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Sample records for microbial genes transforming

  1. Functional Analysis and Discovery of Microbial Genes Transforming Metallic and Organic Pollutants: Database and Experimental Tools

    SciTech Connect

    Lawrence P. Wackett; Lynda B.M. Ellis

    2004-12-09

    Microbial functional genomics is faced with a burgeoning list of genes which are denoted as unknown or hypothetical for lack of any knowledge about their function. The majority of microbial genes encode enzymes. Enzymes are the catalysts of metabolism; catabolism, anabolism, stress responses, and many other cell functions. A major problem facing microbial functional genomics is proposed here to derive from the breadth of microbial metabolism, much of which remains undiscovered. The breadth of microbial metabolism has been surveyed by the PIs and represented according to reaction types on the University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD): http://umbbd.ahc.umn.edu/search/FuncGrps.html The database depicts metabolism of 49 chemical functional groups, representing most of current knowledge. Twice that number of chemical groups are proposed here to be metabolized by microbes. Thus, at least 50% of the unique biochemical reactions catalyzed by microbes remain undiscovered. This further suggests that many unknown and hypothetical genes encode functions yet undiscovered. This gap will be partly filled by the current proposal. The UM-BBD will be greatly expanded as a resource for microbial functional genomics. Computational methods will be developed to predict microbial metabolism which is not yet discovered. Moreover, a concentrated effort to discover new microbial metabolism will be conducted. The research will focus on metabolism of direct interest to DOE, dealing with the transformation of metals, metalloids, organometallics and toxic organics. This is precisely the type of metabolism which has been characterized most poorly to date. Moreover, these studies will directly impact functional genomic analysis of DOE-relevant genomes.

  2. Microbial transformation of antimalarial terpenoids.

    PubMed

    Parshikov, Igor A; Netrusov, Alexander I; Sutherland, John B

    2012-01-01

    The fungal and bacterial transformation of terpenoids derived from plant essential oils, especially the sesquiterpenoid artemisinin from Artemisia annua, has produced several new candidate drugs for the treatment of malaria. Obtaining new derivatives of terpenoids, including artemisinin derivatives with increased antimalarial activity, is an important goal of research in microbial biotechnology and medicinal chemistry.

  3. Microbial steroid transformations: current state and prospects.

    PubMed

    Donova, Marina V; Egorova, Olga V

    2012-06-01

    Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.

  4. Microbial sulfur transformations in sediments from Subglacial Lake Whillans.

    PubMed

    Purcell, Alicia M; Mikucki, Jill A; Achberger, Amanda M; Alekhina, Irina A; Barbante, Carlo; Christner, Brent C; Ghosh, Dhritiman; Michaud, Alexander B; Mitchell, Andrew C; Priscu, John C; Scherer, Reed; Skidmore, Mark L; Vick-Majors, Trista J; The Wissard Science Team

    2014-01-01

    Diverse microbial assemblages inhabit subglacial aquatic environments. While few of these environments have been sampled, data reveal that subglacial organisms gain energy for growth from reduced minerals containing nitrogen, iron, and sulfur. Here we investigate the role of microbially mediated sulfur transformations in sediments from Subglacial Lake Whillans (SLW), Antarctica, by examining key genes involved in dissimilatory sulfur oxidation and reduction. The presence of sulfur transformation genes throughout the top 34 cm of SLW sediments changes with depth. SLW surficial sediments were dominated by genes related to known sulfur-oxidizing chemoautotrophs. Sequences encoding the adenosine-5'-phosphosulfate (APS) reductase gene, involved in both dissimilatory sulfate reduction and sulfur oxidation, were present in all samples and clustered into 16 distinct operational taxonomic units. The majority of APS reductase sequences (74%) clustered with known sulfur oxidizers including those within the "Sideroxydans" and Thiobacillus genera. Reverse-acting dissimilatory sulfite reductase (rDSR) and 16S rRNA gene sequences further support dominance of "Sideroxydans" and Thiobacillus phylotypes in the top 2 cm of SLW sediments. The SLW microbial community has the genetic potential for sulfate reduction which is supported by experimentally measured low rates (1.4 pmol cm(-3)d(-1)) of biologically mediated sulfate reduction and the presence of APS reductase and DSR gene sequences related to Desulfobacteraceae and Desulfotomaculum. Our results also infer the presence of sulfur oxidation, which can be a significant energetic pathway for chemosynthetic biosynthesis in SLW sediments. The water in SLW ultimately flows into the Ross Sea where intermediates from subglacial sulfur transformations can influence the flux of solutes to the Southern Ocean.

  5. Microbial Transformations of Actinides and Other Radionuclides

    SciTech Connect

    Francis,A.J.; Dodge, C. J.

    2009-01-07

    Microorganisms can affect the stability and mobility of the actinides and other radionuclides released from nuclear fuel cycle and from nuclear fuel reprocessing plants. Under appropriate conditions, microorganisms can alter the chemical speciation, solubility and sorption properties and thus could increase or decrease the concentrations of radionuclides in solution in the environment and the bioavailability. Dissolution or immobilization of radionuclides is brought about by direct enzymatic action or indirect non-enzymatic action of microorganisms. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of radionuclides have been extensively investigated, we have only limited information on the effects of microbial processes and biochemical mechanisms which affect the stability and mobility of radionuclides. The mechanisms of microbial transformations of the major and minor actinides U, Pu, Cm, Am, Np, the fission products and other radionuclides such as Ra, Tc, I, Cs, Sr, under aerobic and anaerobic conditions in the presence of electron donors and acceptors are reviewed.

  6. Nitrogen transformations in stratified aquatic microbial ecosystems.

    PubMed

    Revsbech, Niels Peter; Risgaard-Petersen, Nils; Schramm, Andreas; Nielsen, Lars Peter

    2006-11-01

    New analytical methods such as advanced molecular techniques and microsensors have resulted in new insights about how nitrogen transformations in stratified microbial systems such as sediments and biofilms are regulated at a microm-mm scale. A large and ever-expanding knowledge base about nitrogen fixation, nitrification, denitrification, and dissimilatory reduction of nitrate to ammonium, and about the microorganisms performing the processes, has been produced by use of these techniques. During the last decade the discovery of anammmox bacteria and migrating, nitrate accumulating bacteria performing dissimilatory reduction of nitrate to ammonium have given new dimensions to the understanding of nitrogen cycling in nature, and the occurrence of these organisms and processes in stratified microbial communities will be described in detail.

  7. Microbial transformation of uranium in wastes

    SciTech Connect

    Francis, A.J.; Dodge, C.J.; Gillow, J.B.; Cline, J.E.; Oak Ridge Y-12 Plant, TN )

    1989-01-01

    Contamination of soils, water, and sediments by radionuclides and toxic metals from the disposal of uranium processing wastes is a major national concern. Although much is known about the physico- chemical aspects of U, we have little information on the effects of aerobic and anaerobic microbial activities on the mobilization or immobilization of U and other toxic metals in mixed wastes. In order to understand the mechanisms of microbial transformations of uranium, we examined a contaminated pond sediment and a sludge sample from the uranium processing facility at Y-12 Plant, Oak Ridge, TN. The uranium concentration in the sediment and sludge samples was 923 and 3080 ug/g dry wt, respectively. In addition to U, the sediment and sludge samples contained high levels of toxic metals such as Cd, Cr, Cu, Hg, Pb, Ni, and Zn. The association of uranium with the various mineral fractions of the sediment and sludge was determined by selective chemical extraction techniques. Uranium was associated to varying degrees with the exchangeable carbonate, iron oxide, organic, and inert fractions in both samples. Initial results in samples amended with carbon and nitrogen indicate immobilization of U due to enhanced indigenous microbial activity under anaerobic conditions. 23 refs., 4 figs., 5 tabs.

  8. Microbial transformation of elements: the case of arsenic and selenium

    USGS Publications Warehouse

    Stolz, J.; Basu, P.; Oremland, R.

    2002-01-01

    Microbial activity is responsible for the transformation of at least one third of the elements in the periodic table. These transformations are the result of assimilatory, dissimilatory, or detoxification processes and form the cornerstones of many biogeochemical cycles. Arsenic and selenium are two elements whose roles in microbial ecology have only recently been recognized. Known as "essential toxins", they are required in trace amounts for growth and metabolism but are toxic at elevated concentrations. Arsenic is used as an osmolite in some marine organisms while selenium is required as selenocysteine (i.e. the twenty-first amino acid) or as a ligand to metal in some enzymes (e.g. FeNiSe hydrogenase). Arsenic resistance involves a small-molecular-weight arsenate reductase (ArsC). The use of arsenic and selenium oxyanions for energy is widespread in prokaryotes with representative organisms from the Crenarchaeota, thermophilic bacteria, low and high G+C gram-positive bacteria, and Proteobacteria. Recent studies have shown that both elements are actively cycled and play a significant role in carbon mineralization in certain environments. The occurrence of multiple mechanisms involving different enzymes for arsenic and selenium transformation indicates several different evolutionary pathways (e.g. convergence and lateral gene transfer) and underscores the environmental significance and selective impact in microbial evolution of these two elements.

  9. Microbial transformation of elements: the case of arsenic and selenium.

    PubMed

    Stolz, J F; Basu, P; Oremland, R S

    2002-12-01

    Microbial activity is responsible for the transformation of at least one third of the elements in the periodic table. These transformations are the result of assimilatory, dissimilatory, or detoxification processes and form the cornerstones of many biogeochemical cycles. Arsenic and selenium are two elements whose roles in microbial ecology have only recently been recognized. Known as "essential toxins", they are required in trace amounts for growth and metabolism but are toxic at elevated concentrations. Arsenic is used as an osmolite in some marine organisms while selenium is required as selenocysteine (i.e. the twenty-first amino acid) or as a ligand to metal in some enzymes (e.g. FeNiSe hydrogenase). Arsenic resistance involves a small-molecular-weight arsenate reductase (ArsC). The use of arsenic and selenium oxyanions for energy is widespread in prokaryotes with representative organisms from the Crenarchaeota, thermophilic bacteria, low and high G+C gram-positive bacteria, and Proteobacteria. Recent studies have shown that both elements are actively cycled and play a significant role in carbon mineralization in certain environments. The occurrence of multiple mechanisms involving different enzymes for arsenic and selenium transformation indicates several different evolutionary pathways (e.g. convergence and lateral gene transfer) and underscores the environmental significance and selective impact in microbial evolution of these two elements.

  10. Phylogenetic Analysis of an Anaerobic, Trichlorobenzene-Transforming Microbial Consortium

    PubMed Central

    von Wintzingerode, Friedrich; Selent, Burkhard; Hegemann, Werner; Göbel, Ulf B.

    1999-01-01

    A culture-independent phylogenetic survey for an anaerobic trichlorobenzene-transforming microbial community was carried out. Small-subunit rRNA genes were PCR amplified from community DNA by using primers specific for Bacteria or Euryarchaeota and were subsequently cloned. Application of a new hybridization-based screening approach revealed 51 bacterial clone families, one of which was closely related to dechlorinating Dehalobacter species. Several clone sequences clustered to rDNA sequences obtained from a molecular study of an anaerobic aquifer contaminated with hydrocarbons and chlorinated solvents (Dojka et al., Appl. Env. Microbiol. 64:3869–3877, 1998). PMID:9872791

  11. Uses of antimicrobial genes from microbial genome

    DOEpatents

    Sorek, Rotem; Rubin, Edward M.

    2013-08-20

    We describe a method for mining microbial genomes to discover antimicrobial genes and proteins having broad spectrum of activity. Also described are antimicrobial genes and their expression products from various microbial genomes that were found using this method. The products of such genes can be used as antimicrobial agents or as tools for molecular biology.

  12. [Soil microbial ecological process and microbial functional gene diversity].

    PubMed

    Zhang, Jing; Zhang, Huiwen; Li, Xinyu; Su, Zhencheng; Zhang, Chenggang

    2006-06-01

    Soil microbes in terrestrial ecosystem carry out a series of important ecological functions, such as geo-chemical cycling of elements, degradation of pollutants, and buffering to the acute changes of environment, etc. Soil microbial ecological function has a close relation with soil function, and the changes in the structure and composition of soil microbial populations can directly affect the realization of soil function. Through their produced enzymes, soil microbes take part in a series of metabolic activities, and the functional genes of coded enzymes are the functional markers of microbes. In recent ten years, molecular ecology focusing on the functional gene diversity has been developed rapidly, which gives us a new cut-in point to understand soil microbial ecological function from the point of functional gene. This paper reviewed the research advances in the functional gene diversity correlated to soil microbial ecological function, with the perspectives in this field discussed.

  13. Microbial monoterpene transformations-a review.

    PubMed

    Marmulla, Robert; Harder, Jens

    2014-01-01

    Isoprene and monoterpenes constitute a significant fraction of new plant biomass. Emission rates into the atmosphere alone are estimated to be over 500 Tg per year. These natural hydrocarbons are mineralized annually in similar quantities. In the atmosphere, abiotic photochemical processes cause lifetimes of minutes to hours. Microorganisms encounter isoprene, monoterpenes, and other volatiles of plant origin while living in and on plants, in the soil and in aquatic habitats. Below toxic concentrations, the compounds can serve as carbon and energy source for aerobic and anaerobic microorganisms. Besides these catabolic reactions, transformations may occur as part of detoxification processes. Initial transformations of monoterpenes involve the introduction of functional groups, oxidation reactions, and molecular rearrangements catalyzed by various enzymes. Pseudomonas and Rhodococcus strains and members of the genera Castellaniella and Thauera have become model organisms for the elucidation of biochemical pathways. We review here the enzymes and their genes together with microorganisms known for a monoterpene metabolism, with a strong focus on microorganisms that are taxonomically validly described and currently available from culture collections. Metagenomes of microbiomes with a monoterpene-rich diet confirmed the ecological relevance of monoterpene metabolism and raised concerns on the quality of our insights based on the limited biochemical knowledge.

  14. Microbial Aldicarb Transformation in Aquifer, Lake, and Salt Marsh Sediments

    PubMed Central

    Kazumi, J.; Capone, D. G.

    1995-01-01

    The microbial transformation of [N-methyl-(sup14)C]aldicarb, a carbamate pesticide, occurred in aquifer, lake, and salt marsh sediments. Microbial degradation of aldicarb took place within 21 days in aquifer sediments from sites previously exposed to aldicarb (Jamesport, Long Island, N.Y.) but did not occur in sediments which were not previously exposed (Connetquot State Park, Long Island, N.Y.). At the Jamesport sites, higher aldicarb transformation rates occurred in deep, anoxic sediments than in shallow, oxic sediments. There was a significant negative relationship (P < 0.05) between transformation rates and ambient dissolved O(inf2) levels. Aldicarb hydrolysis rates in Jamesport sediments were 10- to 1,000-fold lower than rates previously reported for soils. In addition, aldicarb degradation rates were not significantly correlated with measurements of bacterial activity and density previously determined in the same sediments. Substantially higher aldicarb degradation rates were found in anoxic lake and salt marsh than in aquifer sediments. Furthermore, we investigated the anaerobic microbial processes involved in aldicarb transformation by adding organic substrates (acetate, glucose), an alternative electron acceptor (nitrate), and microbial inhibitors (molybdate, 2-bromoethanesulfonic acid) to anoxic aquifer, lake, and salt marsh sediments. The results suggest that a methanogenic consortium was important in aldicarb transformation or in the use of aldicarb-derived products such as methylamine. In addition, microbial aldicarb transformation proceeded via different pathways under oxic and anoxic conditions. In the presence of O(inf2), aldicarb transformation was mainly via an oxidation pathway, while in the absence of O(inf2), degradation took place through a hydrolytic pathway (including the formation of methylamine precursors). Under anoxic conditions, therefore, aldicarb can be transformed by microbial consortia to yield products which can be of direct

  15. Microbial Transformation of Quercetin by Bacillus cereus

    PubMed Central

    Rao, Koppaka V.; Weisner, Nghe T.

    1981-01-01

    Biotransformation of quercetin was examined with a number of bacterial cultures. In the presence of a bacterial culture (Bacillus cereus), quercetin was transformed into two crystalline products, identified as protocatechuic acid and quercetin-3-glucoside (isoquercitrin). PMID:16345844

  16. MICROBIAL TRANSFORMATIONS OF URANIUM COMPLEXED WITH ORGANIC AND INORGANIC LIGANDS.

    SciTech Connect

    FRANCIS,A.J.

    2002-09-15

    Biotransformation of various chemical forms of uranium present in wastes, contaminated soils and materials by microorganisms under different process conditions such as aerobic and anaerobic (denitrifying, iron-reducing, fermentative, and sulfate-reducing) conditions will affect the solubility, bioavailability, and mobility of uranium in the natural environment. Fundamental understanding of the mechanisms of microbial transformations of uranium under a variety of environmental conditions will be useful in developing appropriate remediation and waste management strategies as well as predicting the microbial impacts on the long-term stewardship of contaminated sites.

  17. Microbial Fuel Cell Transformation of Recalcitrant Organic Compounds in Support of Biosensor Research

    DTIC Science & Technology

    2014-03-27

    Microbial Fuel Cell Transformation of Recalcitrant Organic Compounds in Support of Biosensor ...in the United States. AFIT-ENV-14-M-62 Microbial Fuel Cell Transformation of Recalcitrant Organic Compounds in Support of Biosensor Research...DISTRIBUTION UNLIMITED AFIT-ENV-14-M-62 Microbial Fuel Cell Transformation of Recalcitrant Organic Compounds in Support of Biosensor Research Marc

  18. Mass effects meet species sorting: transformations of microbial assemblages in epiphreatic subsurface karst water pools.

    PubMed

    Shabarova, Tanja; Widmer, Franco; Pernthaler, Jakob

    2013-09-01

    We investigated the transformations of the microbial communities in epiphreatic karst cave pools with different flooding frequencies. Fingerprinting of 16S rRNA genes was combined with microscopic and sequence analysis to examine if source water would transport comparable microbial inocula into the pools at consecutive flood events, and to assess possible effects of residence time on the microbial assemblages during stagnant periods. Variability in the concentrations of dissolved organic carbon and conductivity indicated differences between floods and changes of pool water over time. High numbers of Betaproteobacteria affiliated with Methylophilaceae and Comamonadaceae were introduced into the pools during floodings. While the former persisted in the pools, the latter exhibited considerable microdiversification. These Betaproteobacteria might thus represent core microbial groups in karst water. A decrease in the estimated total diversity of the remaining bacterial taxa was apparent after a few weeks of residence: Some were favoured by stagnant conditions, whereas the majority was rapidly outcompeted. Thus, the microbial communities consisted of different components governed by complementary assembly mechanisms (dispersal versus environmental filtering) upon introduction into the pools. High overlap of temporary and persistent community members between samplings from two winters, moreover, reflected the seasonal recurrence of the studied microbial assemblages.

  19. Anaerobic microbial transformations of radioactive wastes in subsurface environments

    SciTech Connect

    Francis, A.J.

    1984-01-01

    Radioactive wastes disposed of in subsurface environments contain a variety of radionuclides and organic compounds. Microorganisms play a major role in the transformation of organic and inorganic constituents of the waste and are partly responsible for the problems encountered at the waste disposal sites. These include microbial degradation of waste forms resulting in trench cover subsidence, migration of radionuclides, and production of radioactive gases such as /sup 14/CO/sub 2/, /sup 14/CH/sub 4/, HT, and CH/sub 3/T. Microbial processes involved in solubilization, mobilization, and immobilization of toxic metals under aerobic and anaerobic conditions are reviewed. Complexing agents and several organic acids produced by microbial action affect mobilization of radionuclides and heavy metals from the wastes. Microorganisms play a significant role in the transformation and cycling of tritium in the environment by (i) oxidation of tritium and tritiated methane under aerobic conditions and (ii) production of tritium and tritiated methane from wastes containing tritiated water and organic compounds under anaerobic conditions. 23 references, 2 figures, 2 tables.

  20. MICROBIAL TRANSFORMATIONS OF RADIONUCLIDES RELEASED FROM NUCLEAR FUEL REPROCESSING PLANTS.

    SciTech Connect

    FRANCIS,A.J.

    2006-10-18

    Microorganisms can affect the stability and mobility of the actinides U, Pu, Cm, Am, Np, and the fission products Tc, I, Cs, Sr, released from nuclear fuel reprocessing plants. Under appropriate conditions, microorganisms can alter the chemical speciation, solubility and sorption properties and thus could increase or decrease the concentrations of radionuclides in solution and the bioavailability. Dissolution or immobilization of radionuclides is brought about by direct enzymatic action or indirect non-enzymatic action of microorganisms. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of radionuclides have been investigated, we have only limited information on the effects of microbial processes. The mechanisms of microbial transformations of the major and minor actinides and the fission products under aerobic and anaerobic conditions in the presence of electron donors and acceptors are reviewed.

  1. Microbially driven Fenton reaction for transformation of pentachlorophenol

    SciTech Connect

    McKinzi, A.M.; Dichristina, T.J.

    1999-06-01

    A microbially driven transformation system was developed for the oxidative degradation of pentachlorophenol (PCP). The system was based on a free radical-generating Fenton reaction between bacterially produced Fe(II) and H{sub 2}O{sub 2}. The Fe(III)-reducing, facultative anaerobe Shewanella putrefaciens strain 200 was used as a catalyst for both Fe(III) reduction and H{sub 2}O{sub 2} production by alternating between anaerobic and aerobic conditions in liquid batch cultures supplemented with Fe(III). The highest observed PCP degradation rate was approximately 0.31 {micro}M h{sup {minus}1}. Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC) were formed as the principal PCP transformation products, indicating that PCP oxidation proceeded via hydroxyl radical ({sup {sm_bullet}}OH) attack on the ortho and para positions of the aromatic ring. PCP was degraded, and TCHQ and TCC were produced in a chemically driven (biomimetic) system where H{sub 2}O{sub 2} and Fe(II) were supplied at concentrations comparable to those detected in the microbially driven system. PCP was not degraded (and PCP transformation products were not produced) in a set of control experiments that included (1) the presence of Fe(II)-chelating agents or radical scavenging compounds, (2) strict aerobic or anaerobic conditions, (3) the substitution of NO{sub 3}{sup {minus}} for Fe(III) as anaerobic electron acceptor, and (4) the omission of S. putrefaciens. The microbially driven Fenton reaction system operated at neutral pH and required neither addition of exogenous H{sub 2}O{sub 2} nor UV irradiation to regenerate Fe(II). The newly developed system may provide the basis for novel Fenton-type bioremediation strategies.

  2. Microbial transformations of halolactones with p-menthane system.

    PubMed

    Mazur, Marcelina; Grudniewska, Aleksandra; Wawrzeńczyk, Czesław

    2015-01-01

    Biologically active piperitone-derived racemic iodo-, bromo- and chlorolactones (1-3) were transformed with the use of microbial enzymatic systems. Four strains of filamentous fungi Absidia glauca AM254, Absidia cylindrospora AM336, Mortierella vinaceae AM149 and Nigrospora oryzae AM8 transformed halolactones (1-3) to four new halohydroxylactones (4-7). In all biotransformations the hydroxy group was incorporated in inactivated methine carbon atom at isopropyl substituent. In N. oryzae AM8 culture the bromolactone with additional hydroxy group in α-position, relative to CO bond in γ-lactone ring, was also formed as a product. The structures of new compounds were established on the basis of spectral data.

  3. Microbial transformations of arsenic: Mobilization from glauconitic sediments to water

    USGS Publications Warehouse

    Mumford, Adam C.; Barringer, Julia L.; Benzel, William M.; Reilly, Pamela A.; Young, L.Y.

    2012-01-01

    In the Inner Coastal Plain of New Jersey, arsenic (As) is released from glauconitic sediment to carbon- and nutrient-rich shallow groundwater. This As-rich groundwater discharges to a major area stream. We hypothesize that microbes play an active role in the mobilization of As from glauconitic subsurface sediments into groundwater in the Inner Coastal Plain of New Jersey. We have examined the potential impact of microbial activity on the mobilization of arsenic from subsurface sediments into the groundwater at a site on Crosswicks Creek in southern New Jersey. The As contents of sediments 33–90 cm below the streambed were found to range from 15 to 26.4 mg/kg, with siderite forming at depth. Groundwater beneath the streambed contains As at concentrations up to 89 μg/L. Microcosms developed from site sediments released 23 μg/L of As, and active microbial reduction of As(V) was observed in microcosms developed from site groundwater. DNA extracted from site sediments was amplified with primers for the 16S rRNA gene and the arsenate respiratory reductase gene, arrA, and indicated the presence of a diverse anaerobic microbial community, as well as the presence of potential arsenic-reducing bacteria. In addition, high iron (Fe) concentrations in groundwater and the presence of iron-reducing microbial genera suggests that Fe reduction in minerals may provide an additional mechanism for release of associated As, while arsenic-reducing microorganisms may serve to enhance the mobility of As in groundwater at this site.

  4. Microbial Transformations of Selenium Species of Relevance to Bioremediation

    PubMed Central

    Eswayah, Abdurrahman S.; Smith, Thomas J.

    2016-01-01

    Selenium species, particularly the oxyanions selenite (SeO32−) and selenate (SeO42−), are significant pollutants in the environment that leach from rocks and are released by anthropogenic activities. Selenium is also an essential micronutrient for organisms across the tree of life, including microorganisms and human beings, particularly because of its presence in the 21st genetically encoded amino acid, selenocysteine. Environmental microorganisms are known to be capable of a range of transformations of selenium species, including reduction, methylation, oxidation, and demethylation. Assimilatory reduction of selenium species is necessary for the synthesis of selenoproteins. Dissimilatory reduction of selenate is known to support the anaerobic respiration of a number of microorganisms, and the dissimilatory reduction of soluble selenate and selenite to nanoparticulate elemental selenium greatly reduces the toxicity and bioavailability of selenium and has a major role in bioremediation and potentially in the production of selenium nanospheres for technological applications. Also, microbial methylation after reduction of Se oxyanions is another potentially effective detoxification process if limitations with low reaction rates and capture of the volatile methylated selenium species can be overcome. This review discusses microbial transformations of different forms of Se in an environmental context, with special emphasis on bioremediation of Se pollution. PMID:27260359

  5. Microbial Transformation of Ibuprofen by a Nocardia Species

    PubMed Central

    Chen, Yijun; Rosazza, John P. N.

