot et al., 2012). EBW constitutes the major lignocellulosic waste generated during debarking of the Eucalyptus wood in pulp mills.It was initially used as a sole carbon and energy source under solid state fermentation conditions without any chemical pretreatment while corn cob (CC) was taken as a conventional solid substrate for comparative study. The dried and milled substrates were moistened with mineral solution in wide mouth Erlenmeyer flasks (1:5 w/v) and kept for incubation in humidity chamber at 30°C for 7 days. The fermented mass containing mycelia and unutilized substrate was harvested, dried till constant weight and used to extract total cellular lipids as SCOs in presence of chloroform : methanol (2:1) by cryopulverization in a mortar and pestle using liquid nitrogen. The extracted fungal SCOs were analyzed for fatty acid composition by GC-FID and found to be rich in saturated fatty acids for both the substrates with 33% and 24% relative contents for EBW and CC, respectively. In particular, the SCO from EBW was rich in lignoceric acid (C24:0) indicating the direct transformation of lignin fractions of the waste into the fatty acids while oleic acid (C18:1) was the major dominant monounsaturated fatty acid. The PUFAs with ≥ 4 double bonds were not detected for SCOs obtained on both the substrates. A few biodiesel fuel properties (density, kinematic viscosity, iodine value and cetane number) were determined by prediction models for fungal SCO obtained on EBW and CC found to be in accordance with the national and international biodiesel standard specifications. The submerged fungal cultures were grown in shake flasks using EBW (1% w/v) and the SCO yield was determined to be approx. 1g/L after 120 h incubation. The results obtained suggest the suitability of bark waste obtained from Eucalyptus wood mills for the production of SCO with potential for biodiesel production.Background & objectives: Acute tubular necrosis (ATN) caused by renal ischaemia, renal hypo-perfusion, or nephrotoxic substances is the most common form of acute kidney injury (AKI). There are a few treatment options for this life-threatening disease and the mortality rate exceeds 50 per cent. In critical cases of AKI the only option is renal transplantation. In the present study we evaluated whether bone marrow cells (BMCs) are involved in regeneration of kidney tubules following acute tubular necrosis in the mouse. Methods: Six to eight week old C57BL6/J and congenic enhanced green fluorescence protein (eGFP) mice were used. The relative contributions of eGFP-expressing BMCs were compared in two different approaches to kidney regeneration in the mercuric chloride (HgCl2)-induced mouse model of AKI: induced engraftment and forced engraftment. In vitro differentiation of lineage-depleted (Lin-) BMCs into renal epithelial cells was also studied. Results: In the forced engraftment approach, BMCs were found to play a role in the regeneration of tubules of renal cortex and outer medulla regions. About 70 per cent of donor-derived cells expressed megalin. In vitro culture revealed that Lin- BMCs differentiated into megalin, E-cadherin and cytokeratin-19 (CK-19) expressing renal epithelial cells. Interpretation & conclusions: The present results demonstrate that Lin- BMCs may contribute in the regeneration of renal tubular epithelium of HgCl2-induced AKI. This study may also suggest a potential role of BMCs in treating AKI.T engineering is the process of creating functional 3-D tissue combining cells with scaffolds that facilitate cell growth, organization and differentiation. The most important aspect of tissue engineering is the adhesion and proliferation of cells on scaffold material. The cells are grown on the scaffold that functions to provide mechanical support for cells and synchronize the functions of cells in a manner analogous to extracellular matrix. Carbon as an inert element has advantages over other materials because it is a basic constituent of tissues. The high proportion of the tissues of living organisms is composed of carbon compounds so it should be tolerated by the tissues. The fibroblasts are common cells present in the connective tissue that synthesizes and continuously secretes precursors of extra cellular matrix. Fibroblasts provide a structural framework for many tissues and play a critical role in wound healing. To develop a novel biomaterial for wound healing in-vitro biocompatibility of carbon mesh is tested. In the present study, carbon mesh is cut in desired size and after sterilization placed in six well cell culture plates. The mesh was co-cultured with mouse embryonic fibroblast (MEF) cells. At different time intervals the viability and proliferation of the MEF cells was evaluated using Phase contrast inverted microscopy, Light microscopy and Scanning electron microscopy. The results will be discussed in detail at the time of presentation.T work reports on the isolation of Lactic acid bacteria (LAB) from non-broiler chicken, other than commonly useed broiler chicken. Lactic acid bacteria (LAB) were isolated from the ceca, intestine, gizzard and bile of Malaysian non-broiler chicken ‘ayam kampung’. Following the incubation on MRS agar plates, 56 LAB isolates were collected. Based on agar disk diffusion method, 5 out of 56 isolates; L3, L4, L5, L7 and L7 show inhibition against the indicator bacteria Methylene-resistant Staphylococcus aureus MRSA. All of the five isolates show typical characteristics of Enterococcus family which are Gram positive coccus morphology, non-spore forming, catalase negative, producing acid from glucose and able to grow on 4, 6 and 10% NaCl. Ability to produce inhibitory proteins using agar disk diffusion method were tested using three phase partitioning (TPP) purified fraction employing t-butanol and ammonium sulphate. The purified fraction produced zones of inhibition (approx. 