    1994-01-01

    The carboxylic acid functional group of ibuprofen [α-methyl-4-(2-methylpropyl) benzene acetic acid] is reduced to the corresponding alcohol and subsequently esterified to the acetate derivative by cultures of Nocardia species strain NRRL 5646. The alcohol and ester microbial transformation products were isolated, and their structures were determined by 1H and 13C nuclear magnetic resonance spectroscopy and mass spectrometry. By derivatization of synthetic and microbiologically produced ibuprofen alcohols with S(+)-O-acetylmandelic acid, nuclear magnetic resonance analysis indicated that the carboxylic acid reductase of Nocardia sp. is R enantioselective, giving alcohol products with an enantiomeric excess of 61.2%. The R enantioselectivity of the carboxylic acid reductase enzyme system was confirmed by using cell extracts together with ATP and NADPH in the reduction of isomeric ibuprofens. PMID:16349237

  6. Microbial Transformation of TRU and Mixed Wastes: Actinide Speciation and Waste Volume Reduction

    SciTech Connect

    Halada, Gary P.

    2004-12-01

    I. To characterize the biodegradation of cellulosic materials using Fourier Transform Infrared (FTIR) Spectroscopy. II. To develop an electrochemical/spectroscopic methodology to characterize TRU waste microbial transformation III. To develop molecular models of TRU complexes in order to understand microbial transformation In all cases, objectives are designed to compliment the efforts from other team members, and will be periodically coordinated through the lead P.I. at Brookhaven National Laboratory (BNL), A.J. Francis.

  7. Microbial transformation from normal oral microbiota to acute endodontic infections

    PubMed Central

    2012-01-01

    Background Endodontic infections are a leading cause of oro-facial pain and tooth loss in western countries, and may lead to severe life-threatening infections. These infections are polymicrobial with high bacterial diversity. Understanding the spatial transition of microbiota from normal oral cavities through the infected root canal to the acute periapical abscess can improve our knowledge of the pathogenesis of endodontic infections and lead to more effective treatment. We obtained samples from the oral cavity, infected root canal and periapical abscess of 8 patients (5 with localized and 3 with systemic infections). Microbial populations in these samples were analyzed using next-generation sequencing of 16S rRNA amplicons. Bioinformatics tools and statistical tests with rigorous criteria were used to elucidate the spatial transition of the microbiota from normal to diseased sites. Results On average, 10,000 partial 16S rRNA gene sequences were obtained from each sample. All sequences fell into 11 different bacterial phyla. The microbial diversity in root canal and abscess samples was significantly lower than in the oral samples. Streptococcus was the most abundant genus in oral cavities while Prevotella and Fusobacterium were most abundant in diseased samples. The microbiota community structures of root canal and abscess samples were, however, more similar to each other than to the oral cavity microbiota. Using rigorous criteria and novel bioinformatics tools, we found that Granulicatella adiacens, Eubacterium yurii, Prevotella melaninogenica, Prevotella salivae, Streptococcus mitis, and Atopobium rimae were over-represented in diseased samples. Conclusions We used a novel approach and high-throughput methodologies to characterize the microbiota associated normal and diseased oral sites in the same individuals. PMID:22839737

  8. Microbial transformation of low-level radioactive waste

    SciTech Connect

    Francis, A.J.

    1980-06-01

    Microorganisms play a significant role in the transformation of the radioactive waste and waste forms disposed of at shallow-land burial sites. Microbial degradation products of organic wastes may influence the transport of buried radionuclides by leaching, solubilization, and formation of organoradionuclide complexes. The ability of indigenous microflora of the radioactive waste to degrade the organic compounds under aerobic and anaerobic conditions was examined. Leachate samples were extracted with methylene chloried and analyzed for organic compounds by gas chromatography and mass spectrometry. In general, several of the organic compounds in the leachates were degraded under aerobic conditions. Under anaerobic conditions, the degradation of the organics was very slow, and changes in concentrations of several acidic compounds were observed. Several low-molecular-weight organic acids are formed by breakdown of complex organic materials and are further metabolized by microorganisms; hence these compounds are in a dynamic state, being both synthesized and destroyed. Tributyl phosphate, a compound used in the extraction of metal ions from solutions of reactor products, was not degraded under anaerobic conditions.

  9. New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone

    PubMed Central

    Bano, Saira; Wahab, Atia-tul-; Yousuf, Sammer; Jabeen, Almas; Mesaik, Mohammad Ahmed; Rahman, Atta-ur-; Choudhary, M. Iqbal

    2016-01-01

    Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC50 = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC50 values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC50 < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC50 = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines. PMID:27104348

  10. ras gene Amplification and malignant transformation.

    PubMed Central

    Pulciani, S; Santos, E; Long, L K; Sorrentino, V; Barbacid, M

    1985-01-01

    Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia. Images PMID:3915535

  11. Plant transformation via pollen tube-mediated gene transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  12. Predictions of Gene Family Distributions in Microbial Genomes: Evolution by Gene Duplication and Modification

    NASA Astrophysics Data System (ADS)

    Yanai, Itai; Camacho, Carlos J.; Delisi, Charles

    2000-09-01

    A universal property of microbial genomes is the considerable fraction of genes that are homologous to other genes within the same genome. The process by which these homologues are generated is not well understood, but sequence analysis of 20 microbial genomes unveils a recurrent distribution of gene family sizes. We show that a simple evolutionary model based on random gene duplication and point mutations fully accounts for these distributions and permits predictions for the number of gene families in genomes not yet complete. Our findings are consistent with the notion that a genome evolves from a set of precursor genes to a mature size by gene duplications and increasing modifications.

  13. IFIM: a database of integrated fitness information for microbial genes

    PubMed Central

    Wei, Wen; Ye, Yuan-Nong; Luo, Sen; Deng, Yan-Yan; Lin, Dan; Guo, Feng-Biao

    2014-01-01

    Knowledge of an organism’s fitness for survival is important for a complete understanding of microbial genetics and effective drug design. Current essential gene databases provide only binary essentiality data from genome-wide experiments. We therefore developed a new database that Integrates quantitative Fitness Information for Microbial genes (IFIM). The IFIM database currently contains data from 16 experiments and 2186 theoretical predictions. The highly significant correlation between the experiment-derived fitness data and our computational simulations demonstrated that the computer-generated predictions were often as reliable as the experimental data. The data in IFIM can be accessed easily, and the interface allows users to browse through the gene fitness information that it contains. IFIM is the first resource that allows easy access to fitness data of microbial genes. We believe this database will contribute to a better understanding of microbial genetics and will be useful in designing drugs to resist microbial pathogens, especially when experimental data are unavailable. Database URL: http://cefg.uestc.edu.cn/ifim/ or http://cefg.cn/ifim/ PMID:24923821

  14. MICROBIAL TRANSFORMATIONS OF TRU AND MIXED WASTES: ACTINIDE SPECIATION AND WASTE VOLUME REDUCTION.

    SciTech Connect

    FRANCIS, A.J.; DODGE, C.J.

    2006-11-16

    The overall goals of this research project are to determine the mechanism of microbial dissolution and stabilization of actinides in Department of Energy's (DOE) TRU wastes, contaminated sludges, soils, and sediments. This includes (1) investigations on the fundamental aspects of microbially catalyzed radionuclide and metal transformations (oxidation/reduction reactions, dissolution, precipitation, chelation); (2) understanding of the microbiological processes that control speciation and alter the chemical forms of complex inorganic/organic contaminant mixtures; and (3) development of new and improved microbially catalyzed processes resulting in immobilization of metals and radionuclides in the waste with concomitant waste volume reduction.

  15. MICROBIAL TRANSFORMATIONS OF TRU AND MIXED WASTES: ACTINIDE SPECIATION AND WASTE VOLUME REDUCTION

    SciTech Connect

    Francis, A.J.; Dodge, C.J.

    2006-06-01

    The overall goals of this research project are to determine the mechanism of microbial dissolution and stabilization of actinides in Department of Energy’s (DOE) TRU wastes, contaminated sludges, soils, and sediments. This includes (i) investigations on the fundamental aspects of microbially catalyzed radionuclide and metal transformations (oxidation/reduction reactions, dissolution, precipitation, chelation); (ii) understanding of the microbiological processes that control speciation and alter the chemical forms of complex inorganic/organic contaminant mixtures; and (iii) development of new and improved microbially catalyzed processes resulting in immobilization of metals and radionuclides in the waste with concomitant waste volume reduction.

  16. MICROBIAL TRANSFORMATIONS OF TRU AND MIXED WASTES: ACTINIDE SPECIATION AND WASTE VOLUME REDUCTION

    SciTech Connect

    Francis, A.J.; Dodge, C.J.

    2006-06-01

    The overall goals of this research project are to determine the mechanism of microbial dissolution and stabilization of actinides in Department of Energy's (DOE) TRU wastes, contaminated sludges, soils, and sediments. This includes (1) investigations on the fundamental aspects of microbially catalyzed radionuclide and metal transformations (oxidation/reduction reactions, dissolution, precipitation, chelation); (2) understanding of the microbiological processes that control speciation and alter the chemical forms of complex inorganic/organic contaminant mixtures; and (3) development of new and improved microbially catalyzed processes resulting in immobilization of metals and radionuclides in the waste with concomitant waste volume reduction.

  17. MICROBIAL TRANSFORMATIONS OF PLUTONIUM AND IMPLICATIONS FOR ITS MOBILITY.

    SciTech Connect

    FRANCIS, A.J.

    2000-09-30

    The current state of knowledge of the effect of plutonium on microorganisms and microbial activity is reviewed, and also the microbial processes affecting its mobilization and immobilization. The dissolution of plutonium is predominantly due to their production of extracellular metabolic products, organic acids, such as citric acid, and sequestering agents, such as siderophores. Plutonium may be immobilized by the indirect actions of microorganisms resulting in changes in Eh and its reduction from a higher to lower oxidation state, with the precipitation of Pu, its bioaccumulation by biomass, and bioprecipitation reactions. In addition, the abundance of microorganisms in Pu-contaminated soils, wastes, natural analog sites, and backfill materials that will be used for isolating the waste and role of microbes as biocolloids in the transport of Pu is discussed.

  18. Biased gene transfer in microbial evolution.

    PubMed

    Andam, Cheryl P; Gogarten, J Peter

    2011-06-13

    Horizontal gene transfer (HGT) is an important evolutionary process that allows the spread of innovations between distantly related organisms. We present evidence that prokaryotes (bacteria and archaea) are more likely to transfer genetic material with their close relatives than with distantly related lineages. This bias in transfer partners can create phylogenetic signals that are difficult to distinguish from the signal created through shared ancestry. Preferences for transfer partners can be revealed by studying the distribution patterns of divergent genes with identical functions. In many respects, these genes are similar to alleles in a population, except that they coexist only in higher taxonomic groupings and are acquired by a species through HGT. We also discuss the role of biased gene transfer in the formation of taxonomically recognizable natural groups in the tree or net of life.

  19. Long- and short-term temperature responses of microbially-mediated boreal soil organic matter transformations

    NASA Astrophysics Data System (ADS)

    Min, K.; Buckeridge, K. M.; Edwards, K. A.; Ziegler, S. E.; Billings, S. A.

    2015-12-01

    Microorganisms use exoenzymes to decay soil organic matter into assimilable substrates, some of which are transformed into CO2. Microbial CO2 efflux contributes up to 60% of soil respiration, a feature that can change with temperature due to altered exoenzyme activities (short-term) and microbial communities producing different exoenzymes (longer-term). Often, however, microbial temperature responses are masked by factors that also change with temperature in soil, making accurate projections of microbial CO2 efflux with warming challenging. Using soils along a natural climate gradient similar in most respects except for temperature regime (Newfoundland Labrador Boreal Ecosystem Latitudinal Transect), we investigated short-vs. long-term temperature responses of microbially-mediated organic matter transformations. While incubating soils at 5, 15, and 25°C for 84 days, we measured exoenzyme activities, CO2 efflux rates and biomass, and extracted DNA at multiple times. We hypothesized that short-term, temperature-induced increases in exoenzyme activities and CO2 losses would be smaller in soils from warmer regions, because microbes presumably adapted to warmer regions should use assimilable substrates more efficiently and thus produce exoenzymes at a lower rate. While incubation temperature generally induced greater exoenzyme activities (p<0.001), exoenzymes' temperature responses depended on enzymes and regions (p<0.001). Rate of CO2 efflux was affected by incubation temperature (P<0.001), but not by region. Microbial biomass and DNA sequencing will reveal how microbial community abundance and composition change with short-vs. longer-term temperature change. Though short-term microbial responses to temperature suggest higher CO2 efflux and thus lower efficiency of resource use with warming, longer-term adaptations of microbial communities to warmer climates remain unknown; this work helps fill that knowledge gap.

  20. Microbial Transformation of β-Ionone and β-Methylionone

    PubMed Central

    Mikami, Yoichi; Fukunaga, Yumiko; Arita, Masatoshi; Kisaki, Takuro

    1981-01-01

    Aspergillus niger JTS 191 was selected from many microorganisms tested as capable of converting ionones to other compounds having aromas. The individual transformation products from β-ionone were isolated and identified by comparison with synthetically derived compounds. The major products were (R)-4-hydroxy-β-ionone and (S)-2-hydroxy-β-ionone. 2-Oxo-, 4-oxo-, 3,4-dehydro-, 2,3-dehydro-4-oxo-, 3,4-dehydro-2-oxo-, (S)-2-acetoxy-, (R)-4-acetoxy-, and 5,6-epoxy-β-ionone and 4-(2,3,6-trimethylphenyl)-but-3-en-2-one were also identified. Analogous transformation products of β-methylionone also were identified. Based on gas-liquid chromatographic analysis during the fermentation, we propose two main oxidative pathways of β-ionone. The results of this study suggest that these transformations of β-ionones may be useful as tobacco-flavoring compounds. PMID:16345730

  1. Microbial exudate promoted dissolution and transformation of chromium containing minerals

    NASA Astrophysics Data System (ADS)

    Saad, E. M.; Sun, J.; Tang, Y.

    2015-12-01

    Because of its utility in many industrial processes, chromium has become the second most common metal contaminant in the United States. The two most common oxidation states of chromium in nature are Cr(III), which is highly immobile, and Cr(VI), which is highly mobile and toxic. In both natural and engineered environments, the most common remediation of Cr(VI) is through reduction, which results in chromium sequestration in the low solubility mixed Cr(III)-Fe(III) (oxy)hydroxide phases. Consequently, the stability of these minerals must be examined to assess the fate of chromium in the subsurface. We examined the dissolution of mixed Cr(III)-Fe(III) (oxy)hydroxides in the presence of common microbial exudates, including the siderophore desferrioxamine B (DFOB; a common organic ligand secreted by most microbes with high affinity for ferric iron and other trivalent metal ions) and oxalate (a common organic acid produced by microbes). The solids exhibited incongruent dissolution with preferential leaching of Fe from the solid phase. Over time, this leads to a more Cr rich mineral, which is known to be more soluble than the corresponding mixed mineral phase. We are currently investigating the structure of the reacted mineral phases and soluble Cr(III) species, as well as the potential oxidation and remobilization of the soluble Cr species. Results from this study will provide insights regarding the long term transport and fate of chromium in the natural environment in the presence of microbial activities.

  2. Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation.

    PubMed

    Keen, Eric C; Bliskovsky, Valery V; Malagon, Francisco; Baker, James D; Prince, Jeffrey S; Klaus, James S; Adhya, Sankar L

    2017-01-17

    Bacteriophages infect an estimated 10(23) to 10(25) bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub "superspreaders," releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications.

  3. Microbial Transformations of Nitrogen, Sulfur, and Iron Dictate Vegetation Composition in Wetlands: A Review

    PubMed Central

    Lamers, Leon P. M.; van Diggelen, Josepha M. H.; Op den Camp, Huub J. M.; Visser, Eric J. W.; Lucassen, Esther C. H. E. T.; Vile, Melanie A.; Jetten, Mike S. M.; Smolders, Alfons J. P.; Roelofs, Jan G. M.

    2012-01-01

    The majority of studies on rhizospheric interactions focus on pathogens, mycorrhizal symbiosis, or carbon transformations. Although the biogeochemical transformations of N, S, and Fe have profound effects on vegetation, these effects have received far less attention. This review, meant for microbiologists, biogeochemists, and plant scientists includes a call for interdisciplinary research by providing a number of challenging topics for future ecosystem research. Firstly, all three elements are plant nutrients, and microbial activity significantly changes their availability. Secondly, microbial oxidation with oxygen supplied by radial oxygen loss from roots in wetlands causes acidification, while reduction using alternative electron acceptors leads to generation of alkalinity, affecting pH in the rhizosphere, and hence plant composition. Thirdly, reduced species of all three elements may become phytotoxic. In addition, Fe cycling is tightly linked to that of S and P. As water level fluctuations are very common in wetlands, rapid changes in the availability of oxygen and alternative terminal electron acceptors will result in strong changes in the prevalent microbial redox reactions, with significant effects on plant growth. Depending on geological and hydrological settings, these interacting microbial transformations change the conditions and resource availability for plants, which are both strong drivers of vegetation development and composition by changing relative competitive strengths. Conversely, microbial composition is strongly driven by vegetation composition. Therefore, the combination of microbiological and plant ecological knowledge is essential to understand the biogeochemical and biological key factors driving heterogeneity and total (i.e., microorganisms and vegetation) community composition at different spatial and temporal scales. PMID:22539932

  4. Microbial transformations of natural organic compounds and radionuclides in subsurface environments

    SciTech Connect

    Francis, A.J.

    1985-10-01

    A major national concern in the subsurface disposal of energy wastes is the contamination of ground and surface waters by waste leachates containing radionuclides, toxic metals, and organic compounds. Microorganisms play an important role in the transformation of organic compounds, radionuclides, and toxic metals present in the waste and affect their mobility in subsurface environments. Microbial processes involved in dissolution, mobilization, and immobilization of toxic metals under aerobic and anaerobic conditions are briefly reviewed. Metal complexing agents and several organic acids produced by microbial action affect mobilization of radionuclides and toxic metals in subsurface environments. Information on the persistence of and biodegradation rates of synthetic as well as microbiologically produced complexing agents is scarce but important in determining the mobility of metal organic complexes in subsoils. Several gaps in knowledge in the area of microbial transformation of naturally occurring organics, radionuclides, and toxic metals have been identified, and further basic research has been suggested. 31 refs., 1 fig., 3 tabs.

  5. Microbial transformation and sorption of anthracene in liquid culture.

    PubMed

    Hadibarata, Tony; Zubir, Meor Mohd Fikri Ahmad; Rubiyatno; Chuang, Teh Zee

    2013-09-01

    Armillaria sp. F022, a white-rot fungus isolated from decayed wood in tropical rain forest was used to biodegrade anthracene in cultured medium. The percentage of anthracene removal by Armillaria sp. F022 reached 13 % after 7 days and at the end of the experiment, anthracene removal level was at 87 %. The anthracene removal through sorption and transformation was investigated. 69 % of eliminated anthracene was transformed by Armillaria sp. F022 to form other organic structure, while only 18 % was absorbed in the mycelia. In the kinetic experiment, anthracene dissipation will not stop even though the biomass had stopped growing. Anthracene removal by Armillaria sp. F022 was correlated with protein concentration (whole biomass) in the culture. The production of enzyme was affected by biomass production. Anthracene was transformed to two stable metabolic products. The metabolites were extracted in ethyl-acetate, isolated by column chromatography, and then identified using gas chromatography-mass spectrometry (GC-MS).

  6. The landscape of microbial phenotypic traits and associated genes

    PubMed Central

    Brbić, Maria; Piškorec, Matija; Vidulin, Vedrana; Kriško, Anita; Šmuc, Tomislav; Supek, Fran

    2016-01-01

    Bacteria and Archaea display a variety of phenotypic traits and can adapt to diverse ecological niches. However, systematic annotation of prokaryotic phenotypes is lacking. We have therefore developed ProTraits, a resource containing ∼545 000 novel phenotype inferences, spanning 424 traits assigned to 3046 bacterial and archaeal species. These annotations were assigned by a computational pipeline that associates microbes with phenotypes by text-mining the scientific literature and the broader World Wide Web, while also being able to define novel concepts from unstructured text. Moreover, the ProTraits pipeline assigns phenotypes by drawing extensively on comparative genomics, capturing patterns in gene repertoires, codon usage biases, proteome composition and co-occurrence in metagenomes. Notably, we find that gene synteny is highly predictive of many phenotypes, and highlight examples of gene neighborhoods associated with spore-forming ability. A global analysis of trait interrelatedness outlined clusters in the microbial phenotype network, suggesting common genetic underpinnings. Our extended set of phenotype annotations allows detection of 57 088 high confidence gene-trait links, which recover many known associations involving sporulation, flagella, catalase activity, aerobicity, photosynthesis and other traits. Over 99% of the commonly occurring gene families are involved in genetic interactions conditional on at least one phenotype, suggesting that epistasis has a major role in shaping microbial gene content. PMID:27915291

  7. A novel Nrf2 activator from microbial transformation inhibits radiation-induced dermatitis in mice.

    PubMed

    Nakagami, Yasuhiro; Masuda, Kayoko

    2016-09-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that regulates many antioxidants, and we have recently succeeded in obtaining a novel Nrf2 activator, RS9, from microbial transformation. RS9 is categorized as a triterpenoid, and well-known triterpenoids such as RTA 402 (bardoxolone methyl) and RTA 408 have been tested in clinical trials. RTA 408 lotion is currently being tested in patients at risk for radiation dermatitis. This prompted us to study the profiles of RS9 in the skin. All the above triterpenoids increased the level of an Nrf2-targeted gene, NADPH:quinone oxidoreductase-1, in normal human epidermal keratinocytes. Among them, the activity of RS9 was prominent; furthermore, the cellular toxicity was less compared with RTA compounds. BALB/c mice were irradiated with 30 Gy/day on Day 0, and compounds were topically applied on the back once daily from Day 1 to Day 30. Dermatitis scores peaked on Day 18, with a score of 2.6 in vehicle-treated mice, and topical applications of 0.1% RTA 402, RTA 408 and RS9 reduced the scores to 1.8, 2.0 and 1.4, respectively. Moreover, the percentage of animals with scores ≥2 was analyzed, and 0.1% RS9 suppressed the percentage from 100% to 47%. These results imply that RS9 has potential efficacy for treating radiation dermatitis.

  8. A novel Nrf2 activator from microbial transformation inhibits radiation-induced dermatitis in mice

    PubMed Central

    Nakagami, Yasuhiro; Masuda, Kayoko

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that regulates many antioxidants, and we have recently succeeded in obtaining a novel Nrf2 activator, RS9, from microbial transformation. RS9 is categorized as a triterpenoid, and well-known triterpenoids such as RTA 402 (bardoxolone methyl) and RTA 408 have been tested in clinical trials. RTA 408 lotion is currently being tested in patients at risk for radiation dermatitis. This prompted us to study the profiles of RS9 in the skin. All the above triterpenoids increased the level of an Nrf2-targeted gene, NADPH:quinone oxidoreductase-1, in normal human epidermal keratinocytes. Among them, the activity of RS9 was prominent; furthermore, the cellular toxicity was less compared with RTA compounds. BALB/c mice were irradiated with 30 Gy/day on Day 0, and compounds were topically applied on the back once daily from Day 1 to Day 30. Dermatitis scores peaked on Day 18, with a score of 2.6 in vehicle-treated mice, and topical applications of 0.1% RTA 402, RTA 408 and RS9 reduced the scores to 1.8, 2.0 and 1.4, respectively. Moreover, the percentage of animals with scores ≥2 was analyzed, and 0.1% RS9 suppressed the percentage from 100% to 47%. These results imply that RS9 has potential efficacy for treating radiation dermatitis. PMID:27242339

  9. Toll-like receptors and microbial exposure: gene-gene and gene-environment interaction in the development of atopy.

    PubMed

    Reijmerink, N E; Kerkhof, M; Bottema, R W B; Gerritsen, J; Stelma, F F; Thijs, C; van Schayck, C P; Smit, H A; Brunekreef, B; Postma, D S; Koppelman, G H

    2011-10-01

    Environmental and genetic factors contribute to atopy development. High microbial exposure may confer a protective effect on atopy. Toll-like receptors (TLRs) bind microbial products and are important in activating the immune system. To assess whether interactions between microbial exposures and genes encoding TLRs (and related genes) result in atopy, genes, environmental factors and gene-environment interactions of 66 single-nucleotide polymorphisms (SNPs) of 12 genes (TLR 1-6, 9 and 10, CD14, MD2, lipopolysaccharide-binding protein (LBP) and Dectin-1), and six proxy parameters of microbial exposure (sibship size, pets (three different parameters), day-care and intrauterine and childhood tobacco smoke exposure) were analysed for association with atopic phenotypes in 3,062 Dutch children (the Allergenic study). The presence of two or more older siblings increased the risk of developing high total immunoglobulin (Ig)E levels at different ages. This risk increased further in children aged 1-2 yrs carrying the minor allele of TLR6 SNP rs1039559. Furthermore, novel two- and three-factor gene-gene and gene-environment interactions were found (e.g. between sibship size, day-care and LBP SNP rs2232596). Larger sibship size is associated with increased total IgE levels. Furthermore, complex two- and three-factor interactions exist between genes and the environment. The TLRs and related genes interact with proxy parameters of high microbial exposure in atopy development.

  10. Microbial transformations of antimicrobial quinolones and related drugs.

    PubMed

    Parshikov, Igor A; Sutherland, John B

    2012-12-01

    The quinolones are an important group of synthetic antimicrobial drugs used for treating bacterial diseases of humans and animals. Microorganisms transform antimicrobial quinolones (including fluoroquinolones) and the pharmacologically related naphthyridones, pyranoacridones, and cinnolones to a variety of metabolites. The biotransformation processes involve hydroxylation of methyl groups; hydroxylation of aliphatic and aromatic rings; oxidation of alcohols and amines; reduction of carboxyl groups; removal of methyl, carboxyl, fluoro, and cyano groups; addition of formyl, acetyl, nitrosyl, and cyclopentenone groups; and cleavage of aliphatic and aromatic rings. Most of these reactions greatly reduce or eliminate the antimicrobial activity of the quinolones.

  11. Predictions of Gene Family Distributions in Microbial Genomes: Evolution by Gene Duplication and Modification

    SciTech Connect

    Yanai, Itai; Camacho, Carlos J.; DeLisi, Charles

    2000-09-18

    A universal property of microbial genomes is the considerable fraction of genes that are homologous to other genes within the same genome. The process by which these homologues are generated is not well understood, but sequence analysis of 20 microbial genomes unveils a recurrent distribution of gene family sizes. We show that a simple evolutionary model based on random gene duplication and point mutations fully accounts for these distributions and permits predictions for the number of gene families in genomes not yet complete. Our findings are consistent with the notion that a genome evolves from a set of precursor genes to a mature size by gene duplications and increasing modifications. (c) 2000 The American Physical Society.

  12. Impacts of Pristine and Transformed Ag and Cu Engineered Nanomaterials on Surficial Sediment Microbial Communities Appear Short-Lived.

    PubMed

    Moore, Joe D; Stegemeier, John P; Bibby, Kyle; Marinakos, Stella M; Lowry, Gregory V; Gregory, Kelvin B

    2016-03-01

    Laboratory-based studies have shown that many soluble metal and metal oxide engineered nanomaterials (ENM) exert strong toxic effects on microorganisms. However, laboratory-based studies lack the complexity of natural systems and often use "as manufactured" ENMs rather than more environmentally relevant transformed ENMs, leaving open the question of whether natural ligands and seasonal variation will mitigate ENM impacts. Because ENMs will accumulate in subaquatic sediments, we examined the effects of pristine and transformed Ag and Cu ENMs on surficial sediment microbial communities in simulated freshwater wetlands. Five identical mesocosms were dosed through the water column with either Ag(0), Ag2S, CuO or CuS ENMs (nominal sizes of 4.67 ± 1.4, 18.1 ± 3.2, 31.1 ± 12, and 12.4 ± 4.1, respectively) or Cu(2+). Microbial communities were examined at 0, 7, 30, 90, 180, and 300 d using qPCR and high-throughput 16S rRNA gene sequencing. Results suggest differential short-term impacts of Ag(0) and Ag2S, similarities between CuO and CuS, and differences between Cu ENMs and Cu(2+). PICRUSt-predicted metagenomes displayed differential effects of Ag treatments on photosynthesis and of Cu treatments on methane metabolism. By 300 d, all metrics pointed to reconvergence of ENM-dosed mesocosm microbial community structure and composition, suggesting that the long-term microbial community impacts from a pulse of Ag or Cu ENMs are limited.

  13. Dynamics of gene duplication and transposons in microbial genomes following a sudden environmental change

    NASA Astrophysics Data System (ADS)

    Chia, Nicholas; Goldenfeld, Nigel

    2011-02-01

    A variety of genome transformations can occur as a microbial population adapts to a large environmental change. In particular, genomic surveys indicate that, following the transition to an obligate, host-dependent symbiont, the density of transposons first rises, then subsequently declines over evolutionary time. Here we show that these observations can be accounted for by a class of generic stochastic models for the evolution of genomes in the presence of continuous selection and gene duplication. The models use a fitness function that allows for partial contributions from multiple gene copies, is an increasing but bounded function of copy number, and is optimal for one fully adapted gene copy. We use Monte Carlo simulation to show that the dynamics result in an initial rise in gene copy number followed by a subsequent falloff due to adaptation to the new environmental parameters. These results are robust for reasonable gene duplication and mutation parameters when adapting to a novel target sequence. Our model provides a generic explanation for the dynamics of microbial transposon density following a large environmental change such as host restriction.

  14. Bacterial gene transfer by natural genetic transformation in the environment.

    PubMed Central

    Lorenz, M G; Wackernagel, W

    1994-01-01

    Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation. PMID:7968924

  15. Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains

    PubMed Central

    Velayudhan, Vimalkumar; Murphy, Ronan A.; Achtman, Mark

    2012-01-01

    Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

  16. MICROBIAL TRANSFORMATIONS OF URANIUM AND ENVIRONMENTAL RESTORATION THROUGH BIOREMEDIATION.

    SciTech Connect

    FRANCIS,A.J.

    2002-09-10

    Microorganisms present in the natural environment play a significant role in the mobilization and immobilization of uranium. Fundamental understanding of the mechanisms of microbiological transformations of various chemical forms of uranium present in wastes and contaminated soils and water has led to the development of novel bioremediation processes. One process uses anaerobic bacteria to stabilize the radionuclides and toxic metals from the waste, with a concurrent reduction in volume due to the dissolution and removal of nontoxic elements from the waste matrix. In an another process, uranium and other toxic metals are removed from contaminated soils and wastes by extracting with the chelating agent citric acid. Uranium is recovered from the citric acid extract after biodegradation/photodegradation in a concentrated form as UO{sub 3} {center_dot} 2H{sub 2}O for recycling or appropriate disposal.

  17. Microbial transformations of arsenic in the environment: From soda lakes to aquifers

    USGS Publications Warehouse

    Lloyd, J.R.; Oremland, R.S.

    2006-01-01

    Arsenic is a highly toxic element that supports a surprising range of biogeochemical transformations. The biochemical basis of these microbial interactions is described, with an emphasis on energy-yielding redox biotransformations that cycle between the As5+ and As3+ oxidation states. The subsequent impact of As3+-oxidising and As 5+-reducing prokaryotes on the chemistry of selected environments is also described, focusing on soda lakes with naturally high concentrations of the metalloid and on Southeast Asian aquifer sediments, where the microbial reduction of sorbed As5+ and subsequent mobilisation of As 3+ into water abstracted for drinking and irrigation threaten the lives of millions.

  18. Novel Nrf2 activators from microbial transformation products inhibit blood–retinal barrier permeability in rabbits

    PubMed Central

    Nakagami, Yasuhiro; Masuda, Kayoko; Hatano, Emiko; Inoue, Tatsuya; Matsuyama, Takuya; Iizuka, Mayumi; Ono, Yasunori; Ohnuki, Takashi; Murakami, Yoko; Iwasaki, Masaru; Yoshida, Kazuhiro; Kasuya, Yuji; Komoriya, Satoshi

    2015-01-01

    Background and Purpose Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator. Experimental Approach The potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood–retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation. Key Results Bardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively. Conclusion and Implications Nrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease. PMID:25363737

  19. Interspecific evolution: microbial symbiosis, endosymbiosis and gene transfer.

    PubMed

    Hoffmeister, Meike; Martin, William

    2003-08-01

    Microbial symbioses are interesting in their own right and also serve as exemplary models to help biologists to understand two important symbioses in the evolutionary past of eukaryotic cells: the origins of chloroplasts and mitochondria. Most, if not all, microbial symbioses have a chemical basis: compounds produced by one partner are useful for the other. But symbioses can also entail the transfer of genes from one partner to the other, which in some cases cements two cells into a bipartite, co-evolving unit. Here, we discuss some microbial symbioses in which progress is being made in uncovering the nature of symbiotic interactions: anaerobic methane-oxidizing consortia, marine worms that possess endosymbionts instead of a digestive tract, amino acid-producing endosymbionts of aphids, prokaryotic endosymbionts living within a prokaryotic host within mealybugs, endosymbionts of an insect vector of human disease and a photosynthetic sea slug that steals chloroplasts from algae. In the case of chloroplasts and mitochondria, examples of recent and ancient gene transfer to the chromosomes of their host cell illustrate the process of genetic merger in the wake of organelle origins.

  20. Microbial production and chemical transformation of poly-γ-glutamate

    PubMed Central

    Ashiuchi, Makoto

    2013-01-01

    Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of d-glutamate (D-PGA), a homo polymer of l-glutamate (L-PGA), and a random copolymer consisting of d- and l-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented. PMID:23855427

  1. Differential microbial transformation of nitrosamines by an inducible propane monooxygenase.

    PubMed

    Homme, Carissa L; Sharp, Jonathan O

    2013-07-02

    The toxicity of N-nitrosamines, their presence in drinking and environmental water supplies, and poorly understood recalcitrance collectively necessitate a better understanding of their potential for bioattenuation. Here, we show that the bacterial strain Rhodococcus jostii RHA1 can biotransform N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopyrrolidine (NPYR), and possibly N-nitrosomorpholine (NMOR) in addition to N-nitrosodimethylamine (NDMA). Growth of cells on propane as the sole carbon source greatly enhanced degradation rates when contrasted with cells grown on complex organics. Propane-induced rates in order of fastest to slowest were NDMA > NDEA > NDPA > NPYR > NMOR at concentrations <2000 μg/L. Removal rates for linear functional groups scaled inversely with mass and cyclic nitrosamines were more recalcitrant than linear nitrosamines. Controls demonstrated significant NDEA and NDPA losses independent of biomass, suggesting abiotic processes may play a role in attenuation of these two compounds under experimental conditions tested here. In contrast to NDMA, a transition from first to zero order kinetics was not observed for the other nitrosamines included in this study over a concentration range of 20-2000 μg/L. A genetic knockout for the propane monooxygenase enzyme (PrMO) confirmed the role of this enzyme in the biotransformation of NDEA and NPYR. This study furthers our understanding of environmental nitrosamine attenuation by revealing an enzymatic mechanism for the biotransformation of multiple nitrosamines, their relative recalcitrance to transformation, and potential for abiotic loss.

  2. MICROBIAL TRANSFORMATIONS OF RADIONUCLIDES AND ENVIRONMENTAL RESTORATION THROUGH BIOREMEDIATION.

    SciTech Connect

    FRANCIS, A.J.

    2006-09-29

    Treatment of waste streams containing radionuclides, the remediation of contaminated materials, soils, and water, and the safe and economical disposal of radionuclides and toxic metals containing wastes is a major concern. Radionuclides may exist in various oxidation states and may be present as oxide, coprecipitates, inorganic, and organic complexes depending on the process and waste stream. Unlike organic contaminants, the metals cannot be destroyed, but must either be converted to a stable form or removed. Microorganisms present in the natural environment play a major role in the mobilization and immobilization of radionuclides and toxic metals by direct enzymatic or indirect non-enzymatic actions and could affect the chemical nature of the radionuclides by altering the speciation, solubility and sorption properties and thus could increase or decrease the concentrations of radionuclides in solution. Fundamental understanding of the mechanisms of microbiological transformations of various chemical forms of uranium present in wastes and contaminated soils and water has led to the development of novel bioremediation processes. One process uses anaerobic bacteria to stabilize the radionuclides by reductive precipitation from higher to lower oxidation state with a concurrent reduction in volume due to the dissolution and removal of nontoxic elements from the waste matrix. In an another process, uranium and other toxic metals are removed from contaminated surfaces, soils, and wastes by extracting with the chelating agent citric acid. Uranium is recovered from the citric acid extract after biodegradation followed by photodegradation in a concentrated form as UO{sub 3} {center_dot} 2H{sub 2}O for recycling or appropriate disposal. These processes use all naturally occurring materials, common soil bacteria, naturally occurring organic compound citric acid and sunlight.

  3. Fourier transform infrared spectroscopy for molecular analysis of microbial cells.

    PubMed

    Ojeda, Jesús J; Dittrich, Maria

    2012-01-01

    A rapid and inexpensive method to characterise chemical cell properties and identify the functional groups present in the cell wall is Fourier transform infrared spectroscopy (FTIR). Infrared spectroscopy is a well-established technique to identify functional groups in organic molecules based on their vibration modes at different infrared wave numbers. The presence or absence of functional groups, their protonation states, or any changes due to new interactions can be monitored by analysing the position and intensity of the different infrared absorption bands. Additionally, infrared spectroscopy is non-destructive and can be used to monitor the chemistry of living cells. Despite the complexity of the spectra, the elucidation of functional groups on Gram-negative and Gram-positive bacteria has been already well documented in the literature. Recent advances in detector sensitivity have allowed the use of micro-FTIR spectroscopy as an important analytical tool to analyse biofilm samples without the need of previous treatment. Using FTIR spectroscopy, the infrared bands corresponding to proteins, lipids, polysaccharides, polyphosphate groups, and other carbohydrate functional groups on the bacterial cells can now be identified and compared along different conditions. Despite some differences in FTIR spectra among bacterial strains, experimental conditions, or changes in microbiological parameters, the IR absorption bands between approximately 4,000 and 400 cm(-1) are mainly due to fundamental vibrational modes and can often be assigned to the same particular functional groups. In this chapter, an overview covering the different sample preparation protocols for infrared analysis of bacterial cells is given, alongside the basic principles of the technique, the procedures for calculating vibrational frequencies based on simple harmonic motion, and the advantages and disadvantages of FTIR spectroscopy for the analysis of microorganisms.

  4. Microbial functional gene diversity with a shift of subsurface redox conditions during In Situ uranium reduction.

    PubMed

    Liang, Yuting; Van Nostrand, Joy D; N'guessan, Lucie A; Peacock, Aaron D; Deng, Ye; Long, Philip E; Resch, C Tom; Wu, Liyou; He, Zhili; Li, Guanghe; Hazen, Terry C; Lovley, Derek R; Zhou, Jizhong

    2012-04-01

    To better understand the microbial functional diversity changes with subsurface redox conditions during in situ uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of dsrAB genes (dissimilatory sulfite reductase genes) and methane generation-related mcr genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from Geobacter sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (E(h)) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the in situ redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.

  5. Microbial Functional Gene Diversity with a Shift of Subsurface Redox Conditions during In Situ Uranium Reduction

    PubMed Central

    Liang, Yuting; Van Nostrand, Joy D.; N′Guessan, Lucie A.; Peacock, Aaron D.; Deng, Ye; Long, Philip E.; Resch, C. Tom; Wu, Liyou; He, Zhili; Li, Guanghe; Hazen, Terry C.; Lovley, Derek R.

    2012-01-01

    To better understand the microbial functional diversity changes with subsurface redox conditions during in situ uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of dsrAB genes (dissimilatory sulfite reductase genes) and methane generation-related mcr genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from Geobacter sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (Eh) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the in situ redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation. PMID:22327592

  6. Anaerobic transformation of carbon monoxide by microbial communities of Kamchatka hot springs.

    PubMed

    Kochetkova, Tatiana V; Rusanov, Igor I; Pimenov, Nikolay V; Kolganova, Tatyana V; Lebedinsky, Alexander V; Bonch-Osmolovskaya, Elizaveta A; Sokolova, Tatyana G

    2011-05-01

    Carbon monoxide (CO) is one of the common gaseous compounds found in hot volcanic environments. It is known to serve as the growth substrate for a number of thermophilic prokaryotes, both aerobic and anaerobic. The goal of this work was to study the process of anaerobic transformation of CO by microbial communities inhabiting natural thermal environments: hot springs of Uzon Caldera, Kamchatka. The anaerobic microbial community of Treshchinny Spring (80°C, pH 6.5) was found to exhibit two peaks of affinity for CO (K (S1) = 54 nM and K (S2) = 1 μM). The actual rate of anaerobic CO transformation by the microbial community of this spring, calculated after obtaining the concentration dependence curve and extrapolated to the natural concentration of CO dissolved in the hot spring water (20 nM), was found to be 120 μmol l(-1) of sediment day(-1). In all the hot springs studied, more than 90% of the carbon of (14)CO upon anaerobic incubation was recovered as (14)CO(2). From 1 to 5% of (14)CO was transformed to volatile fatty acids (VFA). The number of microorganisms capable of anaerobic CO oxidation determined by dilution-to-extinction method reached 10(6) cells ml(-1) of sediment. CO-transforming anaerobic thermophilic microorganisms isolated from the springs under study exhibited hydrogenogenic type of CO oxidation and belonged to the bacterial genera Carboxydocella and Dictyoglomus. These data suggest a significant role of hydrogenogenic carboxydotrophic prokaryotes in anaerobic CO transformation in Uzon Caldera hot springs.

  7. Microbial sucrose isomerases: producing organisms, genes and enzymes.

    PubMed

    Goulter, Ken C; Hashimi, Saeed M; Birch, Robert G

    2012-01-05

    Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production. This analysis shows that microbial SIs can be considered in 5 structural classes with corresponding functional distinctions that broadly align with the taxonomic differences between producing organisms. The most widely used bacterial strain for industrial production of isomaltulose, widely referred to as "Protaminobacter rubrum" CBS 574.77, is identified as Serratia plymuthica. The strain producing the most structurally divergent SI, with a high product specificity for trehalulose, widely referred to as "Pseudomonas mesoacidophila" MX-45, is identified as Rhizobium sp. Each tested SI-producer is shown to have a single SI gene and enzyme, so the properties reported previously for the isolated proteins can reasonably be associated with the products of the genes subsequently cloned from the same isolates and SI classes. Some natural isolates with potent SI activity do not catabolize the isomer under usual production conditions. The results indicate that their industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate.

  8. Microbial metabolism fuels ecosystem-scale organic matter transformations: an integrated biological and chemical perspective

    NASA Astrophysics Data System (ADS)

    Wrighton, K. C.; Narrowe, A. B.; Angle, J.; Stefanik, K. S.; Daly, R. A.; Johnston, M.; Miller, C. S.

    2014-12-01

    Freshwater saturated sediments and soils represent vital ecosystems due to their nutrient cycling capacities and their prominent contribution to global greenhouse gas emissions. However, the diversity of microorganisms and metabolic pathways involved in carbon cycling, and the impacts of these processes on other biogeochemical cycles remain poorly understood. Major advances in DNA sequencing have helped forge linkages between the previously disconnected biological and chemical components of these systems. Here, we present data on the use of assembly-based metagenomics to generate hypotheses on microbial carbon degradation and biogeochemical cycling in waterlogged sediments and soils. DNA sequencing from a fresh water aquifer adjacent to the Colorado River in Rifle, CO yielded extensive genome recovery from multiple previously unknown bacterial lineages. Fermentative metabolisms encoded by these genomes drive nitrogen, hydrogen, and sulfur cycling in this subsurface system. We are also applying a similar approach to identify microbial processes in a freshwater wetland on Lake Erie, OH. Given the increased diversity (increased richness, decreased evenness, and strain variation) of wetland sediment microbial communities, we modified methods for specialized assembly of long taxonomic marker gene amplicons (EMIRGE) to create a biogeographical map of Fungi, Archaea, and Bacteria along depth and hydrological transects. This map reveals that the microbial community associated with the top two depths (>7 cm) is significantly different from bottom depths (7-40 cm). Dissolved organic matter (DOM) molecular weight and the presence of oxidized terminal electron acceptors best predict differences in microbial community structure. Laboratory mesocosms amended with pore-water DOM, in situ soil communities, and variable oxygen conditions link DOM composition and redox to microbial metabolic networks, biogeochemical cycles, and green house gas emission. Organism identities from

  9. Enhancement of microbial 2,4,6-trinitrotoluene transformation with increased toxicity by exogenous nutrient amendment.

    PubMed

    Liang, Shih-Hsiung; Hsu, Duen-Wei; Lin, Chia-Ying; Kao, Chih-Ming; Huang, Da-Ji; Chien, Chih-Ching; Chen, Ssu-Ching; Tsai, Isheng Jason; Chen, Chien-Cheng

    2017-04-01

    In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation.

  10. Microbial toxicity and characterization of DNAN (bio)transformation product mixtures.

    PubMed

    Olivares, Christopher I; Sierra-Alvarez, Reyes; Alvarez-Nieto, Cristina; Abrell, Leif; Chorover, Jon; Field, Jim A

    2016-07-01

    2,4-Dinitroanisole (DNAN) is an emerging insensitive munitions compound. It undergoes rapid (bio)transformation in soils and anaerobic sludge. The primary transformation pathway catalyzed by a combination of biotic and abiotic factors is nitrogroup reduction followed by coupling of reactive intermediates to form azo-dimers. Additional pathways include N-acetylation and O-demethoxylation. Toxicity due to (bio)transformation products of DNAN has received little attention. In this study, the toxicity of DNAN (bio)transformation monomer products and azo-dimer and trimer surrogates to acetoclastic methanogens and the marine bioluminescent bacterium, Allivibrio fischeri, were evaluated. Methanogens were severely inhibited by 3-nitro-4-methoxyaniline (MENA), with a 50%-inhibiting concentration (IC50) of 25 μM, which is more toxic than DNAN with the same assay, but posed a lower toxicity to Allivibrio fischeri (IC50 = 219 μM). On the other hand, N-(5-amino-2-methoxyphenyl) acetamide (Ac-DAAN) was the least inhibitory test-compound for both microbial targets. Azo-dimer and trimer surrogates were very highly toxic to both microbial systems, with a toxicity similar or stronger than that of DNAN. A semi-quantitative LC-QTOF-MS method was employed to determine product mixture profiles at different stages of biotransformation, and compared with the microbial toxicity of the product-mixtures formed. Methanogenic toxicity increased due to putative reactive nitroso-intermediates as DNAN was reduced. However, the inhibition later attenuated as dimers became the predominant products in the mixtures. In contrast, A. fischeri tolerated the initial biotransformation products but were highly inhibited by the predominant azo-dimer products formed at longer incubation times, suggesting these ultimate products are more toxic than DNAN.

  11. Genetic transformation and gene expression in white pine (pinus strobus)

    SciTech Connect

    Minocha, R.

    1987-10-01

    The objectives of the study were: (1) to develop protocols for transformation of white pine (Pinus strobus) embryonic tissue; and (2) to analyze the regulation of foreign gene expression in Pinus strobus. A number of Agrobacterium tumefaciens strains containing chimeric genes for neomycin phosphotransferase (NPTII for kanamycin resistance) and chloramphenicol acetyl transferase (CAT) under the control of either a constitutive promoter (NOS-nopaline synthase) or light-inducible promoters (RuBisCO small subunit and chlorophyll a/b binding protein) were used. A variety of tissues from white pine seedlings and mature trees was used. The techniques for transformation were modified from those used for tobacco transformation. The results show that white pine tissue from young seedlings is high suitable for transformation by A. tumefaciens. Whereas the normal tissues are very sensitive to kanamycin, transformed callus was quite resistant to this antibiotic.

  12. ipt Gene transformation in petunia by an Agrobacterium mediated method.

    PubMed

    Bai, L J; Ye, C J; Lu, J Y; Yang, D E; Xue, H; Pan, Y; Cao, P X; Wang, B; Liu, M

    2009-01-01

    To prevent leaf senescence of petunia, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of 35S promoter and introduced into petunia. PCR analysis showed an expected 0.5 Kb fragment of ipt gene in transgenic petunia. RT-PCR analysis indicated the expression of ipt gene in the transgenic lines. Leaves from transgenic plants remained green and healthy in normal culture condition, while the non-transformed plants turned to yellow. Transgenic plants showed a reduction in height and smaller leaf sizes. In transgenic lines, the internodes were shorter, and the roots grew slower than the non-transformed plants.