16-18 mm in diameter) on indicator organism Staphylococcus aureus MRSA. The purified fraction contained putative low molecular weight proteins of about 8-10 kDa in size, as determined by SDS-PAGE. These putative proteins are stable upon heat treatments and inactivated by trypsin. The partial rDNA sequences from the 5 isolates showed to be at least 98% similarity with Enterococcus family. Isolates L3, L4, and L5 belonged to the E. faecum. Meanwhile, L7 and L10 belonged to E. faecalis and E. mundtii. These strains were exclusively isolated from non-broiler chicken; and they produce putative inhibitory proteins or bacteriocins against Staphylococcus aureus MRSA are useful as probiotic for chicken.R (Oryza sativa L.) is the staple food for almost 50% of the world’s population. Rice genome is fully sequenced but sharing of genomic information with the world scientific community is one of the important hallenges. Many databases are maintaining rice genomic information but Vanshanudhan is very different because of its queries based searches. Vanshanudhan database (http://125.18.242.23:8080/genome/Login.jsp) facilitates search based on complete genome data including gene, cDNA and protein sequence. Present Vanshanudhan data is based on rice pseudomolecule version 3.0 released from Michigan State University with a unique gene nomenclature e.g. 01-0001, which defines chromosome number as well as gene number. We have upgraded the complete genomic information from pseudomolecule version 3.0 to version 7.0 and integrated it with the recently developed NRCPB rice marker information system which provides specific SSR and SNP marker information for each rice chromosome. We simultaneously incorporated NRCPB trait information system with it which is broadly classified into six different categories such as yield, quality, biotic stress, abiotic stress, plant architecture, biomass and further these are again classified into sub category . These marker and trait information systems are fully integrated with each other and are able to provide relevant query based information. The database platform is developed using MySQL in relational database management system and JSP is used as a middle ware. The entire database is implemented on high performance computing server with UNIX Sun solaris version 5.9 and easily accessed through links. This database aims to provide useful information for the researcher community engaged in rice crop improvement programme.M than 100 heterologous proteins from various phylogenetic origins have been successfully produced in Yarrowia lipolytica yeast expression system, which offers reliable performances in glycosylation, folding and secretion of complex proteins. We present here a short review of the major tools developed in this yeast, with focus on vectors for targeted monocopy integration (YLEX expression kit, Yeastern Biotech Co, Taiwan), a system particularly adapted to genetic engineering of heterologous proteins. In order to examplify the various applications of Yarrowia expression system, a number of current collaborative works on wholecell biocatalysis, genetic engineering of secreted enzymes, and surface-display of heterologous proteins (arming yeasts) will be briefly resumed. Some applications, like high throughput screening, require improving Yarrowia transformation methods for higher efficiency, without sacrificing the high percentage of correctly targeted integrations. For that purpose, we have developed a sensor system for homologous integration, based on E. coli lacZ gene expression, which allowed us to evaluate some chemicals increasing transformation efficiency for maintenance of a high percentage of correct integration. Dithiothreitol was found to be not only more efficient than dimethyl-sulfoxide in increasing transformation efficiency, but also compatible with homologous integration, which was not the case of the latter that promoted out-of-site integration. An optimized transformation protocol using dithiothreitol has been developed, which ensures very high transformation efficiency.During the last three decades numerous bioactive compounds have been isolated from marine microorganisms and used as sources of therapeutic agents. Marine yeasts are considered to be an important category of marine microorganisms. As a unicellular organism yeast has wide range of potential application from ancient fermentation industry to modern molecular biology. Yeast enzymes were found to be useful in various industrial and biomedical fields there by indicating their importance in day to day human activities. Yeast belonging to the genera Candida, Debaryomyces, Pichia, Cryptococcus, Rhodotorula, and Trichosporon were isolated from the Arabian Sea and examined for their hydrolytic enzyme production. The result showed that majority of the isolates were lipolytic (89.91%) followed by gelatinolytic (22.94%) and amylolytic (14.68%). Generic wise analysis of the hydrolytic enzyme potential showed that Trichosporon were found to be the most potent isolate for enzyme production, 56.25% produced amylase, 