  13. Microbial Transformation of Biomacromolecules in a Membrane Bioreactor: Implications for Membrane Fouling Investigation

    PubMed Central

    Zhou, Zhongbo; Meng, Fangang; Chae, So-Ryong; Huang, Guocheng; Fu, Wenjie; Jia, Xiaoshan; Li, Shiyu; Chen, Guang-Hao

    2012-01-01

    Background The complex characteristics and unclear biological fate of biomacromolecules (BMM), including colloidal and soluble microbial products (SMP), extracellular polymeric substances (EPS) and membrane surface foulants (MSF), are crucial factors that limit our understanding of membrane fouling in membrane bioreactors (MBRs). Findings In this study, the microbial transformation of BMM was investigated in a lab-scale MBR by well-controlled bioassay tests. The results of experimental measurements and mathematical modeling show that SMP, EPS, and MSF had different biodegradation behaviors and kinetic models. Based on the multi-exponential G models, SMP were mainly composed of slowly biodegradable polysaccharides (PS), proteins (PN), and non-biodegradable humic substances (HS). In contrast, EPS contained a large number of readily biodegradable PN, slowly biodegradable PS and HS. MSF were dominated by slowly biodegradable PS, which had a degradation rate constant similar to that of SMP-PS, while degradation behaviors of MSF-PN and MSF-HS were much more similar to those of EPS-PN and EPS-HS, respectively. In addition, the large-molecular weight (MW) compounds (>100 kDa) in BMM were found to have a faster microbial transformation rate compared to the small-MW compounds (<5 kDa). The parallel factor (PARAFAC) modeling of three-dimensional fluorescence excitation-emission matrix (EEM) spectra showed that the tryptophan-like PN were one of the major fractions in the BMM and they were more readily biodegradable than the HS. Besides microbial mineralization, humification and hydrolysis could be viewed as two important biotransformation mechanisms of large-MW compounds during the biodegradation process. Significance The results of this work can aid in tracking the origin of membrane foulants from the perspective of the biotransformation behaviors of SMP, EPS, and MSF. PMID:22912694

  14. Adapting rice anther culture to gene transformation and RNA interference.

    PubMed

    Chen, Caiyan; Xiao, Han; Zhang, Wenli; Wang, Aiju; Xia, Zhihui; Li, Xiaobing; Zhai, Wenxue; Cheng, Zhukuan; Zhu, Lihuang

    2006-10-01

    Anther culture offers a rapid method of generating homozygous lines for breeding program and genetic analysis. To produce homozygous transgenic lines of rice (Oryza sativa L.) in one step, we developed an efficient protocol of anther-callus-based transformation mediated by Agrobacterium after optimizing several factors influencing efficient transformation, including callus induction and Agrobacterium density for co-cultivation. Using this protocol, we obtained 145 independent green transformants from five cultivars of japonica rice by transformation with a binary vector pCXK1301 bearing the rice gene, Xa21 for resistance to bacterial blight, of which 140 were further confirmed by PCR and Southern hybridization analysis, including haploids (32.1%), diploids (62.1%) and mixoploids (7.5%). Fifteen diploids were found to be doubled haploids, which accounted for 10.7% of the total positive lines. Finally, by including 28 from colchicine induced or spontaneous diploidization of haploids later after transformation, a total of 43 doubled haploids (30.7%) of Xa21 transgenic lines were obtained. We also generated two RNAi transgenic haploids of the rice OsMADS2 gene, a putative redundant gene of OsMADS4 based on their sequence similarity, to investigate its possible roles in rice flower development by this method. Flowers from the two OsMADS2 RNAi transgenic haploids displayed obvious homeotic alternations, in which lodicules were transformed into palea/lemma-like tissues, whereas identities of other floral organs were maintained. The phenotypic alternations were proved to result from specific transcriptional suppression of OsMADS2 gene by the introduced RNAi transgene. The results confirmed that OsMADS2 is involved in lodicule development of rice flower and functionally redundant with OsMADS4 gene. Our results demonstrated that rice anther culture could be adapted to gene transformation and RNAi analysis in rice.

  15. Stereoselective Microbial Transformation of Triadimefon to Triadimenol in Soils: Varying Production Rates of Triadimenol Stereoisomers Could Impact Risk Assessment

    EPA Science Inventory

    The microbial transformation of triadimefon, an agricultural fungicide of the 1,2,4-triazole class, was followed over several months under aerobic conditions in 3 different soil types to observe rates and products of transformation as well as enantiomer fractions of parent and pr...

  16. Microbial Transformation of Triadimefon to Triadimenol in Soils: Selective Production Rates of Triadimenol Stereoisomers Affect Exposure and Risk

    EPA Science Inventory

    The microbial transformation of triadimefon, an agricultural fungicide of the 1,2,4-triazole class, was followed at a nominal concentration of 50 μg/mL over 4 months under aerobic conditions in three different soil types. Rates and products of transformation were measured, as wel...

  17. Land use type significantly affects microbial gene transcription in soil.

    PubMed

    Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

    2014-05-01

    Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

  18. Stable transformation of maize after gene transfer by electroporation.

    PubMed

    Fromm, M E; Taylor, L P; Walbot, V

    The graminaceous monocots, including the economically important cereals, seem to be refractory to infection by Agrobacterium tumefaciens, a natural gene transfer system that has been successfully exploited for transferring foreign genes into higher plants. Therefore, direct transfer techniques that are potentially applicable to all plant species have been developed using a few dicot and monocot species as model systems. One of these techniques, electroporation, uses electrical pulses of high field strength to permeabilize cell membranes reversibly so as to facilitate the transfer of DNA into cells. Electroporation-mediated gene transfer has resulted in stably transformed animal cells and transient gene expression in monocot and dicot plant cells. Here we report that electroporation-mediated DNA transfer of a chimaeric gene encoding neomycin phosphotransferase results in stably transformed maize cells that are resistant to kanamycin.

  19. Photochemical and microbial transformation of emerging flame retardants: cause for concern?

    PubMed

    Chen, Da; Hale, Robert C; Letcher, Robert J

    2015-04-01

    Among anthropogenic chemicals, flame retardants have attracted mounting environmental concerns. In recent years, an increasing number of studies have been conducted worldwide to investigate flame-retardant sources, environmental distribution, wildlife and human exposure, and toxicity. Data generated have demonstrated that some flame-retardant substances such as polybrominated diphenyl ethers (PBDE) are persistent, bioaccumulative, and toxic to exposed organisms. However, comparatively much less attention has been paid to the mechanisms and products of environmental transformation of flame retardants. This lack of information undermines our understanding of the environmental behavior and fate of flame retardants, as well as the associated risks to environmental and human health. Photochemical and microbial transformation of flame retardants in various matrices and environmental compartments can elevate the toxicological significance of flame retardant exposure, via the formation of, for example, lesser halogenated but more bioaccumulative degradation products and toxic radicals. Such pathways raise concerns related to the environmental safety of some alternative flame retardants that are presumably safe and used to replace PBDEs. To fully assess the environmental risks, more research is needed to investigate the environmental transformation potential of emerging flame retardants including polymeric flame retardants. Enhanced analytical efforts are needed to better characterize transformation products and transient radicals. Additional mesocosm and field studies are needed to elucidate transformation kinetics and consequences under environmentally relevant conditions.

  20. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    USGS Publications Warehouse

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  1. [Effects of selective microbial inhibitors on the microbial transformation of phosphorous in aggregates of highly weathered red soil with rice straw amendment].

    PubMed

    Ding, Long-jun; Xiao, He-ai; Wu, Jin-shui; Ge, Ti-da

    2010-07-01

    In order to further understand the mechanisms of microbial immobilization of phosphorous (P) in highly weathered red soil with organic amendment, an incubation test was conducted to investigate the roles of microbial functional groups in the transformation of P in 0.2-2 mm soil aggregates. Throughout the 90-day incubation period, amendment with rice straw induced a substantial increase in the amounts of microbial biomass C and P, Olsen-P, and organic P in the aggregates. Comparing with rice straw amendment alone, the amendment with rice straw plus fungal inhibitor actidione decreased the amount of microbial biomass C in the aggregates by 10.5%-31.8% in the first 30 days. Such a decrement was significantly larger than that (6.8%-11.6%) in the treatment amended with rice straw plus bacterial inhibitors tetracycline and streptomycin sulphate (P<0.01). After the first 30 days, the microbial biomass C remained constant. In the first 20 days, the amount of microbial biomass P in the aggregates was 10.0%-28.8% higher in the treatment amended with bacterial inhibitors than in the treatment amended with fungal inhibitor (P<0.01). All the results suggested that that both the fungal and the bacterial groups were involved in the microbial immobilization of P in the soil aggregates, and the fungal group played a relatively larger role.

  2. Spectral induced polarization and electrodic potential monitoring of microbially mediated iron sulfide transformations

    NASA Astrophysics Data System (ADS)

    Personna, Yves Robert; Ntarlagiannis, Dimitrios; Slater, Lee; Yee, Nathan; O'Brien, Michael; Hubbard, Susan

    2008-06-01

    Stimulated sulfate-reduction is a bioremediation technique utilized for the sequestration of heavy metals in the subsurface. We performed laboratory column experiments to investigate the geoelectrical response of iron sulfide transformations by Desulfovibrio vulgaris. Two geoelectrical methods, (1) spectral induced polarization (SIP), and (2) electrodic potential measurements, were investigated. Aqueous geochemistry (sulfate, lactate, sulfide, and acetate), observations of precipitates (identified from electron microscopy as iron sulfide), and electrodic potentials on bisulfide ion (HS-) sensitive silver-silver chloride (Ag-AgCl) electrodes (˜-630 mV) were diagnostic of induced transitions between anaerobic iron sulfide forming conditions and aerobic conditions promoting iron sulfide dissolution. The SIP data showed ˜10 mrad anomalies during iron sulfide mineralization accompanying microbial activity under an anaerobic transition. These anomalies disappeared during iron sulfide dissolution under the subsequent aerobic transition. SIP model parameters based on a Cole-Cole relaxation model of the polarization at the mineral-fluid interface were converted to (1) estimated biomineral surface area to pore volume (Sp), and (2) an equivalent polarizable sphere diameter (d) controlling the relaxation time. The temporal variation in these model parameters is consistent with filling and emptying of pores by iron sulfide biofilms, as the system transitions between anaerobic (pore filling) and aerobic (pore emptying) conditions. The results suggest that combined SIP and electrodic potential measurements might be used to monitor spatiotemporal variability in microbial iron sulfide transformations in the field.

  3. Microbial Transformation of Multiwalled Carbon Nanotubes by Mycobacterium vanbaalenii PYR-1.

    PubMed

    You, Yaqi; Das, Kamol K; Guo, Huiyuan; Chang, Che-Wei; Navas-Moreno, Maria; Chan, James W; Verburg, Paul; Poulson, Simon R; Wang, Xilong; Xing, Baoshan; Yang, Yu

    2017-02-21

    Carbonaceous nanomaterials are widely used in industry and consumer products, but concerns have been raised regarding their release into the environment and subsequent impacts on ecosystems and human health. Although many efforts have been devoted to understanding the environmental fate of carbonaceous nanomaterials, information about their microbial transformation is still rare. In this study, we found that within 1 month a polycyclic aromatic hydrocarbon-degrading bacterium, Mycobacterium vanbaalenii PYR-1, was able to degrade both pristine and carboxyl-functionalized multiwalled carbon nanotubes (p-MWCNT and c-MWCNT), as demonstrated by consistent results from high resolution transmission electron microscopy, Raman spectroscopy, and confocal Raman microspectroscopy. Statistical analysis of Raman spectra identified a significant increase in the density of disordered or amorphous carbon in p-MWCNT and c-MWCNT after biodegradation. Microbial respiration further suggested potential mineralization of MWCNTs within about 1 month. All of our analyses consistently showed higher degradation or mineralization of c-MWCNT compared to p-MWCNT. These results highlight the potential of using bacteria in engineered systems to remove residual carbonaceous nanomaterials and reduce risk of human exposure and environmental impact. Meanwhile, our finding suggests possible transformation of carbonaceous nanomaterials by polycyclic aromatic hydrocarbon-degrading bacteria in the natural environment, which should be accounted for in predicting the environmental fate of these emerging contaminants and in nanotechnology risk regulation.

  4. Spectral induced polarization and electrodic potential monitoring of microbially mediated iron sulfide transformations

    SciTech Connect

    Hubbard, Susan; Personna, Y.R.; Ntarlagiannis, D.; Slater, L.; Yee, N.; O'Brien, M.; Hubbard, S.

    2008-02-15

    Stimulated sulfate-reduction is a bioremediation technique utilized for the sequestration of heavy metals in the subsurface.We performed laboratory column experiments to investigate the geoelectrical response of iron sulfide transformations by Desulfo vibriovulgaris. Two geoelectrical methods, (1) spectral induced polarization (SIP), and (2) electrodic potential measurements, were investigated. Aqueous geochemistry (sulfate, lactate, sulfide, and acetate), observations of precipitates (identified from electron microscopy as iron sulfide), and electrodic potentials on bisulfide ion (HS) sensitive silver-silver chloride (Ag-AgCl) electrodes (630 mV) were diagnostic of induced transitions between an aerobic iron sulfide forming conditions and aerobic conditions promoting iron sulfide dissolution. The SIP data showed 10m rad anomalies during iron sulfide mineralization accompanying microbial activity under an anaerobic transition. These anomalies disappeared during iron sulfide dissolution under the subsequent aerobic transition. SIP model parameters based on a Cole-Cole relaxation model of the polarization at the mineral-fluid interface were converted to (1) estimated biomineral surface area to pore volume (Sp), and (2) an equivalent polarizable sphere diameter (d) controlling the relaxation time. The temporal variation in these model parameters is consistent with filling and emptying of pores by iron sulfide biofilms, as the system transitions between anaerobic (pore filling) and aerobic (pore emptying) conditions. The results suggest that combined SIP and electrodic potential measurements might be used to monitor spatiotemporal variability in microbial iron sulfide transformations in the field.

  5. Transformation of hexabromocyclododecane in contaminated soil in association with microbial diversity.

    PubMed

    Le, Thao Thanh; Son, Min-Hui; Nam, In-Huyn; Yoon, Hakwon; Kang, Yu-Gyeong; Chang, Yoon-Seok

    2017-03-05

    This study evaluated the transformation of 1,2,5,6,9,10-hexabromocyclododecane (HBCD) in soil under various conditions. Under anaerobic conditions for 21days, 34% of the total HBCD was reduced from rhizosphere soil containing humic acid, and 35% of the total HBCD was reduced from the non-rhizosphere soil; under aerobic conditions, 29% and 57-60% of the total HBCD were reduced from the same soil types after 40days. Three HBCD isomers (α-, β-, and γ-HBCD) were separately analyzed for their isomeric effects on transformation. In the soils with added glucose as a carbon and energy source, the fraction of γ-HBCD was reduced due to the blooming microbial activity. The population of Gram-positive bacteria decreased during the aerobic treatments of HBCD, whereas the population of several Gram-negative bacteria (e.g., Brassia rhizosphere, Sphingomonas sp.) increased. Humic acid and glucose increased the HBCD removal efficiency and microbial diversity in both rhizosphere and non-rhizosphere soils.

  6. Gene expression profiling of microbial activities and interactions in sediments under haloclines of E. Mediterranean deep hypersaline anoxic basins.

    PubMed

    Edgcomb, Virginia P; Pachiadaki, Maria G; Mara, Paraskevi; Kormas, Konstantinos A; Leadbetter, Edward R; Bernhard, Joan M

    2016-11-01

    Deep-sea hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea are considered some of the most polyextreme habitats on Earth. In comparison to microbial activities occurring within the haloclines and brines of these unusual water column habitats near the Mediterranean seafloor, relatively little is known about microbial metabolic activities in the underlying sediments. In addition, it is not known whether activities are shaped by the unique chemistries of the different DHAB brines and whether evidence exists for active microbial eukaryotes in those sediments. Metatranscriptome analysis was applied to sediment samples collected using ROV Jason from underneath the haloclines of Urania, Discovery and L'Atalante DHABs and a control site. We report on expression of genes associated with sulfur and nitrogen cycling, putative osmolyte biosynthetic pathways and ion transporters, trace metal detoxification, selected eukaryotic activities (particularly of fungi), microbe-microbe interactions, and motility in sediments underlying the haloclines of three DHABs. Relative to our control sediment sample collected outside of Urania Basin, microbial communities (including eukaryotes) in the Urania and Discovery DHAB sediments showed upregulation of expressed genes associated with nitrogen transformations, osmolyte biosynthesis, heavy metals resistance and metabolism, eukaryotic organelle functions, and cell-cell interactions. Sediments underlying DHAB haloclines that have cumulative physico-chemical stressors within the limits of tolerance for microoorganisms can therefore be hotspots of activity in the deep Mediterranean Sea.

  7. Differential transformation of mammary epithelial cells by Wnt genes.

    PubMed Central

    Wong, G T; Gavin, B J; McMahon, A P

    1994-01-01

    The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent. Images PMID:8065359

  8. Transformation of chloroform in model treatment wetlands: from mass balance to microbial analysis.

    PubMed

    Chen, Yi; Wen, Yue; Zhou, Junwei; Zhou, Qi; Vymazal, Jan; Kuschk, Peter

    2015-05-19

    Chloroform is one of the common disinfection byproducts, which is not susceptible to degradation and poses great health concern. In this study, the chloroform removal efficiencies and contributions of sorption, microbial degradation, plant uptake, and volatilization were evaluated in six model constructed wetlands (CWs). The highest chloroform removal efficiency was achieved in litter-added CWs (99%), followed by planted (46-54%) and unplanted CWs (39%). Mass balance study revealed that sorption (73.5-81.2%) and microbial degradation (17.6-26.2%) were the main chloroform removal processes in litter-added CWs, and that sorption (53.6-66.1%) and plant uptake (25.3-36.2%) were the primary contributors to chloroform removal in planted CWs. Around 60% of chloroform got accumulated in the roots after plant uptake, and both transpiration and gas-phase transport were expected to be the drivers for the plant uptake. Sulfate-reducing bacteria and methanogens were found to be the key microorganisms for chloroform biodegradation through cometabolic dechlorination, and positive correlations were observed between functional genes (dsrA, mcrA) and biodegradation rates. Overall, this study suggests that wetland is an efficient ecosystem for sustainable chloroform removal, and that plant and litter can enhance the removal performance through root uptake and microbial degradation stimulation, respectively.

  9. Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession

    NASA Astrophysics Data System (ADS)

    Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

    2015-05-01

    The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth’s biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P < 0.05) higher than those of CF and MBF, rendering their microbial community compositions markedly different. Consistently, microbial functional diversity was also highest in the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession.

  10. Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession

    PubMed Central

    Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

    2015-01-01

    The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth’s biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P < 0.05) higher than those of CF and MBF, rendering their microbial community compositions markedly different. Consistently, microbial functional diversity was also highest in the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession. PMID:25943705

  11. Analyses of soil microbial community compositions and functional genes reveal potential consequences of natural forest succession.

    PubMed

    Cong, Jing; Yang, Yunfeng; Liu, Xueduan; Lu, Hui; Liu, Xiao; Zhou, Jizhong; Li, Diqiang; Yin, Huaqun; Ding, Junjun; Zhang, Yuguang

    2015-05-06

    The succession of microbial community structure and function is a central ecological topic, as microbes drive the Earth's biogeochemical cycles. To elucidate the response and mechanistic underpinnings of soil microbial community structure and metabolic potential relevant to natural forest succession, we compared soil microbial communities from three adjacent natural forests: a coniferous forest (CF), a mixed broadleaf forest (MBF) and a deciduous broadleaf forest (DBF) on Shennongjia Mountain in central China. In contrary to plant communities, the microbial taxonomic diversity of the DBF was significantly (P < 0.05) higher than those of CF and MBF, rendering their microbial community compositions markedly different. Consistently, microbial functional diversity was also highest in the DBF. Furthermore, a network analysis of microbial carbon and nitrogen cycling genes showed the network for the DBF samples was relatively large and tight, revealing strong couplings between microbes. Soil temperature, reflective of climate regimes, was important in shaping microbial communities at both taxonomic and functional gene levels. As a first glimpse of both the taxonomic and functional compositions of soil microbial communities, our results suggest that microbial community structure and function potentials will be altered by future environmental changes, which have implications for forest succession.

  12. TRANSFORMATION

    SciTech Connect

    LACKS,S.A.

    2003-10-09

    Transformation, which alters the genetic makeup of an individual, is a concept that intrigues the human imagination. In Streptococcus pneumoniae such transformation was first demonstrated. Perhaps our fascination with genetics derived from our ancestors observing their own progeny, with its retention and assortment of parental traits, but such interest must have been accelerated after the dawn of agriculture. It was in pea plants that Gregor Mendel in the late 1800s examined inherited traits and found them to be determined by physical elements, or genes, passed from parents to progeny. In our day, the material basis of these genetic determinants was revealed to be DNA by the lowly bacteria, in particular, the pneumococcus. For this species, transformation by free DNA is a sexual process that enables cells to sport new combinations of genes and traits. Genetic transformation of the type found in S. pneumoniae occurs naturally in many species of bacteria (70), but, initially only a few other transformable species were found, namely, Haemophilus influenzae, Neisseria meningitides, Neisseria gonorrheae, and Bacillus subtilis (96). Natural transformation, which requires a set of genes evolved for the purpose, contrasts with artificial transformation, which is accomplished by shocking cells either electrically, as in electroporation, or by ionic and temperature shifts. Although such artificial treatments can introduce very small amounts of DNA into virtually any type of cell, the amounts introduced by natural transformation are a million-fold greater, and S. pneumoniae can take up as much as 10% of its cellular DNA content (40).

  13. Lateral gene transfer in a heavy metal-contaminated-groundwater microbial community

    DOE PAGES

    Hemme, Christopher L.; Green, Stefan J.; Rishishwar, Lavanya; ...

    2016-04-05

    Here, unraveling the drivers controlling the response and adaptation of biological communities to environmental change, especially anthropogenic activities, is a central but poorly understood issue in ecology and evolution. Comparative genomics studies suggest that lateral gene transfer (LGT) is a major force driving microbial genome evolution, but its role in the evolution of microbial communities remains elusive.

  14. Microbial transformations of uranium in wastes and implication on its mobility

    SciTech Connect

    Suzuki,Y.; Nankawa, T.; Ozaki, T.; Ohnuki, T.; Francis, A.J.; Enokida, Y.; Yamamoto, I.

    2008-09-14

    Uranium exists in several chemical forms in mining and mill tailings and in nuclear and weapons production wastes. Under appropriate conditions, microorganisms can affect the stability and mobility of U in wastes by altering the chemical speciation, solubility and sorption properties and thus could increase or decrease the concentrations of U in solution and the bioavailability. Dissolution or immobilization of U is brought about by direct enzymatic action or indirect nonenzymatic action of microorganisms. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of U have been extensively investigated, we have only limited information on the mechanisms of microbial transformations of various chemical forms of U in the presence of electron donors and acceptors.

  15. Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites.

    PubMed

    Gliszczyńska, Anna; Łysek, Agnieszka; Janeczko, Tomasz; Świtalska, Marta; Wietrzyk, Joanna; Wawrzeńczyk, Czesław

    2011-04-01

    Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.

  16. Microbial Internal Storage Alters the Carbon Transformation in Dynamic Anaerobic Fermentation.

    PubMed

    Ni, Bing-Jie; Batstone, Damien; Zhao, Bai-Hang; Yu, Han-Qing

    2015-08-04

    Microbial internal storage processes have been demonstrated to occur and play an important role in activated sludge systems under both aerobic and anoxic conditions when operating under dynamic conditions. High-rate anaerobic reactors are often operated at a high volumetric organic loading and a relatively dynamic profile, with large amounts of fermentable substrates. These dynamic operating conditions and high catabolic energy availability might also facilitate the formation of internal storage polymers by anaerobic microorganisms. However, so far information about storage under anaerobic conditions (e.g., anaerobic fermentation) as well as its consideration in anaerobic process modeling (e.g., IWA Anaerobic Digestion Model No. 1, ADM1) is still sparse. In this work, the accumulation of storage polymers during anaerobic fermentation was evaluated by batch experiments using anaerobic methanogenic sludge and based on mass balance analysis of carbon transformation. A new mathematical model was developed to describe microbial storage in anaerobic systems. The model was calibrated and validated by using independent data sets from two different anaerobic systems, with significant storage observed, and effectively simulated in both systems. The inclusion of the new anaerobic storage processes in the developed model allows for more successful simulation of transients due to lower accumulation of volatile fatty acids (correction for the overestimation of volatile fatty acids), which mitigates pH fluctuations. Current models such as the ADM1 cannot effectively simulate these dynamics due to a lack of anaerobic storage mechanisms.

  17. Microbial Transformation of TRU and Mixed Waste: Actinide Speciation and Waste Volume

    SciTech Connect

    Halada, Gary P

    2008-04-10

    In order to understand the susceptibility of transuranic and mixed waste to microbial degradation (as well as any mechanism which depends upon either complexation and/or redox of metal ions), it is essential to understand the association of metal ions with organic ligands present in mixed wastes. These ligands have been found in our previous EMSP study to limit electron transfer reactions and strongly affect transport and the eventual fate of radionuclides in the environment. As transuranic waste (and especially mixed waste) will be retained in burial sites and in legacy containment for (potentially) many years while awaiting treatment and removal (or remaining in place under stewardship agreements at government subsurface waste sites), it is also essential to understand the aging of mixed wastes and its implications for remediation and fate of radionuclides. Mixed waste containing actinides and organic materials are especially complex and require extensive study. The EMSP program described in this report is part of a joint program with the Environmental Sciences Department at Brookhaven National Laboratory. The Stony Brook University portion of this award has focused on the association of uranium (U(VI)) and transuranic analogs (Ce(III) and Eu(III)) with cellulosic materials and related compounds, with development of implications for microbial transformation of mixed wastes. The elucidation of the chemical nature of mixed waste is essential for the formulation of remediation and encapsulation technologies, for understanding the fate of contaminant exposed to the environment, and for development of meaningful models for contaminant storage and recovery.

  18. Microbial nitrogen transformation potential in surface run-off leachate from a tropical landfill

    SciTech Connect

    Mangimbulude, Jubhar C.; Straalen, Nico M. van; Roeling, Wilfred F.M.

    2012-01-15

    Highlights: Black-Right-Pointing-Pointer Microbial nitrogen transformations can alleviate toxic ammonium discharge. Black-Right-Pointing-Pointer Aerobic ammonium oxidation was rate-limiting in Indonesian landfill leachate. Black-Right-Pointing-Pointer Organic nitrogen ammonification was most dominant. Black-Right-Pointing-Pointer Anaerobic nitrate reduction and ammonium oxidation potential were also high. Black-Right-Pointing-Pointer A two-stage aerobic-anaerobic nitrogen removal system needs to be implemented. - Abstract: Ammonium is one of the major toxic compounds and a critical long-term pollutant in landfill leachate. Leachate from the Jatibarang landfill in Semarang, Indonesia, contains ammonium in concentrations ranging from 376 to 929 mg N L{sup -1}. The objective of this study was to determine seasonal variation in the potential for organic nitrogen ammonification, aerobic nitrification, anaerobic nitrate reduction and anaerobic ammonium oxidation (anammox) at this landfilling site. Seasonal samples from leachate collection treatment ponds were used as an inoculum to feed synthetic media to determine potential rates of nitrogen transformations. Aerobic ammonium oxidation potential (<0.06 mg N L{sup -1} h{sup -1}) was more than a hundred times lower than the anaerobic nitrogen transformation processes and organic nitrogen ammonification, which were of the same order of magnitude. Anaerobic nitrate oxidation did not proceed beyond nitrite; isolates grown with nitrate as electron acceptor did not degrade nitrite further. Effects of season were only observed for aerobic nitrification and anammox, and were relatively minor: rates were up to three times higher in the dry season. To completely remove the excess ammonium from the leachate, we propose a two-stage treatment system to be implemented. Aeration in the first leachate pond would strongly contribute to aerobic ammonium oxidation to nitrate by providing the currently missing oxygen in the anaerobic

  19. Molecular docking simulation studies on potent butyrylcholinesterase inhibitors obtained from microbial transformation of dihydrotestosterone

    PubMed Central

    2013-01-01

    Background Biotransformation is an effective technique for the synthesis of libraries of bioactive compounds. Current study on microbial transformation of dihydrotestosterone (DHT) (1) was carried out to produce various functionalized metabolites. Results Microbial transformation of DHT (1) by using two fungal cultures resulted in potent butyrylcholinesterase (BChE) inhibitors. Biotransformation with Macrophomina phaseolina led to the formation of two known products, 5α-androstan-3β,17β-diol (2), and 5β-androstan-3α,17β-diol (3), while biotransformation with Gibberella fujikuroi yielded six known metabolites, 11α,17β-dihydroxyandrost-4-en-3-one (4), androst-1,4-dien-3,17-dione (5), 11α-hydroxyandrost-4-en-3,17-dione (6), 11α-hydroxyandrost-1,4-dien-3,17-dione (7), 12β-hydroxyandrost-1,4-dien-3,17-dione (8), and 16α-hydroxyandrost-1,4-dien-3,17-dione (9). Metabolites 2 and 3 were found to be inactive, while metabolite 4 only weakly inhibited the enzyme. Metabolites 5–7 were identified as significant inhibitors of BChE. Furthermore, predicted results from docking simulation studies were in complete agreement with experimental data. Theoretical results were found to be helpful in explaining the possible mode of action of these newly discovered potent BChE inhibitors. Compounds 8 and 9 were not evaluated for enzyme inhibition activity both in vitro and in silico, due to lack of sufficient quantities. Conclusion Biotransformation of DHT (1) with two fungal cultures produced eight known metabolites. Metabolites 5–7 effectively inhibited the BChE activity. Cholinesterase inhibition is among the key strategies in the management of Alzheimer’s disease (AD). The experimental findings were further validated by in silico inhibition studies and possible modes of action were deduced. PMID:24103815

  20. Long-Term Oil Contamination Alters the Molecular Ecological Networks of Soil Microbial Functional Genes

    PubMed Central

    Liang, Yuting; Zhao, Huihui; Deng, Ye; Zhou, Jizhong; Li, Guanghe; Sun, Bo

    2016-01-01

    With knowledge on microbial composition and diversity, investigation of within-community interactions is a further step to elucidate microbial ecological functions, such as the biodegradation of hazardous contaminants. In this work, microbial functional molecular ecological networks were studied in both contaminated and uncontaminated soils to determine the possible influences of oil contamination on microbial interactions and potential functions. Soil samples were obtained from an oil-exploring site located in South China, and the microbial functional genes were analyzed with GeoChip, a high-throughput functional microarray. By building random networks based on null model, we demonstrated that overall network structures and properties were significantly different between contaminated and uncontaminated soils (P < 0.001). Network connectivity, module numbers, and modularity were all reduced with contamination. Moreover, the topological roles of the genes (module hub and connectors) were altered with oil contamination. Subnetworks of genes involved in alkane and polycyclic aromatic hydrocarbon degradation were also constructed. Negative co-occurrence patterns prevailed among functional genes, thereby indicating probable competition relationships. The potential “keystone” genes, defined as either “hubs” or genes with highest connectivities in the network, were further identified. The network constructed in this study predicted the potential effects of anthropogenic contamination on microbial community co-occurrence interactions. PMID:26870020

  1. Repeats in transforming acidic coiled-coil (TACC) genes.

    PubMed

    Trivedi, Seema

    2013-06-01

    Transforming acidic coiled-coil proteins (TACC1, 2, and 3) are essential proteins associated with the assembly of spindle microtubules and maintenance of bipolarity. Dysregulation of TACCs is associated with tumorigenesis, but studies of microsatellite instability in TACC genes have not been extensive. Microsatellite or simple sequence repeat instability is known to cause many types of cancer. The present in silico analysis of SSRs in human TACC gene sequences shows the presence of mono- to hexa-nucleotide repeats, with the highest densities found for mono- and di-nucleotide repeats. Density of repeats is higher in introns than in exons. Some of the repeats are present in regulatory regions and retained introns. Human TACC genes show conservation of many repeat classes. Microsatellites in TACC genes could be valuable markers for monitoring numerical chromosomal aberrations and or cancer.

  2. Nickel-induced heritable alterations in retroviral transforming gene expression.

    PubMed Central

    Biggart, N W; Gallick, G E; Murphy, E C

    1987-01-01

    Determination of the mutagenic effects of carcinogenic nickel compounds has been difficult because, like many metals, nickel is poorly or nonmutagenic in procaryotic mutagenicity assays. We attempted to characterize nickel-induced genetic lesions by assessing the effect of nickel chloride on the conditionally defective expression of the v-mos transforming gene in normal rat kidney cells infected with the Murine sarcoma virus mutant ts110 (MuSVts110) retrovirus. MuSVts110 contains an out-of-frame gag gene-mos gene junction that prevents the expression of the v-mos gene at the nonpermissive temperature (39 degrees C). In MuSVts110-infected cells (6m2 cells) grown at 33 degrees C, however, this defect can be suppressed by a splicing event that restores the mos reading frame, allowing the expression of a gag-mos fusion protein which induces the transformed phenotype. The capacity to splice the viral transcript at 33 degrees C, but not at 39 degrees C, is an intrinsic property of the viral RNA. This property allowed us to target the MuSVts110 genome using a positive selection scheme whereby nickel was used to induce genetic changes which resulted in expression of the transformed phenotype at 39 degrees C. We treated 6m2 cells with NiCl2 and isolated foci consisting of cells which had reverted to the transformed phenotype at 39 degrees C. We found that brief nickel treatment increased the reversion frequency of 6m2 cells grown at 39 degrees C sevenfold over the spontaneous reversion frequency. The nickel-induced revertants displayed the following heritable characteristics: They stably maintained the transformed phenotype at 39 degrees C; unlike the MuSVts110 RNA in 6m2 cells, the nickel-induced revertant viral RNA could be spliced efficiently at 39 degrees C; as a consequence of the enhanced accumulation of spliced viral RNA, the nickel-induced revertants produced substantial amounts of the transforming v-mos protein P85gag-mos at 39 degrees C; the nickel

  3. Functional gene diversity of soil microbial communities from five oil-contaminated fields in China

    PubMed Central

    Liang, Yuting; Van Nostrand, Joy D; Deng, Ye; He, Zhili; Wu, Liyou; Zhang, Xu; Li, Guanghe; Zhou, Jizhong

    2011-01-01

    To compare microbial functional diversity in different oil-contaminated fields and to know the effects of oil contaminant and environmental factors, soil samples were taken from typical oil-contaminated fields located in five geographic regions of China. GeoChip, a high-throughput functional gene array, was used to evaluate the microbial functional genes involved in contaminant degradation and in other major biogeochemical/metabolic processes. Our results indicated that the overall microbial community structures were distinct in each oil-contaminated field, and samples were clustered by geographic locations. The organic contaminant degradation genes were most abundant in all samples and presented a similar pattern under oil contaminant stress among the five fields. In addition, alkane and aromatic hydrocarbon degradation genes such as monooxygenase and dioxygenase were detected in high abundance in the oil-contaminated fields. Canonical correspondence analysis indicated that the microbial functional patterns were highly correlated to the local environmental variables, such as oil contaminant concentration, nitrogen and phosphorus contents, salt and pH. Finally, a total of 59% of microbial community variation from GeoChip data can be explained by oil contamination, geographic location and soil geochemical parameters. This study provided insights into the in situ microbial functional structures in oil-contaminated fields and discerned the linkages between microbial communities and environmental variables, which is important to the application of bioremediation in oil-contaminated sites. PMID:20861922

  4. RELATIONSHIPS BETWEEN CULTURABLE SOIL MICROBIAL POPULATIONS AND GROSS NITROGEN TRANSFORMATION PROCESSES IN A CLAY LOAM SOIL ACROSS ECOSYSTEMS

    EPA Science Inventory

    The size and quality of soil organic matter (SOM) pool can vary between ecosystems and can affect many soil properties. The objective of this study was to examine the relationship between gross N transformation rates and microbial populations and to investigate the role that SOM...

  5. Biotransformation of selected iodinated X-ray contrast media and characterization of microbial transformation pathways.

    PubMed

    Kormos, Jennifer Lynne; Schulz, Manoj; Kohler, Hans-Peter E; Ternes, Thomas A

    2010-07-01

    Iodinated X-ray contrast media (ICM) are commonly detected in the aquatic environment at concentrations up to the low microgram per liter range. In this study, the biotransformation of selected ICM (diatrizoate, iohexol, iomeprol, and iopamidol) in aerobic soil-water and river sediment-water batch systems was investigated. In addition, microbial transformation pathways were proposed. Diatrizoate, an ionic ICM, was not biotransformed, while three nonionic ICM were transformed into several biotransformation products (TPs) at neutral pH. Iohexol and iomeprol were biotransformed to eleven TPs and fifteen TPs, respectively, while eight TPs were detected for iopamidol. Since seven of the TPs detected during biotransformation had not been previously identified, mass fragmentation experiments were completed to elucidate the chemical structures. Oxidation of primary alcoholic moieties, cleavage of the N-C bonds (i.e., deacetylation and removal of hydroxylated propanoic acids), and decarboxylation are potential reactions that can explain the formation of the identified TPs. Iohexol and iomeprol had similar biotransformation rates, while iopamidol was biotransformed slower and to a lesser extent. A LC tandem MS method confirmed the presence of ICM TPs in aqueous environmental samples. Fifteen of the ICM TPs were even detected in drinking water with concentrations up to 120 ng/L.

  6. Abelson murine leukemia virus: structural requirements for transforming gene function.

    PubMed Central

    Srinivasan, A; Dunn, C Y; Yuasa, Y; Devare, S G; Reddy, E P; Aaronson, S A

    1982-01-01

    The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage lambda gtWES.lambda B was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments showed that subgenomic clones that lacked the 5' long terminal repeat and adjoining sequences (300 base pairs downstream of the repeat) were not biologically active. In contrast, subgenomic clones that lacked the 3' long terminal repeat and as much as 1.3 kilobase pairs of the A-MuLV cell-derived abl gene were as efficient as wild-type viral DNA in transformation. The A-MuLV-encoded polyprotein P120 and its associated protein kinase activity were detected in transformants obtained by transfection with Cla I, BamHI, and HindIII subgenomic clones. In contrast, individual transformants obtained with subgenomic Sal I clones expressed A-MuLV proteins ranging in size from 82,000 to 95,000 daltons. Each demonstrated an associated protein kinase activity. These results provide direct genetic evidence that only the proximal 40% of abl with its associated 5' helper viral sequences is required for fibroblast transformation. Images PMID:6291048

  7. Dramatic Increases of Soil Microbial Functional Gene Diversity at the Treeline Ecotone of Changbai Mountain.

    PubMed

    Shen, Congcong; Shi, Yu; Ni, Yingying; Deng, Ye; Van Nostrand, Joy D; He, Zhili; Zhou, Jizhong; Chu, Haiyan

    2016-01-01

    The elevational and latitudinal diversity patterns of microbial taxa have attracted great attention in the past decade. Recently, the distribution of functional attributes has been in the spotlight. Here, we report a study profiling soil microbial communities along an elevation gradient (500-2200 m) on Changbai Mountain. Using a comprehensive functional gene microarray (GeoChip 5.0), we found that microbial functional gene richness exhibited a dramatic increase at the treeline ecotone, but the bacterial taxonomic and phylogenetic diversity based on 16S rRNA gene sequencing did not exhibit such a similar trend. However, the β-diversity (compositional dissimilarity among sites) pattern for both bacterial taxa and functional genes was similar, showing significant elevational distance-decay patterns which presented increased dissimilarity with elevation. The bacterial taxonomic diversity/structure was strongly influenced by soil pH, while the functional gene diversity/structure was significantly correlated with soil dissolved organic carbon (DOC). This finding highlights that soil DOC may be a good predictor in determining the elevational distribution of microbial functional genes. The finding of significant shifts in functional gene diversity at the treeline ecotone could also provide valuable information for predicting the responses of microbial functions to climate change.

  8. Dramatic Increases of Soil Microbial Functional Gene Diversity at the Treeline Ecotone of Changbai Mountain

    PubMed Central

    Shen, Congcong; Shi, Yu; Ni, Yingying; Deng, Ye; Van Nostrand, Joy D.; He, Zhili; Zhou, Jizhong; Chu, Haiyan

    2016-01-01

    The elevational and latitudinal diversity patterns of microbial taxa have attracted great attention in the past decade. Recently, the distribution of functional attributes has been in the spotlight. Here, we report a study profiling soil microbial communities along an elevation gradient (500–2200 m) on Changbai Mountain. Using a comprehensive functional gene microarray (GeoChip 5.0), we found that microbial functional gene richness exhibited a dramatic increase at the treeline ecotone, but the bacterial taxonomic and phylogenetic diversity based on 16S rRNA gene sequencing did not exhibit such a similar trend. However, the β-diversity (compositional dissimilarity among sites) pattern for both bacterial taxa and functional genes was similar, showing significant elevational distance-decay patterns which presented increased dissimilarity with elevation. The bacterial taxonomic diversity/structure was strongly influenced by soil pH, while the functional gene diversity/structure was significantly correlated with soil dissolved organic carbon (DOC). This finding highlights that soil DOC may be a good predictor in determining the elevational distribution of microbial functional genes. The finding of significant shifts in functional gene diversity at the treeline ecotone could also provide valuable information for predicting the responses of microbial functions to climate change. PMID:27524983

  9. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance.

    PubMed

    Kembel, Steven W; Wu, Martin; Eisen, Jonathan A; Green, Jessica L

    2012-01-01

    The abundance of different SSU rRNA ("16S") gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly - from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data.

  10. Functional Ecological Gene Networks to Reveal the Changes Among Microbial Interactions Under Elevated Carbon Dioxide Conditions

    SciTech Connect

    Deng, Ye; Zhou, Jizhong; Luo, Feng; He, Zhili; Tu, Qichao; Zhi, Xiaoyang

    2010-05-17

    Biodiversity and its responses to environmental changes is a central issue in ecology, and for society. Almost all microbial biodiversity researches focus on species richness and abundance but ignore the interactions among different microbial species/populations. However, determining the interactions and their relationships to environmental changes in microbial communities is a grand challenge, primarily due to the lack of information on the network structure among different microbial species/populations. Here, a novel random matrix theory (RMT)-based conceptual framework for identifying functional ecological gene networks (fEGNs) is developed with the high throughput functional gene array hybridization data from the grassland microbial communities in a long-term FACE (Free Air CO2 Enrichment) experiment. Both fEGNs under elevated CO2 (eCO2) and ambient CO2 (aCO2) possessed general characteristics of many complex systems such as scale-free, small-world, modular and hierarchical. However, the topological structure of the fEGNs is distinctly different between eCO2 and aCO2, suggesting that eCO2 dramatically altered the interactions among different microbial functional groups/populations. In addition, the changes in network structure were significantly correlated with soil carbon and nitrogen dynamics, and plant productivity, indicating the potential importance of network interactions in ecosystem functioning. Elucidating network interactions in microbial communities and their responses to environmental changes are fundamentally important for research in microbial ecology, systems microbiology, and global change.

  11. Evolutionary Conservation of Ceratitis capitata transformer Gene Function

    PubMed Central

    Pane, Attilio; De Simone, Annamaria; Saccone, Giuseppe; Polito, Catello

    2005-01-01

    Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects. PMID:15998727

  12. Investigating microbial transformations of soil organic matter: synthesizing knowledge from disparate fields to guide new experimentation

    NASA Astrophysics Data System (ADS)

    Billings, S. A.; Tiemann, L. K.; Ballantyne, F., IV; Lehmeier, C. A.; Min, K.

    2015-04-01

    Discerning why some soil organic matter (SOM) leaves soil profiles relatively quickly while other compounds, especially at depth, can be retained for decades to millennia is challenging for a multitude of reasons. Simultaneous with soil-specific advances, multiple other disciplines have enhanced their knowledge bases in ways potentially useful for future investigations of SOM decay. In this article, we highlight observations highly relevant for those investigating SOM decay and retention but often emanating from disparate fields and residing in literature seldom cited in SOM research. We focus on recent work in two key areas. First, we turn to experimental approaches using natural and artificial aquatic environments to investigate patterns of microbially mediated OM transformations as environmental conditions change, and highlight how aquatic microbial responses to environmental change can reveal processes likely important to OM decay and retention in soils. Second, we emphasize the importance of establishing intrinsic patterns of decay kinetics for purified substrates commonly found in soils to develop baseline rates. These decay kinetics - which represent the upper limit of the reaction rates - can then be compared to substrate decay kinetics observed in natural samples, which integrate intrinsic decay reaction rates and edaphic factors essential to the site under study but absent in purified systems. That comparison permits the site-specific factors to be parsed from the fundamental decay kinetics, an important advance in our understanding of SOM decay (and thus persistence) in natural systems. We then suggest ways in which empirical observations from aquatic systems and purified substrate-enzyme reaction kinetics can be used to advance recent theoretical efforts in SOM-focused research. Finally, we suggest how the observations in aquatic and purified substrate-enzyme systems could be used to help unravel the puzzles presented by oft-observed patterns of SOM

  13. Anoxic carbon degradation in Arctic sediments: Microbial transformations of complex substrates

    NASA Astrophysics Data System (ADS)

    Arnosti, C.; Finke, N.; Larsen, O.; Ghobrial, S.

    2005-05-01

    Complex substrates are degraded in anoxic sediments by the concerted activities of diverse microbial communities. To explore the effects of substrate complexity on carbon transformations in permanently cold anoxic sediments, four substrates— Spirulina cells, Isochrysis cells, and soluble high molecular weight carbohydrate-rich extracts of these cells (Spir-Ex and Iso-Ex)—were added to sediments collected from Svalbard. The sediments were homogenized, incubated anaerobically in gas-tight bags at 0°C, and enzyme activities, fermentation, and terminal respiration were monitored over a 1134 h time course. All substrate additions yielded a fraction (8%-13%) of carbon that was metabolized to CO 2 over the first 384 h of incubation. The timecourse of VFA (volatile fatty acid) production and consumption, as well as the suite of VFAs produced, was similar for all substrates. After this phase, pathways of carbon degradation diverged, with an additional 43%, 32%, 33%, and 8% of Isochrysis, Iso-Ex, Spirulina, and Spir-Ex carbon respired to CO 2 over the next 750 h of incubation. Somewhat surprisingly, the soluble, carbohydrate-rich extracts did not prove to be more labile substrates than the whole cells from which they were derived. Although Spirulina and Iso-Ex differed in physical and chemical characteristics (solid/soluble, C/N ratio, lipid and carbohydrate content), nearly identical quantities of carbon were respired to CO 2. In contrast, only 15% of Spir-Ex carbon was respired, despite the initial burst of activity that it fueled, its soluble nature, and its relatively high (50%) carbohydrate content. The microbial community in these cold anoxic sediments clearly has the capacity to react rapidly to carbon input; extent and timecourse of remineralization of added carbon is similar to observations made at much higher temperatures in temperate sediments. The extent of carbon remineralization from these specific substrates, however, would not likely have been predicted

  14. Biofuel Potential of Plants Transformed Genetically with NAC Family Genes

    PubMed Central

    Singh, Sadhana; Grover, Atul; Nasim, M.

    2016-01-01

    NAC genes contribute to enhance survivability of plants under conditions of environmental stress and in secondary growth of the plants, thereby building biomass. Thus, genetic transformation of plants using NAC genes provides a possibility to tailor biofuel plants. Over-expression studies have indicated that NAC family genes can provide tolerance to various biotic and abiotic stresses, either by physiological or biochemical changes at the cellular level, or by affecting visible morphological and anatomical changes, for example, by development of lateral roots in a number of plants. Over-expression of these genes also work as triggers for development of secondary cell walls. In our laboratory, we have observed a NAC gene from Lepidium latifolium contributing to both enhanced biomass as well as cold stress tolerance of model plants tobacco. Thus, we have reviewed all the developments of genetic engineering using NAC genes which could enhance the traits required for biofuel plants, either by enhancing the stress tolerance or by enhancing the biomass of the plants. PMID:26858739

  15. Effects of soil type and farm management on soil ecological functional genes and microbial activities

    SciTech Connect

    Reeve, Jennifer; Schadt, Christopher Warren; Carpenter-Boggs, Lynne; Kang, S.; Zhou, Jizhong; Reganold, John P.

    2010-01-01

    Relationships between soil microbial diversity and soil function are the subject of much debate. Process-level analyses have shown that microbial function varies with soil type and responds to soil management. However, such measurements cannot determine the role of community structure and diversity in soil function. The goal of this study was to investigate the role of gene frequency and diversity, measured by microarray analysis, on soil processes. The study was conducted in an agro-ecosystem characterized by contrasting management practices and soil types. Eight pairs of adjacent commercial organic and conventional strawberry fields were matched for soil type, strawberry variety, and all other environmental conditions. Soil physical, chemical and biological analyses were conducted including functional gene microarrays (FGA). Soil physical and chemical characteristics were primarily determined by soil textural type (coarse vs fine-textured), but biological and FGA measures were more influenced by management (organic vs conventional). Organically managed soils consistently showed greater functional activity as well as FGA signal intensity (SI) and diversity. Overall FGA SI and diversity were correlated to total soil microbial biomass. Functional gene group SI and/or diversity were correlated to related soil chemical and biological measures such as microbial biomass, cellulose, dehydrogenase, ammonium and sulfur. Management was the dominant determinant of soil biology as measured by microbial gene frequency and diversity, which paralleled measured microbial processes.

  16. Investigating microbial transformations of soil organic matter: synthesizing knowledge from disparate fields to guide new experimentation

    NASA Astrophysics Data System (ADS)

    Billings, S. A.; Tiemann, L. K.; Ballantyne, F., IV; Lehmeier, C.; Min, K.

    2014-12-01

    Investigators of soil organic matter (SOM) transformations struggle with a deceptively simple-sounding question: "Why does some SOM leave the soil profile relatively quickly, while other compounds, especially those at depth, appear to be retained on timescales ranging from the decadal to the millennial?" This question is important on both practical and academic levels, but addressing it is challenging for a multitude of reasons. Simultaneous with soil-specific advances, multiple other disciplines have enhanced their knowledge bases in ways potentially useful for future investigations of SOM decay. In this article, we highlight observations highly relevant for those investigating SOM decay and retention but often emanating from disparate fields and residing in literature seldom cited in SOM research. We focus on recent work in two key areas. First, we turn to experimental approaches using natural and artificial aquatic environments to investigate patterns of microbially-mediated OM transformations as environmental conditions change, and highlight how aquatic microbial responses to environmental change can reveal processes likely important to OM decay and retention in soils. Second, we emphasize the importance of establishing intrinsic patterns of decay kinetics for purified substrates commonly found in soils to develop baseline rates. These decay kinetics - which represent the upper limit of the reaction rates - can then be compared to substrate decay kinetics observed in natural samples, which integrate intrinsic decay reaction rates and edaphic factors essential to the site under study but absent in purified systems. That comparison permits the site-specific factors to be parsed from the fundamental decay kinetics, an important advance in our understanding of SOM decay (and thus persistence) in natural systems. We then suggest ways in which empirical observations from aquatic systems and purified enzyme-substrate reaction kinetics can be used to advance recent

  17. Microbial transformations of azaarenes in creosite-contaminated soil and ground water: Laboratory and field studies

    USGS Publications Warehouse

    Pereira, W.E.; Rostad, C.E.; Updegraff, D.M.; Bennett, J.L.

    1988-01-01

    Azaarenes or aromatic nitrogen heterocycles are a class of compounds found in wood-preservative wastes containing creosote. The fate and movement of these compounds in contaminated aquifers is not well understood. Water-quality studies in an aquifer contaminated with creosote near Pensacola, Florida, indicated that ground water was contaminated with several azaarenes and their oxygenated and alkylated derivatives, suggesting that these oxygenated compounds may be products of microbial transformation reactions. Accordingly, laboratory studies were designed to investigate the fate of these compounds. Under aerobic conditions, soil pseudomonads isolated from creosote-contaminated soil converted quinoline to 2(1H)quinoline that subsequently was degraded to unknown products. A methanogenic consortium isolated from an anaerobic sewage digestor, in presence of ground-water and creosote-contaminated soil, converted quinoline, isoquinoline, and 4-methylquinoline to their respective oxygenated analogs. In addition, N-, C-, and O-methylated analogs of oxygenated azaarenes were identified by gas chromatography-mass spectrometry (GC-MS) in aerobic cultures. Under the experimental conditions, 2-methylquinoline was biorefractory. Presence of similar biotransformation products in anaerobic cultures and contaminated ground water from the Pensacola site provided further evidence that these compounds indeed were mivrobial transformation products. Stable isotope labeling studies indicated that the source of the oxygen atom for this hydroxylation reaction under aerobic and anaerobic conditions was water. A mechanism was proposed for this hydroxylation reaction. Whereas parent azaarenes are biodegradable in both anaerobic and aerobic zones, oxygenated and alkylated analogs are more biorefractory and, hence, persistent in anaerobic zones of contaminated aquifers.