32% were gelatinolytic and 9.18% exhibited lipolytic activity. Among Cryptococcus strains 18.75% produced amylase, 16% gelatinase and 9.18% produced lipase. Of Candida 12.5% produced amylase, 12% gelatinase and 39.8% lipase. Among Rhodotorula 11.22% of produced lipase and none of this isolate produced amylase or gelatinase. Among Debaryomyces strains 6.25% produced amylase, 40% gelatinase and 18.31% lipase. Among Pichia 6.25% produced amylase and 12.25% lipases and none of this isolate produced gelatinase. Among the isolated species 23.85% of strains showed urease activity. The present study highlights the importance of marine yeast as a potent source of hydrolytic enzymes.Immobilization technique is used for preservation of enzymes. Here we are using different colors of natural dye in this technique. Our major emphasis would be to create beads of different colors which will act as different carriers for essential enzymes. The need for using colorful dye is because when we need to preserve more than one enzyme then it is helpful in identification of that which enzyme is to be preserved and in which color. Here we used natural dye because most of the chemical dyes are carcinogenic in nature and may alter the nature of preserved enzyme. For this different plant products like mint leaf, rose petal, beat root and Carrot are used for the extraction of dye. In our research studies, we have identified, extracted, characterized, optimized and standardized the natural dyes from plant and microbial sources and we did a comparative study between natural dyes and artificial dyes with respect to different solvent systems like petroleum ether, diethyl ether, acetone, chloroform, ethanol and water systems. The extraction methodologies, characterization, MIC (minimum inhibitory concentration), and solubility studies will be discussed. These immobilization studies will help us to use this application in a variety of fields like in wine stabilization, in modifying the shelf life of food and other natural products which degrade quickly and are difficult to preserve under natural conditions. Here sodium Alginate beads are being used so that there is good number of beads formation and that will help for the proper entrapment of the essential enzymes required for an important reaction in Bio-systems.T investigation of biofilm formation is critical to reducing infection rates as more than 60% of the clinically infectious diseases are associated with biofilm formation. Here, we used a plasmid pRL27, having an R6Kγ origin of replication, to generate transposon mutants altered in biofilm development in Escherichia coli 2443 (K-12 strain). The R6Kγ origin of replication requires the pir gene encoding the π protein, which binds at a 22-bp recognition sequence to initiate DNA replication. As the Escherichia coli 2443 is a pir negative strain, the plasmid pRL27 cannot replicate in it and this makes the plasmid an effective suicide vector for genome wide transposon mutagenesis. The plasmid was maintained in MFDpir strain, which is a diaminopimelate (DAP) auxotrophic mutant (dap–) of Escherichia coli and produces the π protein. The plasmid pRL27 was conjugatively transferred from MFDpir to Escherichia coli 2443 on minimal medium plates. Transconjugants were selected by plating the mating mixtures onto LB plates supplemented with dap (300 μg/ml) and kanamycin (50 μg/ml). The transposon insertion mutants obtained were screened for altered biofilm formation using crystal violet staining. The biofilm formation was normalized to planktonic growth, thus obtaining the biofilm formation index (BFI). Based on the results two mutants RM 56 and RM 69, showing significantly lower and higher BFI respectively, were selected. These mutants will be characterized further to identify the site of the transposon insertions and the underlying mechanism altering biofilm formation. This may be of benefit to develop a better understanding of biofilm formation. Isolation of transposon mutant of Escherichia coli with altered biofilm formationThe antibacterial effect of aqueous garlic and cinnamon extract at five different temperatures (40O C, 60O C, 80O C, 100O C, 120O C) against five multidrug resistant bacterial isolates (2 gram negative and 3 gram positive), including Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, E. Coli and Proteus mirabilis were studied by well diffusion method. The maximum antibacterial effect of aqueous garlic and cinnamon extract of different temperature obtained in Enterococcus faecalis and E. Coli at 60O C (1.041) and in Enterococcus faecalis at 60O C (0.87) respectively. This antibacterial property was applied on preservation of fish. A time dependent antibacterial study was done using the gum acacia coating with garlic and cinnamon paste. It is observed that the microbial load present on the fish was totally reduced on second day. In short, the aqueous garlic and cinnamon extract and pastes show a wide range of antibacterial activity at 40O C to 60O C and satisfy all the criteria for antibacterial agent as compared to antibiotic Gentamicin. These results suggests that garlic and cinnamon can be used as food preservative and thus the use of other chemical preservatives can be minimized, which would be beneficial for environment and consumer health; or a plastic for food preservation can be invented using the antibacterial activity of garlic and cinnamon, the inner wall of the plastic coated with garlic and cinnamon paste.