  18. Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance

    PubMed Central

    Bokulich, Nicholas A; Bergsveinson, Jordyn; Ziola, Barry; Mills, David A

    2015-01-01

    Distinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments. DOI: http://dx.doi.org/10.7554/eLife.04634.001 PMID:25756611

  19. Taxa-area Relationship (TAR) of Microbial Functional Genes with Long-TGerm Fertilization

    SciTech Connect

    Liang, Yuting; Wu, Liyou; Clark, Ian; Xue, Kai; Van Nostrand, Joy D.; Deng, Ye; He, Zhili; Hirsch, Penny; Mcgrath, Steve; Zhou, Jizhong

    2010-05-17

    Diversity and spatial patterns in plant and animal communities are well documented as a positive-power law of a taxa-area relationship (TAR). At present little is known whether this also applies to soil microbial communities and whether long-term fertilization has an influence on the underlying microbial diversity. To test the effects of long-term fertilization on above-ground botanical diversity and below-ground microbial diversity, a nested sampling approach on Park Grass plots (12d& 11/2c) of Rothamsted Reseach in United Kingdom, both at ~;; pH 5 but with plant diversities of between 42 and 13 respectively were used. GeoChip 3.0, covering approximately 57, 000 gene sequences of 292 gene families involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation, was used to determine the gene area relationships for both functional and phylogenetic groups and the relationship to plant diversity. Our analysis indicated that the microbial communities were separated by different plant diversity based on DCA. The soil microbial diversity was in accord with plant diversity. Soil microbial community exhibited different z value with different plant diversity, z = 0.0449 with higher plant diversity and z = 0.0583 with lower plant diversity (P< 0.0001). These results suggest that the turnover in space of microorganisms may be higher with long-term fertilization.

  20. Bioremediation of petroleum hydrocarbons: catabolic genes, microbial communities, and applications.

    PubMed

    Fuentes, Sebastián; Méndez, Valentina; Aguila, Patricia; Seeger, Michael

    2014-06-01

    Bioremediation is an environmental sustainable and cost-effective technology for the cleanup of hydrocarbon-polluted soils and coasts. In spite of that longer times are usually required compared with physicochemical strategies, complete degradation of the pollutant can be achieved, and no further confinement of polluted matrix is needed. Microbial aerobic degradation is achieved by the incorporation of molecular oxygen into the inert hydrocarbon molecule and funneling intermediates into central catabolic pathways. Several families of alkane monooxygenases and ring hydroxylating dioxygenases are distributed mainly among Proteobacteria, Actinobacteria, Firmicutes and Fungi strains. Catabolic routes, regulatory networks, and tolerance/resistance mechanisms have been characterized in model hydrocarbon-degrading bacteria to understand and optimize their metabolic capabilities, providing the basis to enhance microbial fitness in order to improve hydrocarbon removal. However, microbial communities taken as a whole play a key role in hydrocarbon pollution events. Microbial community dynamics during biodegradation is crucial for understanding how they respond and adapt to pollution and remediation. Several strategies have been applied worldwide for the recovery of sites contaminated with persistent organic pollutants, such as polycyclic aromatic hydrocarbons and petroleum derivatives. Common strategies include controlling environmental variables (e.g., oxygen availability, hydrocarbon solubility, nutrient balance) and managing hydrocarbon-degrading microorganisms, in order to overcome the rate-limiting factors that slow down hydrocarbon biodegradation.

  1. Impacts of gene bioaugmentation with pJP4-harboring bacteria of 2,4-D-contaminated soil slurry on the indigenous microbial community.

    PubMed

    Inoue, Daisuke; Yamazaki, Yuji; Tsutsui, Hirofumi; Sei, Kazunari; Soda, Satoshi; Fujita, Masanori; Ike, Michihiko

    2012-04-01

    Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.

  2. The functional gene composition and metabolic potential of coral-associated microbial communities

    PubMed Central

    Zhang, Yanying; Ling, Juan; Yang, Qingsong; Wen, Chongqing; Yan, Qingyun; Sun, Hongyan; Van Nostrand, Joy D.; Shi, Zhou; Zhou, Jizhong; Dong, Junde

    2015-01-01

    The phylogenetic diversity of coral-associated microbes has been extensively examined, but some contention remains regarding whether coral-associated microbial communities are species-specific or site-specific. It is suggested that corals may associate with microbes in terms of function, although little is known about the differences in coral-associated microbial functional gene composition and metabolic potential among coral species. Here, 16S rRNA Illumina sequencing and functional gene array (GeoChip 5.0) were used to assess coral-associated microbial communities. Our results indicate that both host species and environmental variables significantly correlate with shifts in the microbial community structure and functional potential. Functional genes related to key biogeochemical cycles including carbon, nitrogen, sulfur and phosphorus cycling, metal homeostasis, organic remediation, antibiotic resistance and secondary metabolism were shown to significantly vary between and among the four study corals (Galaxea astreata, Porites lutea, Porites andrewsi and Pavona decussata). Genes specific for anammox were also detected for the first time in the coral holobiont and positively correlated with ammonium. This study reveals that variability in the functional potential of coral-associated microbial communities is largely driven by changes in environmental factors and further demonstrates the importance of linking environmental parameters with genomic data in complex environmental systems. PMID:26536917

  3. Functional gene differences in soil microbial communities from conventional, low-input, and organic farmlands.

    PubMed

    Xue, Kai; Wu, Liyou; Deng, Ye; He, Zhili; Van Nostrand, Joy; Robertson, Philip G; Schmidt, Thomas M; Zhou, Jizhong

    2013-02-01

    Various agriculture management practices may have distinct influences on soil microbial communities and their ecological functions. In this study, we utilized GeoChip, a high-throughput microarray-based technique containing approximately 28,000 probes for genes involved in nitrogen (N)/carbon (C)/sulfur (S)/phosphorus (P) cycles and other processes, to evaluate the potential functions of soil microbial communities under conventional (CT), low-input (LI), and organic (ORG) management systems at an agricultural research site in Michigan. Compared to CT, a high diversity of functional genes was observed in LI. The functional gene diversity in ORG did not differ significantly from that of either CT or LI. Abundances of genes encoding enzymes involved in C/N/P/S cycles were generally lower in CT than in LI or ORG, with the exceptions of genes in pathways for lignin degradation, methane generation/oxidation, and assimilatory N reduction, which all remained unchanged. Canonical correlation analysis showed that selected soil (bulk density, pH, cation exchange capacity, total C, C/N ratio, NO(3)(-), NH(4)(+), available phosphorus content, and available potassium content) and crop (seed and whole biomass) variables could explain 69.5% of the variation of soil microbial community composition. Also, significant correlations were observed between NO(3)(-) concentration and denitrification genes, NH(4)(+) concentration and ammonification genes, and N(2)O flux and denitrification genes, indicating a close linkage between soil N availability or process and associated functional genes.

  4. Competitive Oxidation Kinetics and Microbial Ecology: Intermediate Sulfur Transformations in Acid Mine Drainage Environments

    NASA Astrophysics Data System (ADS)

    Druschel, G. K.; Hamers, R. J.; Banfield, J. F.

    2001-12-01

    Experimental studies have demonstrated that oxidation of pyrite proceeds through several intermediate sulfur species, notably elemental sulfur, thiosulfate, and polythionates (Schippers et al., 1996). However, detailed sampling and analysis of flowing waters and pore waters failed to detect intermediate sulfur species in the 5-way area of the Richmond metal sulfide deposit at the Iron Mountain Mine in northern California. Potential energy available from the oxidation of intermediate sulfur species is considerable, so microbial activity may explain absence of intermediate sulfur compounds at the site. However, the abundance of sulfur-oxidizing microorganisms in areas of active pyrite oxidation at the 5-way is generally low (Bond et al. 2000). Rapid inorganic oxidation rates may prevent microorganisms from utilizing these intermediate sulfur species, thus shaping the structure of microbial communities in acid mine drainage (AMD) environments. Rates and mechanisms of oxidation for tetrathionate and elemental sulfur have been experimentally determined. Batch and flow-through experiments have indicated very slow oxidation of elemental sulfur in inorganic solutions analogous to AMD environments. Results for tetrathionate indicate the importance of non-metabolic and inorganic processes, including surface catalysis and the generation of hydroxyl radicals. Surface catalysis occurs through trithionate on iron oxide surfaces. Hydroxyl radicals may be formed directly by microbes living in proximity to pyrite surfaces, or at pyrite surfaces undergoing wetting and drying cycles. Further experiments investigating the importance of organic compounds associated with iron-oxidizing microorganisms acting as electron transport shuttles and/or wetting agents and ab initio calculations of the electronic structure of potential reactants and intermediates are currently being performed. It is suggested that inorganic processes involved with seasonal wetting and drying of pyritic sediment

  5. Microbial co-habitation and lateral gene transfer: what transposases can tell us

    SciTech Connect

    Hooper, Sean D.; Mavromatis, Konstantinos; Kyrpides, Nikos C.

    2009-03-01

    Determining the habitat range for various microbes is not a simple, straightforward matter, as habitats interlace, microbes move between habitats, and microbial communities change over time. In this study, we explore an approach using the history of lateral gene transfer recorded in microbial genomes to begin to answer two key questions: where have you been and who have you been with? All currently sequenced microbial genomes were surveyed to identify pairs of taxa that share a transposase that is likely to have been acquired through lateral gene transfer. A microbial interaction network including almost 800 organisms was then derived from these connections. Although the majority of the connections are between closely related organisms with the same or overlapping habitat assignments, numerous examples were found of cross-habitat and cross-phylum connections. We present a large-scale study of the distributions of transposases across phylogeny and habitat, and find a significant correlation between habitat and transposase connections. We observed cases where phylogenetic boundaries are traversed, especially when organisms share habitats; this suggests that the potential exists for genetic material to move laterally between diverse groups via bridging connections. The results presented here also suggest that the complex dynamics of microbial ecology may be traceable in the microbial genomes.

  6. Arsenic and sulfur transformations in hydrothermal spring waters and microbial mats of Yellowstone National Park

    NASA Astrophysics Data System (ADS)

    Druschel, G. K.; Lorenson, G. W.; Oduro, H.; McDermott, T.

    2006-12-01

    Many Yellowstone National Park hydrothermal waters contain high concentrations of arsenic and sulfur species which support various communities of chemotrophic microorganisms. In order to delineate the spatial and temporal variability of these organisms, which is critical in defining their ecological niche and role in element cycling, both temporal and spatial resolution of arsenic and sulfur speciation is needed. We present results from 2 years of field data and experiments showing the utility of Au-amalgam voltammetric microelectrodes in describing specific arsenic and sulfur speciation in hydrothermal systems. New insights on sulfur cycling in several hydrothermal pools, particularly facilitated by the in situ observation of polysulfides in these waters, are becoming evident and may help to resolve key issues surrounding the activity of organisms in these systems. The additional observation and description of voltammetric signals for dissolved and surfactant-stabilized colloidal forms of elemental sulfur with polysulfides in these systems suggests that sulfur cycling in many springs is largely dependent on the formation and oxidation of polysulfides derived from the interaction of dissolved hydrogen sulfide and elemental sulfur. We will present laboratory and field evidence for these reactions and discuss their importance in sulfur cycling and the potential role of microorganisms in these transformations. Arsenic (As(III)) and sulfide (H2S) oxidation in biofilms of Dragon spring in the Norris Geyser basin have now been described with high spatial resolution (as fine as 25 micron step sizes for vertical profiles). Small-scale coring with immediate freeze preservation and cryomicrotoming of those materials was attempted to develop techniques which will facilitate the description of coupled geochemical and microbiological changes on the micron scale in these systems. We will additionally describe the initial limited success of these microbial sampling techniques and

  7. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

  8. Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning.

    PubMed

    Xue, Kai; Xie, Jianping; Zhou, Aifen; Liu, Feifei; Li, Dejun; Wu, Liyou; Deng, Ye; He, Zhili; Van Nostrand, Joy D; Luo, Yiqi; Zhou, Jizhong

    2016-01-01

    Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward more C4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming.

  9. Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning

    PubMed Central

    Xue, Kai; Xie, Jianping; Zhou, Aifen; Liu, Feifei; Li, Dejun; Wu, Liyou; Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Luo, Yiqi; Zhou, Jizhong

    2016-01-01

    Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward more C4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming. PMID:27199978

  10. Microbial gene functions enriched in the Deepwater Horizon deep-sea oil plume

    SciTech Connect

    Lu, Z.; Deng, Y.; Nostrand, J.D. Van; He, Z.; Voordeckers, J.; Zhou, A.; Lee, Y.-J.; Mason, O.U.; Dubinsky, E.; Chavarria, K.; Tom, L.; Fortney, J.; Lamendella, R.; Jansson, J.K.; D?haeseleer, P.; Hazen, T.C.; Zhou, J.

    2011-06-15

    The Deepwater Horizon oil spill in the Gulf of Mexico is the deepest and largest offshore spill in U.S. history and its impacts on marine ecosystems are largely unknown. Here, we showed that the microbial community functional composition and structure were dramatically altered in a deep-sea oil plume resulting from the spill. A variety of metabolic genes involved in both aerobic and anaerobic hydrocarbon degradation were highly enriched in the plume compared to outside the plume, indicating a great potential for intrinsic bioremediation or natural attenuation in the deep-sea. Various other microbial functional genes relevant to carbon, nitrogen, phosphorus, sulfur and iron cycling, metal resistance, and bacteriophage replication were also enriched in the plume. Together, these results suggest that the indigenous marine microbial communities could play a significant role in biodegradation of oil spills in deep-sea environments.

  11. Genetic transformation of Begonia tuberhybrida by Ri rol genes.

    PubMed

    Kiyokawa, S; Kikuchi, Y; Kamada, H; Harada, H

    1996-04-01

    We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals.

  12. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    SciTech Connect

    Lagrimini, L.M.

    1990-12-31

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  13. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    SciTech Connect

    Lagrimini, L.M.

    1990-01-01

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  14. Detection of catabolic genes in indigenous microbial consortia isolated from a diesel-contaminated soil.

    PubMed

    Milcic-Terzic, J; Lopez-Vidal, Y; Vrvic, M M; Saval, S

    2001-05-01

    Bioremediation is often used for in situ remediation of petroleum-contaminated sites. The primary focus of this study was on understanding the indigenous microbial community which can survive in contaminated environment and is responsible for the degradation. Diesel. toluene and naphthalene-degrading microbial consortia were isolated from diesel-contaminated soil by growing on selective hydrocarbon substrates. The presence and frequency of the catabolic genes responsible for aromatic hydrocarbon biodegradation (xylE, ndoB) within the isolated consortia were screened using polymerase chain reaction PCR and DNA DNA colony hybridization. The diesel DNA-extract possessed both the xy/E catabolic gene for toluene, and the nah catabolic gene for polynuclear aromatic hydrocarbon degradation. The toluene DNA-extract possessed only the xylE catabolic gene, while the naphthalene DNA-extract only the ndoB gene. Restriction enzyme analysis with HaeIII indicated similar restriction patterns for the xylE gene fragment between toluene DNA-extract and a type strain, Pseudomonas putida ATCC 23973. A substantial proportion (74%) of the colonies from the diesel-consortium possessed the xylE gene, and the ndoB gene (78%), while a minority (29%) of the toluene-consortium harbored the xylE gene. 59% of the colonies from the naphthalene-consortium had the ndoB gene, and did not have the xylE gene. These results indicate that the microbial population has been naturally enriched in organisms carrying genes for aromatic hydrocarbon degradation and that significant aromatic biodegradative potential exists at the site. Characterization of the population genotype constitutes a molecular diagnosis which permits the determination of the catabolic potential of the site to degrade the contaminant present.

  15. Organic micropollutants in aerobic and anaerobic membrane bioreactors: Changes in microbial communities and gene expression.

    PubMed

    Harb, Moustapha; Wei, Chun-Hai; Wang, Nan; Amy, Gary; Hong, Pei-Ying

    2016-10-01

    Organic micro-pollutants (OMPs) are contaminants of emerging concern in wastewater treatment due to the risk of their proliferation into the environment, but their impact on the biological treatment process is not well understood. The purpose of this study is to examine the effects of the presence of OMPs on the core microbial populations of wastewater treatment. Two nanofiltration-coupled membrane bioreactors (aerobic and anaerobic) were subjected to the same operating conditions while treating synthetic municipal wastewater spiked with OMPs. Microbial community dynamics, gene expression levels, and antibiotic resistance genes were analyzed using molecular-based approaches. Results showed that presence of OMPs in the wastewater feed had a clear effect on keystone bacterial populations in both the aerobic and anaerobic sludge while also significantly impacting biodegradation-associated gene expression levels. Finally, multiple antibiotic-type OMPs were found to have higher removal rates in the anaerobic MBR, while associated antibiotic resistance genes were lower.

  16. The dynamics of gene duplication and transposons in microbial genome evolution

    NASA Astrophysics Data System (ADS)

    Chia, Nicholas; Goldenfeld, Nigel

    2010-03-01

    Evidence indicates that new functional genes emerge from a process of gene duplication coupled with selection for a novel function. Recently, Bergthorsson et al. proposed a model of continuous selection in order to describe this process. Here, we examine their proposed evolutionary scheme, by modeling gene evolution using a stochastic simulation. Our results indicate that this model, and a related one that includes horizontal gene transfer, can account for the distribution of transposons in microbial genomes, and reproduce the observed environmentally-driven spatial dependence of transposon density in marine bacteria.

  17. Increases in the abundance of microbial genes encoding halotolerance and photosynthesis along a sediment salinity gradient

    NASA Astrophysics Data System (ADS)

    Jeffries, T. C.; Seymour, J. R.; Newton, K.; Smith, R. J.; Seuront, L.; Mitchell, J. G.

    2012-02-01

    Biogeochemical cycles are driven by the metabolic activity of microbial communities, yet the environmental parameters that underpin shifts in the functional potential coded within microbial community genomes are still poorly understood. Salinity is one of the primary determinants of microbial community structure and can vary strongly along gradients within a variety of habitats. To test the hypothesis that shifts in salinity will also alter the bulk biogeochemical potential of aquatic microbial assemblages, we generated four metagenomic DNA sequence libraries from sediment samples taken along a continuous, natural salinity gradient in the Coorong lagoon, Australia, and compared them to physical and chemical parameters. A total of 392483 DNA sequences obtained from four sediment samples were generated and used to compare genomic characteristics along the gradient. The most significant shifts along the salinity gradient were in the genetic potential for halotolerance and photosynthesis, which were more highly represented in hypersaline samples. At these sites, halotolerance was achieved by an increase in genes responsible for the acquisition of compatible solutes - organic chemicals which influence the carbon, nitrogen and methane cycles of sediment. Photosynthesis gene increases were coupled to an increase in genes matching Cyanobacteria, which are responsible for mediating CO2 and nitrogen cycles. These salinity driven shifts in gene abundance will influence nutrient cycles along the gradient, controlling the ecology and biogeochemistry of the entire ecosystem.

  18. Increases in the abundance of microbial genes encoding halotolerance and photosynthesis along a sediment salinity gradient

    NASA Astrophysics Data System (ADS)

    Jeffries, T. C.; Seymour, J. R.; Newton, K.; Smith, R. J.; Seuront, L.; Mitchell, J. G.

    2011-07-01

    Biogeochemical cycles are driven by the metabolic activity of microbial communities, yet the environmental parameters that underpin shifts in the functional potential coded within microbial community genomes are still poorly understood. Salinity is one of the primary determinants of microbial community structure and can vary strongly along gradients within a variety of habitats. To test the hypothesis that shifts in salinity will also alter the bulk biogeochemical potential of aquatic microbial assemblages, we generated four metagenomic DNA sequence libraries from sediment samples taken along a continuous, natural salinity gradient in the Coorong lagoon, Australia, and compared them to physical and chemical parameters. A total of 392483 DNA sequences obtained from four sediment samples were generated and used to compare genomic characteristics along the gradient. The most significant shifts along the salinity gradient were in the genetic potential for halotolerance and photosynthesis, which were more highly represented in hypersaline samples. At these sites, halotolerance was achieved by an increase in genes responsible for the acquisition of compatible solutes - organic chemicals which influence the carbon, nitrogen and methane cycles of sediment. Photosynthesis gene increases were coupled to an increase in genes matching Cyanobacteria, which are responsible for mediating CO2 and nitrogen cycles. These salinity driven shifts in gene abundance will influence nutrient cycles along the gradient, controlling the ecology and biogeochemistry of the entire ecosystem.

  19. Transformation Experiment Using Bioluminescence Genes of "Vibrio fischeri."

    ERIC Educational Resources Information Center

    Slock, James

    1995-01-01

    Bioluminescence transformation experiments show students the excitement and power of recombinant DNA technology. This laboratory experiment utilizes two plasmids of "Vibrio fischeri" in a transformation experiment. (LZ)

  20. Combining Push Pull Tracer Tests and Microbial DNA and mRNA Analysis to Assess In-Situ Groundwater Nitrate Transformations

    NASA Astrophysics Data System (ADS)

    Henson, W.; Graham, W. D.; Huang, L.; Ogram, A.

    2015-12-01

    Nitrogen transformation mechanisms in the Upper Floridan Aquifer (UFA) are still poorly understood because of karst aquifer complexity and spatiotemporal variability in nitrate and carbon loading. Transformation rates have not been directly measured in the aquifer. This study quantifies nitrate-nitrogen transformation potential in the UFA using single well push-pull tracer injection (PPT) experiments combined with microbial characterization of extracted water via qPCR and RT-qPCR of selected nitrate reduction genes. Tracer tests with chloride and nitrate ± carbon were executed in two wells representing anoxic and oxic geochemical end members in a spring groundwater contributing area. A significant increase in number of microbes with carbon addition suggests stimulated growth. Increases in the activities of denitrification genes (nirK and nirS) as measured by RT-qPCR were not observed. However, only microbes suspended in the tracer were obtained, ignoring effects of aquifer material biofilms. Increases in nrfA mRNA and ammonia concentrations were observed, supporting Dissimilatory Reduction of Nitrate to Ammonia (DNRA) as a reduction mechanism. In the oxic aquifer, zero order nitrate loss rates ranged from 32 to 89 nmol /L*hr with no added carbon and 90 to 240 nmol /L*hr with carbon. In the anoxic aquifer, rates ranged from 18 to 95 nmol /L*hr with no added carbon and 34 to 207 nmol /L*hr with carbon. These loss rates are low; 13 orders of magnitude less than the loads applied in the contributing area each year, however they do indicate that losses can occur in oxic and anoxic aquifers with and without carbon. These rates may include, ammonia adsorption, uptake, or denitrification in aquifer material biofilms. Rates with and without carbon addition for both aquifers were similar, suggesting aquifer redox state and carbon availability alone are insufficient to predict response to nutrient additions without characterization of microbial response. Surprisingly, these

  1. Soil microbial respiration (CO2) of natural and anthropogenically-transformed ecosystems in Moscow region, Russia

    NASA Astrophysics Data System (ADS)

    Ivashchenko, Kristina; Ananyeva, Nadezhda; Rogovaya, Sofia; Vasenev, Viacheslav

    2016-04-01

    The CO2 concentration in modern atmosphere is increasing and one of the most reasons of it is land use changing. It is related not only with soil plowing, but also with growing urbanization and, thereby, forming the urban ecosystems. Such conversion of soil cover might be affected by efflux CO2 from soil into atmosphere. The soil CO2 efflux mainly supplies by soil microorganisms respiration (contribution around 70-90%) and plant roots respiration. Soil microbial respiration (MR) is determined in the field (in situ) and laboratory (in vitro) conditions. The measurement of soil MR in situ is labour-consuming, and for district, region and country areas it is difficult carried. We suggest to define the MR of the upper highest active 10 cm mineral soil layer (in vitro) followed by the accounting of area for different ecosystems in large region of Russia. Soils were sampled (autumn, 2011) in natural (forest, meadow) and anthropogenically-transformed (arable, urban) ecosystems of Sergiev-Posad, Taldom, Voskresenk, Shatura, Serpukhov and Serbryanye Prudy districts in Moscow region. In soil samples (total 156) the soil MR (24 h, 22°C, 60% WHC) were measured after preincubation procedure (7 d., 22°C, 55% WHC). The soil MR ranged from 0.13 (urban) to 5.41 μg CO2-C g-1 h-1 (meadow), the difference between these values was 42 times. Then, the soil MR values (per unit soil weight) were calculated per unit soil area (1 m2), the layer thickness of which was 0.1 m (soil volume weight was equaled 1 g cm-3). The high MR values were noted for forests soil (832-1410 g CO2-C m-2 yr-1) of studied districts, and the low MR values were for arable and urban soils (by 1.6-3.2 and 1.3-2.7 times less compared to forests, respectively). The MR rate of urban soil in Voskresenk district was comparable to that of corresponding meadows and it was even higher (in average by 2.3 times) in Serpukhov district. The soil MR rate of studied cities was higher by 20%, than in corresponding arable soils

  2. Functional gene composition, diversity and redundancy in microbial stream biofilm communities.

    PubMed

    Dopheide, Andrew; Lear, Gavin; He, Zhili; Zhou, Jizhong; Lewis, Gillian D

    2015-01-01

    We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition.

  3. Functional Gene Composition, Diversity and Redundancy in Microbial Stream Biofilm Communities

    PubMed Central

    Dopheide, Andrew; Lear, Gavin; He, Zhili; Zhou, Jizhong; Lewis, Gillian D.

    2015-01-01

    We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition. PMID:25849814

  4. Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

    PubMed Central

    Xu, Meiying; Zhang, Qin; Xia, Chunyu; Zhong, Yuming; Sun, Guoping; Guo, Jun; Yuan, Tong; Zhou, Jizhong; He, Zhili

    2014-01-01

    Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition. PMID:24671084

  5. A Multi-omics Approach to Understand the Microbial Transformation of Lignocellulosic Materials in the Digestive System of the Wood-Feeding Beetle Odontotaenius disjunctus

    NASA Astrophysics Data System (ADS)

    Ceja Navarro, J. A.; Karaoz, U.; White, R. A., III; Lipton, M. S.; Adkins, J.; Mayali, X.; Blackwell, M.; Pett-Ridge, J.; Brodie, E.; Hao, Z.

    2015-12-01

    Odontotaenius disjuctus is a wood feeding beetle that processes large amounts of hardwoods and plays an important role in forest carbon cycling. In its gut, plant material is transformed into simple molecules by sequential processing during passage through the insect's digestive system. In this study, we used multiple 'omics approaches to analyze the distribution of microbial communities and their specific functions in lignocellulose deconstruction within the insect's gut. Fosmid clones were selected and sequenced from a pool of clones based on their expression of plant polymer degrading enzymes, allowing the identification of a wide range of carbohydrate degrading enzymes. Comparison of metagenomes of all gut regions demonstrated the distribution of genes across the beetle gut. Cellulose, starch, and xylan degradation genes were particularly abundant in the midgut and posterior hindgut. Genes involved in hydrogenotrophic production of methane and nitrogenases were more abundant in the anterior hindgut. Assembled contigs were binned into 127 putative genomes representing Bacteria, Archaea, Fungi and Nematodes. Eleven complete genomes were reconstructed allowing to identify linked functions/traits, including organisms with cellulosomes, and a combined potential for cellulose, xylan and starch hydrolysis and nitrogen fixation. A metaproteomic study was conducted to test the expression of the pathways identified in the metagenomic study. Preliminary analyses suggest enrichment of pathways related to hemicellulosic degradation. A complete xylan degradation pathway was reconstructed and GC-MS/MS based metabolomics identified xylobiose and xylose as major metabolite pools. To relate microbial identify to function in the beetle gut, Chip-SIP isotope tracing was conducted with RNA extracted from beetles fed 13C-cellulose. Multiple 13C enriched bacterial groups were detected, mainly in the midgut. Our multi-omics approach has allowed us to characterize the contribution of

  6. Cloning of Acetylcholinesterase Gene in a Microbial Vector.

    DTIC Science & Technology

    1983-02-15

    variants of Chinese hamster ovary cells which have amplified the gene for glutamine synthetase some 1500- fold. In these cells, glutamine synthetase accounts...the advice and assistance of Dr. R.H. Wilson, Department of Genetics, University of Glasgow, who has successfully amplified the gene for glutamine ... synthetase . LO 20 TABLE 1 Levels of AChE and ChE in Neuroblastoma Cell Lines IMR-32 and CHP-126 ICell Linel Growth Conditions Igo innibitoir+ Lysivanel I

  7. Novel “Superspreader” Bacteriophages Promote Horizontal Gene Transfer by Transformation

    PubMed Central

    Bliskovsky, Valery V.; Malagon, Francisco; Baker, James D.; Prince, Jeffrey S.; Klaus, James S.; Adhya, Sankar L.

    2017-01-01

    ABSTRACT Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. PMID:28096488

  8. Anaerobic transformation of DDT related to iron(III) reduction and microbial community structure in paddy soils.

    PubMed

    Chen, Manjia; Cao, Fang; Li, Fangbai; Liu, Chengshuai; Tong, Hui; Wu, Weijian; Hu, Min

    2013-03-06

    We studied the mechanisms of microbial transformation in functional bacteria on 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in two different field soils, Haiyan (HY) and Chenghai (CH). The results showed that microbial activities had a steady dechlorination effect on DDT and its metabolites (DDx). Adding lactate or glucose as carbon sources increased the amount of Desulfuromonas, Sedimentibacter, and Clostridium bacteria, which led to an increase in adsorbed Fe(II) and resulted in increased DDT transformation rates. The electron shuttle of anthraquinone-2,6-disulfonic disodium salt resulted in an increase in the negative potential of soil by mediating the electron transfer from the bacteria to the DDT. Moreover, the DDT-degrading bacteria in the CH soil were more abundant than those in the HY soil, which led to higher DDT transformation rates in the CH soil. The most stable compound of DDx was 1,1-dichloro-2,2-bis(p-chloro-phenyl)ethane, which also was the major dechlorination metabolite of DDT, and 1-chloro-2,2-bis-(p-chlorophenyl)ethane and 4,4'-dichlorobenzo-phenone were found to be the terminal metabolites in the anaerobic soils.

  9. Patterns in wetland microbial community composition and functional gene repertoire associated with methane emissions

    SciTech Connect

    He, Shaomei; Malfatti, Stephanie A.; McFarland, Jack W.; Anderson, Frank E.; Pati, Amrita; Huntemann, Marcel; Tremblay, Julien; Glavina del Rio, Tijana; Waldrop, Mark P.; Windham-Myers, Lisamarie; Tringe, Susannah G.

    2015-05-19

    Wetland restoration on peat islands previously drained for agriculture has potential to reverse land subsidence and sequester atmospheric carbon dioxide as peat accretes. However, the emission of methane could potentially offset the greenhouse gas benefits of captured carbon. As microbial communities play a key role in governing wetland greenhouse gas fluxes, we are interested in how microbial community composition and functions are associated with wetland hydrology, biogeochemistry, and methane emission, which is critical to modeling the microbial component in wetland methane fluxes and to managing restoration projects for maximal carbon sequestration. Here, we couple sequence-based methods with biogeochemical and greenhouse gas measurements to interrogate microbial communities from a pilot-scale restored wetland in the Sacramento-San Joaquin Delta of California, revealing considerable spatial heterogeneity even within this relatively small site. A number of microbial populations and functions showed strong correlations with electron acceptor availability and methane production; some also showed a preference for association with plant roots. Marker gene phylogenies revealed a diversity of major methane-producing and -consuming populations and suggested novel diversity within methanotrophs. Methanogenic archaea were observed in all samples, as were nitrate-, sulfate-, and metal-reducing bacteria, indicating that no single terminal electron acceptor was preferred despite differences in energetic favorability and suggesting spatial microheterogeneity and microniches. Notably, methanogens were negatively correlated with nitrate-, sulfate-, and metal-reducing bacteria and were most abundant at sampling sites with high peat accretion and low electron acceptor availability, where methane production was highest. Wetlands are the largest nonanthropogenic source of atmospheric methane but also a key global carbon reservoir. Characterizing belowground microbial communities

  10. Patterns in wetland microbial community composition and functional gene repertoire associated with methane emissions

    DOE PAGES

    He, Shaomei; Malfatti, Stephanie A.; McFarland, Jack W.; ...

    2015-05-19

    Wetland restoration on peat islands previously drained for agriculture has potential to reverse land subsidence and sequester atmospheric carbon dioxide as peat accretes. However, the emission of methane could potentially offset the greenhouse gas benefits of captured carbon. As microbial communities play a key role in governing wetland greenhouse gas fluxes, we are interested in how microbial community composition and functions are associated with wetland hydrology, biogeochemistry, and methane emission, which is critical to modeling the microbial component in wetland methane fluxes and to managing restoration projects for maximal carbon sequestration. Here, we couple sequence-based methods with biogeochemical and greenhousemore » gas measurements to interrogate microbial communities from a pilot-scale restored wetland in the Sacramento-San Joaquin Delta of California, revealing considerable spatial heterogeneity even within this relatively small site. A number of microbial populations and functions showed strong correlations with electron acceptor availability and methane production; some also showed a preference for association with plant roots. Marker gene phylogenies revealed a diversity of major methane-producing and -consuming populations and suggested novel diversity within methanotrophs. Methanogenic archaea were observed in all samples, as were nitrate-, sulfate-, and metal-reducing bacteria, indicating that no single terminal electron acceptor was preferred despite differences in energetic favorability and suggesting spatial microheterogeneity and microniches. Notably, methanogens were negatively correlated with nitrate-, sulfate-, and metal-reducing bacteria and were most abundant at sampling sites with high peat accretion and low electron acceptor availability, where methane production was highest. Wetlands are the largest nonanthropogenic source of atmospheric methane but also a key global carbon reservoir. Characterizing belowground microbial

  11. Microbial Nitrogen-Cycle Gene Abundance in Soil of Cropland Abandoned for Different Periods

    PubMed Central

    Huhe; Borjigin, Shinchilelt; Buhebaoyin; Wu, Yanpei; Li, Minquan; Cheng, Yunxiang

    2016-01-01

    In Inner Mongolia, steppe grasslands face desertification or degradation because of human overuse and abandonment after inappropriate agricultural management. The soils in these abandoned croplands exist in heterogeneous environments characterized by widely fluctuating microbial growth. Quantitative polymerase chain reaction analysis of microbial genes encoding proteins involved in the nitrogen cycle was used to study Azotobacter species, nitrifiers, and denitrifiers in the soils from steppe grasslands and croplands abandoned for 2, 6, and 26 years. Except for nitrifying archaea and nitrous oxide-reducing bacteria, the relative genotypic abundance of microbial communities involved in nitrogen metabolism differed by approximately 2- to 10-fold between abandoned cropland and steppe grassland soils. Although nitrogen-cycle gene abundances varied with abandonment time, the abundance patterns of nitrogen-cycle genes separated distinctly into abandoned cropland versus light-grazing steppe grassland, despite the lack of any cultivation for over a quarter-century. Plant biomass and plant diversity exerted a significant effect on the abundance of microbial communities that mediate the nitrogen cycle (P < 0.002 and P < 0.03, respectively). The present study elucidates the ecology of bacteria that mediate the nitrogen cycle in recently abandoned croplands. PMID:27140199

  12. Diversity and distribution of a key sulpholipid biosynthetic gene in marine microbial assemblages.

    PubMed

    Villanueva, Laura; Bale, Nicole; Hopmans, Ellen C; Schouten, Stefan; Damsté, Jaap S Sinninghe

    2014-03-01

    Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur-containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5'-diphosphate-sulphoquinovose (UDP-SQ) synthase involved in the SQDG biosynthetic pathway to assess potential microbial sources of SQDGs in the marine environment. The phylogeny of the sqdB-coding protein reveals two distinct clusters: one including green algae, higher plants and cyanobacteria, and another one comprising mainly non-photosynthetic bacteria, as well as other cyanobacteria and algal groups. Evolutionary analysis suggests that the appearance of UDP-SQ synthase occurred twice in cyanobacterial evolution, and one of those branches led to the diversification of the protein in members of the phylum Proteobacteria. A search of homologues of sqdB-proteins in marine metagenomes strongly suggested the presence of heterotrophic bacteria potential SQDG producers. Application of newly developed sqdB gene primers in the marine environment revealed a high diversity of sequences affiliated to cyanobacteria and Proteobacteria in microbial mats, while in North Sea surface water, most of the detected sqdB genes were attributed to the cyanobacterium Synechococcus sp. Lipid analysis revealed that specific SQDGs were characteristic of microbial mat depth, suggesting that SQDG lipids are associated with specific producers.

  13. Microbial Nitrogen-Cycle Gene Abundance in Soil of Cropland Abandoned for Different Periods.

    PubMed

    Huhe; Borjigin, Shinchilelt; Buhebaoyin; Wu, Yanpei; Li, Minquan; Cheng, Yunxiang

    2016-01-01

    In Inner Mongolia, steppe grasslands face desertification or degradation because of human overuse and abandonment after inappropriate agricultural management. The soils in these abandoned croplands exist in heterogeneous environments characterized by widely fluctuating microbial growth. Quantitative polymerase chain reaction analysis of microbial genes encoding proteins involved in the nitrogen cycle was used to study Azotobacter species, nitrifiers, and denitrifiers in the soils from steppe grasslands and croplands abandoned for 2, 6, and 26 years. Except for nitrifying archaea and nitrous oxide-reducing bacteria, the relative genotypic abundance of microbial communities involved in nitrogen metabolism differed by approximately 2- to 10-fold between abandoned cropland and steppe grassland soils. Although nitrogen-cycle gene abundances varied with abandonment time, the abundance patterns of nitrogen-cycle genes separated distinctly into abandoned cropland versus light-grazing steppe grassland, despite the lack of any cultivation for over a quarter-century. Plant biomass and plant diversity exerted a significant effect on the abundance of microbial communities that mediate the nitrogen cycle (P < 0.002 and P < 0.03, respectively). The present study elucidates the ecology of bacteria that mediate the nitrogen cycle in recently abandoned croplands.

  14. Tools and Principles for Microbial Gene Circuit Engineering.

    PubMed

    Bradley, Robert W; Buck, Martin; Wang, Baojun

    2016-02-27

    Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks that require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and we describe efficient strategies for rapidly identifying and correcting causes of failure and fine-tuning circuit characteristics.

  15. Metagenomic data utilization and analysis (MEDUSA) and construction of a global gut microbial gene catalogue.

    PubMed

    Karlsson, Fredrik H; Nookaew, Intawat; Nielsen, Jens

    2014-07-01

    Metagenomic sequencing has contributed important new knowledge about the microbes that live in a symbiotic relationship with humans. With modern sequencing technology it is possible to generate large numbers of sequencing reads from a metagenome but analysis of the data is challenging. Here we present the bioinformatics pipeline MEDUSA that facilitates analysis of metagenomic reads at the gene and taxonomic level. We also constructed a global human gut microbial gene catalogue by combining data from 4 studies spanning 3 continents. Using MEDUSA we mapped 782 gut metagenomes to the global gene catalogue and a catalogue of sequenced microbial species. Hereby we find that all studies share about half a million genes and that on average 300,000 genes are shared by half the studied subjects. The gene richness is higher in the European studies compared to Chinese and American and this is also reflected in the species richness. Even though it is possible to identify common species and a core set of genes, we find that there are large variations in abundance of species and genes.

  16. Lateral Gene Transfer in a Heavy Metal-Contaminated-Groundwater Microbial Community

    PubMed Central

    Hemme, Christopher L.; Green, Stefan J.; Rishishwar, Lavanya; Prakash, Om; Pettenato, Angelica; Chakraborty, Romy; Deutschbauer, Adam M.; Van Nostrand, Joy D.; Wu, Liyou; He, Zhili; Jordan, I. King; Arkin, Adam P.; Kostka, Joel E.

    2016-01-01

    ABSTRACT Unraveling the drivers controlling the response and adaptation of biological communities to environmental change, especially anthropogenic activities, is a central but poorly understood issue in ecology and evolution. Comparative genomics studies suggest that lateral gene transfer (LGT) is a major force driving microbial genome evolution, but its role in the evolution of microbial communities remains elusive. To delineate the importance of LGT in mediating the response of a groundwater microbial community to heavy metal contamination, representative Rhodanobacter reference genomes were sequenced and compared to shotgun metagenome sequences. 16S rRNA gene-based amplicon sequence analysis indicated that Rhodanobacter populations were highly abundant in contaminated wells with low pHs and high levels of nitrate and heavy metals but remained rare in the uncontaminated wells. Sequence comparisons revealed that multiple geochemically important genes, including genes encoding Fe2+/Pb2+ permeases, most denitrification enzymes, and cytochrome c553, were native to Rhodanobacter and not subjected to LGT. In contrast, the Rhodanobacter pangenome contained a recombinational hot spot in which numerous metal resistance genes were subjected to LGT and/or duplication. In particular, Co2+/Zn2+/Cd2+ efflux and mercuric resistance operon genes appeared to be highly mobile within Rhodanobacter populations. Evidence of multiple duplications of a mercuric resistance operon common to most Rhodanobacter strains was also observed. Collectively, our analyses indicated the importance of LGT during the evolution of groundwater microbial communities in response to heavy metal contamination, and a conceptual model was developed to display such adaptive evolutionary processes for explaining the extreme dominance of Rhodanobacter populations in the contaminated groundwater microbiome. PMID:27048805

  17. Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

    PubMed Central

    Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

    1999-01-01

    We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

  18. Transforming fusions of FGFR and TACC genes in human glioblastoma.

    PubMed

    Singh, Devendra; Chan, Joseph Minhow; Zoppoli, Pietro; Niola, Francesco; Sullivan, Ryan; Castano, Angelica; Liu, Eric Minwei; Reichel, Jonathan; Porrati, Paola; Pellegatta, Serena; Qiu, Kunlong; Gao, Zhibo; Ceccarelli, Michele; Riccardi, Riccardo; Brat, Daniel J; Guha, Abhijit; Aldape, Ken; Golfinos, John G; Zagzag, David; Mikkelsen, Tom; Finocchiaro, Gaetano; Lasorella, Anna; Rabadan, Raul; Iavarone, Antonio

    2012-09-07

    The brain tumor glioblastoma multiforme (GBM) is among the most lethal forms of human cancer. Here, we report that a small subset of GBMs (3.1%; 3 of 97 tumors examined) harbors oncogenic chromosomal translocations that fuse in-frame the tyrosine kinase coding domains of fibroblast growth factor receptor (FGFR) genes (FGFR1 or FGFR3) to the transforming acidic coiled-coil (TACC) coding domains of TACC1 or TACC3, respectively. The FGFR-TACC fusion protein displays oncogenic activity when introduced into astrocytes or stereotactically transduced in the mouse brain. The fusion protein, which localizes to mitotic spindle poles, has constitutive kinase activity and induces mitotic and chromosomal segregation defects and triggers aneuploidy. Inhibition of FGFR kinase corrects the aneuploidy, and oral administration of an FGFR inhibitor prolongs survival of mice harboring intracranial FGFR3-TACC3-initiated glioma. FGFR-TACC fusions could potentially identify a subset of GBM patients who would benefit from targeted FGFR kinase inhibition.

  19. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences

    PubMed Central

    Langille, Morgan G. I.; Zaneveld, Jesse; Caporaso, J. Gregory; McDonald, Daniel; Knights, Dan; Reyes, Joshua A.; Clemente, Jose C.; Burkepile, Deron E.; Vega Thurber, Rebecca L.; Knight, Rob; Beiko, Robert G.; Huttenhower, Curtis

    2013-01-01

    Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community’s functional capabilities. Here we describe PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this ‘predictive metagenomic’ approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available. PMID:23975157

  20. Patterns in Wetland Microbial Community Composition and Functional Gene Repertoire Associated with Methane Emissions

    PubMed Central

    He, Shaomei; Malfatti, Stephanie A.; McFarland, Jack W.; Anderson, Frank E.; Pati, Amrita; Huntemann, Marcel; Tremblay, Julien; Glavina del Rio, Tijana; Waldrop, Mark P.; Windham-Myers, Lisamarie

    2015-01-01

    ABSTRACT Wetland restoration on peat islands previously drained for agriculture has potential to reverse land subsidence and sequester atmospheric carbon dioxide as peat accretes. However, the emission of methane could potentially offset the greenhouse gas benefits of captured carbon. As microbial communities play a key role in governing wetland greenhouse gas fluxes, we are interested in how microbial community composition and functions are associated with wetland hydrology, biogeochemistry, and methane emission, which is critical to modeling the microbial component in wetland methane fluxes and to managing restoration projects for maximal carbon sequestration. Here, we couple sequence-based methods with biogeochemical and greenhouse gas measurements to interrogate microbial communities from a pilot-scale restored wetland in the Sacramento-San Joaquin Delta of California, revealing considerable spatial heterogeneity even within this relatively small site. A number of microbial populations and functions showed strong correlations with electron acceptor availability and methane production; some also showed a preference for association with plant roots. Marker gene phylogenies revealed a diversity of major methane-producing and -consuming populations and suggested novel diversity within methanotrophs. Methanogenic archaea were observed in all samples, as were nitrate-, sulfate-, and metal-reducing bacteria, indicating that no single terminal electron acceptor was preferred despite differences in energetic favorability and suggesting spatial microheterogeneity and microniches. Notably, methanogens were negatively correlated with nitrate-, sulfate-, and metal-reducing bacteria and were most abundant at sampling sites with high peat accretion and low electron acceptor availability, where methane production was highest. PMID:25991679

  1. Microbial taxa and functional genes shift in degraded soil with bacterial wilt

    PubMed Central

    Zhang, Hongchun; Wang, Rui; Chen, Shu; Qi, Gaofu; He, Zhili; Zhao, Xiuyun

    2017-01-01

    Soil degradation is a serious global problem, but little is known about how soil microbial communities respond to soil degradation as well as their feedback to ecosystem functioning. In this study, we found the microbial community composition, structure and functional potential significantly altered in the degraded soils with bacterial wilt (termed as degraded soils). Compared with healthy soils, OTU richness of beneficial microorganisms were significantly decreased, but OTU richness of pathogenic microorganisms were significantly increased in the degraded soils. Functional gene array (GeoChip 5.0) analysis showed the functional metabolic potential of genes involved in stress, virulence, sulfur cycle, metal resistance, degradation of plant cell wall was significantly increased in the degraded soils. Increased functional metabolic potential of these genes may be related to the acidification and severe plant disease of degraded soils. Biological activity of degraded soils was obviously decreased with weakened soil enzyme activities when compared to the healthy soils. Soil pH and enzyme activities were negatively correlated with the abundance of genes involved in sulfur cycle, virulence, and stress responses. This study provides new insights into our understanding of soil microbial community responses to soil degradation. PMID:28051173

  2. Microbially-mediated transformation and mobilization of soil Fe-organic associations

    NASA Astrophysics Data System (ADS)

    Poggenburg, Christine; Mikutta, Robert; Schippers, Axel; Dohrmann, Reiner; Kaufhold, Stephan; Guggenberger, Georg

    2014-05-01

    Soil organic matter (OM) has been proposed to be stabilized in the long term via sorption to iron((oxy)hydr)oxides under aerobic conditions. However, in an anaerobic environment, Fe-organic associations may be subject to microbial reduction and mobilization, which counteract the suggested stabilizing effect of Fe compounds. Desorption of OM can result in its microbial decomposition causing the emission of greenhouse gases (CO2, CH4, N2O) or release of associated contaminants into the soil solution and groundwater. While the reductive dissolution of pure iron((oxy)hydr)oxides by dissimilatory FeIII reducing bacteria is well established, little is known about the influence of natural OM on microbially mediated mobilization of Fe-organic associations. Therefore, this study aims to elucidate the effect of adsorbed OM on microbial FeIII reduction of Fe-organic associations with regard to (i) the composition of OM, (ii) the carbon loading, and (iii) surface coverage and/or pore blockage by adsorbed OM. Mineral-organic associations with varying carbon contents were synthesized using several iron((oxy)hydr)oxides (Goethite, Lepidocrocite, Ferrihydrite, Hematite, Magnetite) and OM of different origin (dissolved OM extracted from the Oa horizon of a Podzol and Oi horizon of a Cambisol, extracellular polymeric substance extracted from Bacillus subtilis). Incubation experiments under anaerobic conditions were conducted for 16 days using two different strains of dissimilatory FeIII reducing bacteria (Shewanella putrefaciens, Geobacter metallireducens). At five sampling points in time the solution phase was analyzed for pH, Fetotal, and FeII. The initial mineral-organic associations and post-incubation phase were characterized by N2 gas adsorption, FTIR, XRD, and XPS. The results indicate that the composition of OM and carbon loading significantly influence the rate and extend of microbial reduction of Fe-organic associations depending on the type of microbial strain and iron

  3. Potential for microbial H2 and metal transformations associated with novel bacteria and archaea in deep terrestrial subsurface sediments.

    PubMed

    Hernsdorf, Alex W; Amano, Yuki; Miyakawa, Kazuya; Ise, Kotaro; Suzuki, Yohey; Anantharaman, Karthik; Probst, Alexander; Burstein, David; Thomas, Brian C; Banfield, Jillian F

    2017-03-28

    Geological sequestration in deep underground repositories is the prevailing proposed route for radioactive waste disposal. After the disposal of radioactive waste in the subsurface, H2 may be produced by corrosion of steel and, ultimately, radionuclides will be exposed to the surrounding environment. To evaluate the potential for microbial activities to impact disposal systems, we explored the microbial community structure and metabolic functions of a sediment-hosted ecosystem at the Horonobe Underground Research Laboratory, Hokkaido, Japan. Overall, we found that the ecosystem hosted organisms from diverse lineages, including many from the phyla that lack isolated representatives. The majority of organisms can metabolize H2, often via oxidative [NiFe] hydrogenases or electron-bifurcating [FeFe] hydrogenases that enable ferredoxin-based pathways, including the ion motive Rnf complex. Many organisms implicated in H2 metabolism are also predicted to catalyze carbon, nitrogen, iron and sulfur transformations. Notably, iron-based metabolism is predicted in a novel lineage of Actinobacteria and in a putative methane-oxidizing ANME-2d archaeon. We infer an ecological model that links microorganisms to sediment-derived resources and predict potential impacts of microbial activity on H2 consumption and retardation of radionuclide migration.The ISME Journal advance online publication, 28 March 2017; doi:10.1038/ismej.2017.39.

  4. Importance of Mobile Genetic Elements and Conjugal Gene Transfer for Subsurface Microbial Community Adaptation to Biotransformation of Metals

    SciTech Connect

    Sorensen, Soren J.

    2005-06-01

    The overall goal of this project is to investigate the effect of mobile genetic elements and conjugal gene transfer on subsurface microbial community adaptation to mercury and chromium stress and biotransformation. Our studies focus on the interaction between the fate of these metals in the subsurface and the microbial community structure and activity.

  5. Rapid analysis of two food-borne microbial communities at the species level by Fourier-transform infrared microspectroscopy.

    PubMed

    Wenning, Mareike; Theilmann, Vera; Scherer, Siegfried

    2006-05-01

    The species composition of microbial communities in natural habitats may be extremely complex and therefore a quantitative analysis of the fraction each species contributes to the consortium has proven to be difficult. During recent years, the identification of bacterial pure cultures based on their infrared spectra has been established. Fourier-transform infrared microspectroscopy now proceeds a step further and allows identification of microorganisms directly plated from community dilutions. Infrared spectra of microcolonies of 70-250 microm in diameter can be recorded without producing a pure culture of the isolate. We have applied this novel technique for quantitative comparative analysis of two undefined, geographically separated food-borne smear cheese microbial consortia of limited complexity. Due to the high degree of automation, up to 200 microcolonies could be identified in 1 day and, in total, 3170 infrared spectra of microcolonies were recorded. The results obtained have been verified by Fourier-transform infrared macrospectroscopy and 16S rDNA sequencing. Interestingly, although the communities were unrelated, Staphylococcus equorum, Corynebacterium casei, Arthrobacter casei and Brevibacterium linens were found to be part of both consortia, however, with different incidence. In addition, Corynebacterium variabile, Microbacterium gubbeenense, Brachybacterium alimentarium, Enterococcus faecalis and an unknown species were detected in either one of the consortia.

  6. Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.

    PubMed

    Militello, Kevin T; Chang, Ming-Mei; Simon, Robert D; Lazatin, Justine C

    2016-01-01

    The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl β-D-thiogalactopyranoside (IPTG). Over the course of the next three weeks, students use a battery of approaches including plasmid DNA isolation experiments, restriction maps, and PCR to differentiate between mutation and transformation. The students ultimately come to the conclusion that the changes in phenotypes are due to genetic transformation and not mutation based on the evidence generated over the four-week period. Pre-laboratory tests and post-laboratory tests indicate that this set of exercises is successful in helping students differentiate between transformation and mutation. The laboratory is designed for underclassmen and is a good prerequisite for an apprentice-based research opportunity, although it is not designed as a class based research experience. Potential modifications and future directions of the laboratory based upon student experiences and assessment are presented.

  7. New Dimensions in Microbial Ecology—Functional Genes in Studies to Unravel the Biodiversity and Role of Functional Microbial Groups in the Environment

    PubMed Central

    Imhoff, Johannes F.

    2016-01-01

    During the past decades, tremendous advances have been made in the possibilities to study the diversity of microbial communities in the environment. The development of methods to study these communities on the basis of 16S rRNA gene sequences analysis was a first step into the molecular analysis of environmental communities and the study of biodiversity in natural habitats. A new dimension in this field was reached with the introduction of functional genes of ecological importance and the establishment of genetic tools to study the diversity of functional microbial groups and their responses to environmental factors. Functional gene approaches are excellent tools to study the diversity of a particular function and to demonstrate changes in the composition of prokaryote communities contributing to this function. The phylogeny of many functional genes largely correlates with that of the 16S rRNA gene, and microbial species may be identified on the basis of functional gene sequences. Functional genes are perfectly suited to link culture-based microbiological work with environmental molecular genetic studies. In this review, the development of functional gene studies in environmental microbiology is highlighted with examples of genes relevant for important ecophysiological functions. Examples are presented for bacterial photosynthesis and two types of anoxygenic phototrophic bacteria, with genes of the Fenna-Matthews-Olson-protein (fmoA) as target for the green sulfur bacteria and of two reaction center proteins (pufLM) for the phototrophic purple bacteria, with genes of adenosine-5′phosphosulfate (APS) reductase (aprA), sulfate thioesterase (soxB) and dissimilatory sulfite reductase (dsrAB) for sulfur oxidizing and sulfate reducing bacteria, with genes of ammonia monooxygenase (amoA) for nitrifying/ammonia-oxidizing bacteria, with genes of particulate nitrate reductase and nitrite reductases (narH/G, nirS, nirK) for denitrifying bacteria and with genes of methane

  8. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    PubMed

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  9. Microbial signatures of oral dysbiosis, periodontitis and edentulism revealed by Gene Meter methodology.

    PubMed

    Hunter, M Colby; Pozhitkov, Alex E; Noble, Peter A

    2016-12-01

    Conceptual models suggest that certain microorganisms (e.g., the "red" complex) are indicative of a specific disease state (e.g., periodontitis); however, recent studies have questioned the validity of these models. Here, the abundances of 500+ microbial species were determined in 16 patients with clinical signs of one of the following oral conditions: periodontitis, established caries, edentulism, and oral health. Our goal was to determine if the abundances of certain microorganisms reflect dysbiosis or a specific clinical condition that could be used as a 'signature' for dental research. Microbial abundances were determined by the analysis of 138,718 calibrated probes using Gene Meter methodology. Each 16S rRNA gene was targeted by an average of 194 unique probes (n=25nt). The calibration involved diluting pooled gene target samples, hybridizing each dilution to a DNA microarray, and fitting the probe intensities to adsorption models. The fit of the model to the experimental data was used to assess individual and aggregate probe behavior; good fits (R(2)>0.90) were retained for back-calculating microbial abundances from patient samples. The abundance of a gene was determined from the median of all calibrated individual probes or from the calibrated abundance of all aggregated probes. With the exception of genes with low abundances (<2 arbitrary units), the abundances determined by the different calibrations were highly correlated (r~1.0). Seventeen genera were classified as 'signatures of dysbiosis' because they had significantly higher abundances in patients with periodontitis and edentulism when contrasted with health. Similarly, 13 genera were classified as 'signatures of periodontitis', and 14 genera were classified as 'signatures of edentulism'. The signatures could be used, individually or in combination, to assess the clinical status of a patient (e.g., evaluating treatments such as antibiotic therapies). Comparisons of the same patient samples revealed

  10. Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient

    PubMed Central

    Fortunato, Caroline S.; Crump, Byron C.

    2015-01-01

    Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance

  11. Pattern and synchrony of gene expression among sympatric marine microbial populations

    PubMed Central

    Ottesen, Elizabeth A.; Young, Curtis R.; Eppley, John M.; Ryan, John P.; Chavez, Francisco P.; Scholin, Christopher A.; DeLong, Edward F.

    2013-01-01

    Planktonic marine microbes live in dynamic habitats that demand rapid sensing and response to periodic as well as stochastic environmental change. The kinetics, regularity, and specificity of microbial responses in situ, however, are not well-described. We report here simultaneous multitaxon genome-wide transcriptome profiling in a naturally occurring picoplankton community. An in situ robotic sampler using a Lagrangian sampling strategy enabled continuous tracking and repeated sampling of coherent microbial populations over 2 d. Subsequent RNA sequencing analyses yielded genome-wide transcriptome profiles of eukaryotic (Ostreococcus) and bacterial (Synechococcus) photosynthetic picoplankton as well as proteorhodopsin-containing heterotrophs, including Pelagibacter, SAR86-cluster Gammaproteobacteria, and marine Euryarchaea. The photosynthetic picoplankton exhibited strong diel rhythms over thousands of gene transcripts that were remarkably consistent with diel cycling observed in laboratory pure cultures. In contrast, the heterotrophs did not cycle diurnally. Instead, heterotrophic picoplankton populations exhibited cross-species synchronous, tightly regulated, temporally variable patterns of gene expression for many genes, particularly those genes associated with growth and nutrient acquisition. This multitaxon, population-wide gene regulation seemed to reflect sporadic, short-term, reversible responses to high-frequency environmental variability. Although the timing of the environmental responses among different heterotrophic species seemed synchronous, the specific metabolic genes that were expressed varied from taxon to taxon. In aggregate, these results provide insights into the kinetics, diversity, and functional patterns of microbial community response to environmental change. Our results also suggest a means by which complex multispecies metabolic processes could be coordinated, facilitating the regulation of matter and energy processing in a dynamically

  12. Microbial Gene Abundance and Expression Patterns across a River to Ocean Salinity Gradient.

    PubMed

    Fortunato, Caroline S; Crump, Byron C

    2015-01-01

    Microbial communities mediate the biogeochemical cycles that drive ecosystems, and it is important to understand how these communities are affected by changing environmental conditions, especially in complex coastal zones. As fresh and marine waters mix in estuaries and river plumes, the salinity, temperature, and nutrient gradients that are generated strongly influence bacterioplankton community structure, yet, a parallel change in functional diversity has not been described. Metagenomic and metatranscriptomic analyses were conducted on five water samples spanning the salinity gradient of the Columbia River coastal margin, including river, estuary, plume, and ocean, in August 2010. Samples were pre-filtered through 3 μm filters and collected on 0.2 μm filters, thus results were focused on changes among free-living microbial communities. Results from metagenomic 16S rRNA sequences showed taxonomically distinct bacterial communities in river, estuary, and coastal ocean. Despite the strong salinity gradient observed over sampling locations (0 to 33), the functional gene profiles in the metagenomes were very similar from river to ocean with an average similarity of 82%. The metatranscriptomes, however, had an average similarity of 31%. Although differences were few among the metagenomes, we observed a change from river to ocean in the abundance of genes encoding for catabolic pathways, osmoregulators, and metal transporters. Additionally, genes specifying both bacterial oxygenic and anoxygenic photosynthesis were abundant and expressed in the estuary and plume. Denitrification genes were found throughout the Columbia River coastal margin, and most highly expressed in the estuary. Across a river to ocean gradient, the free-living microbial community followed three different patterns of diversity: 1) the taxonomy of the community changed strongly with salinity, 2) metabolic potential was highly similar across samples, with few differences in functional gene abundance

  13. Pattern and synchrony of gene expression among sympatric marine microbial populations.

    PubMed

    Ottesen, Elizabeth A; Young, Curtis R; Eppley, John M; Ryan, John P; Chavez, Francisco P; Scholin, Christopher A; DeLong, Edward F

    2013-02-05

    Planktonic marine microbes live in dynamic habitats that demand rapid sensing and response to periodic as well as stochastic environmental change. The kinetics, regularity, and specificity of microbial responses in situ, however, are not well-described. We report here simultaneous multitaxon genome-wide transcriptome profiling in a naturally occurring picoplankton community. An in situ robotic sampler using a Lagrangian sampling strategy enabled continuous tracking and repeated sampling of coherent microbial populations over 2 d. Subsequent RNA sequencing analyses yielded genome-wide transcriptome profiles of eukaryotic (Ostreococcus) and bacterial (Synechococcus) photosynthetic picoplankton as well as proteorhodopsin-containing heterotrophs, including Pelagibacter, SAR86-cluster Gammaproteobacteria, and marine Euryarchaea. The photosynthetic picoplankton exhibited strong diel rhythms over thousands of gene transcripts that were remarkably consistent with diel cycling observed in laboratory pure cultures. In contrast, the heterotrophs did not cycle diurnally. Instead, heterotrophic picoplankton populations exhibited cross-species synchronous, tightly regulated, temporally variable patterns of gene expression for many genes, particularly those genes associated with growth and nutrient acquisition. This multitaxon, population-wide gene regulation seemed to reflect sporadic, short-term, reversible responses to high-frequency environmental variability. Although the timing of the environmental responses among different heterotrophic species seemed synchronous, the specific metabolic genes that were expressed varied from taxon to taxon. In aggregate, these results provide insights into the kinetics, diversity, and functional patterns of microbial community response to environmental change. Our results also suggest a means by which complex multispecies metabolic processes could be coordinated, facilitating the regulation of matter and energy processing in a dynamically

  14. GC and GC-MS characterization of crude oil transformation in sediments and microbial mat samples after the 1991 oil spill in the Saudi Arabian Gulf coast.

    PubMed

    Garcia de Oteyza, T; Grimalt, J O

    2006-02-01

    The massive oil discharge in the Saudi Arabian coast at the end of the 1991 Gulf War is used here as a natural experiment to study the ability of microbial mats to transform oil residues after major spills. The degree of oil transformation has been evaluated from the analysis of the aliphatic and aromatic hydrocarbons by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The oil-polluted microbial mat samples from coastal environments exhibited an intermediate degree of transformation between that observed in superficial and deep sediments. Evaporation, photo-oxidation and water-washing seemed to lead to more effective and rapid elimination of hydrocarbons than cyanobacteria and its associated microorganisms. Furthermore, comparison of some compounds (e.g. regular isoprenoid hydrocarbons or alkylnaphthalenes) in the oil collected in the area after the spill or in the mixtures retained by cyanobacterial growth gave rise to an apparent effect of hydrocarbon preservation in the microbial mat ecosystems.

  15. Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays

    PubMed Central

    Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

    2017-01-01

    Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T1 and T2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T2 progenies and PCR-identified Apr gene segregation in T2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods. PMID:28377783

  16. Efficient anaerobic transformation of raw wheat straw by a robust cow rumen-derived microbial consortium.

    PubMed

    Lazuka, Adèle; Auer, Lucas; Bozonnet, Sophie; Morgavi, Diego P; O'Donohue, Michael; Hernandez-Raquet, Guillermina

    2015-11-01

    A rumen-derived microbial consortium was enriched on raw wheat straw as sole carbon source in a sequential batch-reactor (SBR) process under strict mesophilic anaerobic conditions. After five cycles of enrichment the procedure enabled to select a stable and efficient lignocellulolytic microbial consortium, mainly constituted by members of Firmicutes and Bacteroidetes phyla. The enriched community, designed rumen-wheat straw-derived consortium (RWS) efficiently hydrolyzed lignocellulosic biomass, degrading 55.5% w/w of raw wheat straw over 15days at 35°C and accumulating carboxylates as main products. Cellulolytic and hemicellulolytic activities, mainly detected on the cell bound fraction, were produced in the earlier steps of degradation, their production being correlated with the maximal lignocellulose degradation rates. Overall, these results demonstrate the potential of RWS to convert unpretreated lignocellulosic substrates into useful chemicals.

  17. Microbially mediated transformations of phosphorus in the sea: new views of an old cycle.

    PubMed

    Karl, David M

    2014-01-01

    Phosphorus (P) is a required element for life. Its various chemical forms are found throughout the lithosphere and hydrosphere, where they are acted on by numerous abiotic and biotic processes collectively referred to as the P cycle. In the sea, microorganisms are primarily responsible for P assimilation and remineralization, including recently discovered P reduction-oxidation bioenergetic processes that add new complexity to the marine microbial P cycle. Human-induced enhancement of the global P cycle via mining of phosphate-bearing rock will likely influence the pace of P-cycle dynamics, especially in coastal marine habitats. The inextricable link between the P cycle and cycles of other bioelements predicts future impacts on, for example, nitrogen fixation and carbon dioxide sequestration. Additional laboratory and field research is required to build a comprehensive understanding of the marine microbial P cycle.

  18. Contrasting microbial functional genes in two distinct saline-alkali and slightly acidic oil-contaminated sites.

    PubMed

    Liang, Yuting; Zhao, Huihui; Zhang, Xu; Zhou, Jizhong; Li, Guanghe

    2014-07-15

    To compare the functional gene structure and diversity of microbial communities in saline-alkali and slightly acidic oil-contaminated sites, 40 soil samples were collected from two typical oil exploration sites in North and South China and analyzed with a comprehensive functional gene array (GeoChip 3.0). The overall microbial pattern was significantly different between the two sites, and a more divergent pattern was observed in slightly acidic soils. Response ratio was calculated to compare the microbial functional genes involved in organic contaminant degradation and carbon, nitrogen, phosphorus, and sulfur cycling. The results indicated a significantly low abundance of most genes involved in organic contaminant degradation and in the cycling of nitrogen and phosphorus in saline-alkali soils. By contrast, most carbon degradation genes and all carbon fixation genes had similar abundance at both sites. Based on the relationship between the environmental variables and microbial functional structure, pH was the major factor influencing the microbial distribution pattern in the two sites. This study demonstrated that microbial functional diversity and heterogeneity in oil-contaminated environments can vary significantly in relation to local environmental conditions. The limitation of nitrogen and phosphorus and the low degradation capacity of organic contaminant should be carefully considered, particularly in most oil-exploration sites with saline-alkali soils.

  19. Microbial transformation of the Deepwater Horizon oil spill-past, present, and future perspectives.

    PubMed

    Kimes, Nikole E; Callaghan, Amy V; Suflita, Joseph M; Morris, Pamela J

    2014-01-01

    The Deepwater Horizon blowout, which occurred on April 20, 2010, resulted in an unprecedented oil spill. Despite a complex effort to cap the well, oil and gas spewed from the site until July 15, 2010. Although a large proportion of the hydrocarbons was depleted via natural processes and human intervention, a substantial portion of the oil remained unaccounted for and impacted multiple ecosystems throughout the Gulf of Mexico. The depth, duration and magnitude of this spill were unique, raising many questions and concerns regarding the fate of the hydrocarbons released. One major question was whether or not microbial communities would be capable of metabolizing the hydrocarbons, and if so, by what mechanisms and to what extent? In this review, we summarize the microbial response to the oil spill as described by studies performed during the past four years, providing an overview of the different responses associated with the water column, surface waters, deep-sea sediments, and coastal sands/sediments. Collectively, these studies provide evidence that the microbial response to the Deepwater Horizon oil spill was rapid and robust, displaying common attenuation mechanisms optimized for low molecular weight aliphatic and aromatic hydrocarbons. In contrast, the lack of evidence for the attenuation of more recalcitrant hydrocarbon components suggests that future work should focus on both the environmental impact and metabolic fate of recalcitrant compounds, such as oxygenated oil components.

  20. Microbial transformation of the Deepwater Horizon oil spill—past, present, and future perspectives

    PubMed Central

    Kimes, Nikole E.; Callaghan, Amy V.; Suflita, Joseph M.; Morris, Pamela J.

    2014-01-01

    The Deepwater Horizon blowout, which occurred on April 20, 2010, resulted in an unprecedented oil spill. Despite a complex effort to cap the well, oil and gas spewed from the site until July 15, 2010. Although a large proportion of the hydrocarbons was depleted via natural processes and human intervention, a substantial portion of the oil remained unaccounted for and impacted multiple ecosystems throughout the Gulf of Mexico. The depth, duration and magnitude of this spill were unique, raising many questions and concerns regarding the fate of the hydrocarbons released. One major question was whether or not microbial communities would be capable of metabolizing the hydrocarbons, and if so, by what mechanisms and to what extent? In this review, we summarize the microbial response to the oil spill as described by studies performed during the past four years, providing an overview of the different responses associated with the water column, surface waters, deep-sea sediments, and coastal sands/sediments. Collectively, these studies provide evidence that the microbial response to the Deepwater Horizon oil spill was rapid and robust, displaying common attenuation mechanisms optimized for low molecular weight aliphatic and aromatic hydrocarbons. In contrast, the lack of evidence for the attenuation of more recalcitrant hydrocarbon components suggests that future work should focus on both the environmental impact and metabolic fate of recalcitrant compounds, such as oxygenated oil components. PMID:25477866

  1. Gene context analysis in the Integrated Microbial Genomes (IMG) data management system.

    PubMed

    Mavromatis, Konstantinos; Chu, Ken; Ivanova, Natalia; Hooper, Sean D; Markowitz, Victor M; Kyrpides, Nikos C

    2009-11-24

    Computational methods for determining the function of genes in newly sequenced genomes have been traditionally based on sequence similarity to genes whose function has been identified experimentally. Function prediction methods can be extended using gene context analysis approaches such as examining the conservation of chromosomal gene clusters, gene fusion events and co-occurrence profiles across genomes. Context analysis is based on the observation that functionally related genes are often having similar gene context and relies on the identification of such events across phylogenetically diverse collection of genomes. We have used the data management system of the Integrated Microbial Genomes (IMG) as the framework to implement and explore the power of gene context analysis methods because it provides one of the largest available genome integrations. Visualization and search tools to facilitate gene context analysis have been developed and applied across all publicly available archaeal and bacterial genomes in IMG. These computations are now maintained as part of IMG's regular genome content update cycle. IMG is available at: http://img.jgi.doe.gov.

  2. Gene context analysis in the Integrated Microbial Genomes (IMG) data management system

    SciTech Connect

    Mavromatis, Konstantinos; Chu, Ken; Ivanova, Natalia; Hooper, Sean D.; Markowitz, Victor M.; Kyrpides, Nikos C.

    2009-05-01

    Computational methods for determining the function of genes in newly sequenced genomes have been traditionally based on sequence similarity to genes whose function has been identified experimentally. Function prediction methods can be extended using gene context analysis approaches such as examining the conservation of chromosomal gene clusters, gene fusion events and co-occurrence profiles across genomes. Context analysis is based on the observation that functionally related genes are often having similar gene context and relies on the identification of such events across a statistically significant and phylogeneticaly diverse collection of genomes. We have used the data management system of the Integrated Microbial Genomes (IMG) as the framework to implement and explore the power of gene context analysis methods because it provides one of the largest available genome integrations. Visualization and search tools to facilitate and explore gene context analysis have been developed and applied across all publicly available archaeal and bacterial genomes in IMG. These computations are now maintained as part of IMG's regular genome content update cycle. IMG is available at: http://img.jgi.doe.gov.

  3. ENANTIOSELECTIVE MICROBIAL TRANSFORMATION OF THE PHENYLPYRAZOLE INSECTICIDE FIPRONIL IN ANOXIC SEDIMENTS

    EPA Science Inventory

    Fipronil, a chiral insecticide, was biotransformed initially to fipronil sulfide in anoxic sediment slurries following a short lag period. Sediment slurries characterized as either sulfidogenic or methanogenic transformed fipronil with half-lives of approximately 35 and 40 days, ...

  4. Microbial mediated retention/transformation of organic and inorganic materials in freshwater and marine ecosystems

    EPA Science Inventory

    Aquatic ecosystems are globally connected by hydrological and biogeochemical cycles. Microorganisms inhabiting aquatic ecosystems form the basis of food webs, mediate essential element cycles, decompose natural organic matter, transform inorganic nutrients and metals, and degrad...

  5. High frequency vector-mediated transformation and gene replacement in Tetrahymena.

    PubMed Central

    Gaertig, J; Gu, L; Hai, B; Gorovsky, M A

    1994-01-01

    Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene

  6. Microbial diversity and PAH catabolic genes tracking spatial heterogeneity of PAH concentrations.

    PubMed

    Bengtsson, Göran; Törneman, Niklas; De Lipthay, Julia R; Sørensen, Søren J

    2013-01-01

    We analyzed the within-site spatial heterogeneity of microbial community diversity, polyaromatic hydrocarbon (PAH) catabolic genotypes, and physiochemical soil properties at a creosote contaminated site. Genetic diversity and community structure were evaluated from an analysis of denaturant gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified sequences of 16S rRNA gene. The potential PAH degradation capability was determined from PCR amplification of a suit of aromatic dioxygenase genes. Microbial diversity, evenness, and PAH genotypes were patchily distributed, and hot and cold spots of their distribution coincided with hot and cold spots of the PAH distribution. The analyses revealed a positive covariation between microbial diversity, biomass, evenness, and PAH concentration, implying that the creosote contamination at this site promotes diversity and abundance. Three patchily distributed PAH-degrading genotypes, NAH, phnA, and pdo1, were identified, and their abundances were positively correlated with the PAH concentration and the fraction of soil organic carbon. The covariation of the PAH concentration with the number and spatial distribution of catabolic genotypes suggests that a field site capacity to degrade PAHs may vary with the extent of contamination.

  7. Organic amendments enhance microbial diversity and abundance of functional genes in Australian Soils

    NASA Astrophysics Data System (ADS)

    Aldorri, Sind; McMillan, Mary; Pereg, Lily

    2016-04-01

    Food and cash crops play important roles in Australia's economy with black, grey and red clay soil, widely use for growing cotton, wheat, corn and other crops in rotation. While the majority of cotton growers use nitrogen and phosphate fertilizers only in the form of agrochemicals, a few experiment with the addition of manure or composted plant material before planting. We hypothesized that the use of such organic amendments would enhance the soil microbial function through increased microbial diversity and abundance, thus contribute to improved soil sustainability. To test the hypothesis we collected soil samples from two cotton-growing farms in close geographical proximity and with mostly similar production practices other than one grower has been using composted plants as organic amendment and the second farmer uses only agrochemicals. We applied the Biolog Ecoplate system to study the metabolic signature of microbial communities and used qPCR to estimate the abundance of functional genes in the soil. The soil treated with organic amendments clearly showed higher metabolic activity of a more diverse range of carbon sources as well as higher abundance of genes involved in the nitrogen and phosphorous cycles. Since microbes undertake a large number of soil functions, the use of organic amendments can contribute to the sustainability of agricultural soils.

  8. The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

    PubMed Central

    2010-01-01

    Background In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their

  9. Transformation studies of Bacillus thuringiensis cryIC gene into a nitrogen-fixing Azospirillum lipoferum.

    PubMed

    Gounder, R; Rajendran, N

    2001-01-01

    A lepidopteran toxin gene, cryIC (pSB607) from entomopathogenic Bacillus thuringiensis subsp. aizawai was introduced into nitrogen-fixing Azospirillum lipoferum by transformation. Regeneration of spheroplasts was achieved at 99% with 39% frequency of regeneration. Transformants were screened on NB kanamycin with ampicillin plates and 4 transformants were selected after ten generations. SDS-PAGE and Western blot analysis confirmed the presence of a 68 kDa protein in the transformants. Studies on utilization of carbon sources indicate that glucose and sucrose are the most favorable carbon sources and 2% molasses is the cheap alternate carbon source for the better growth of parent A. lipoferum and transformants.

  10. MICROBIAL TRANSFORMATIONS OF PLUTONIUM AND OTHER ACTINIDES IN TRANSURANIC AND MIXED WASTES.

    SciTech Connect

    FRANCIS,A.J.

    2003-07-06

    The presence of the actinides Th, U, Np, Pu, and Am in transuranic (TRU) and mixed wastes is a major concern because of their potential for migration from the waste repositories and long-term contamination of the environment. The toxicity of the actinide elements and the long half-lives of their isotopes are the primary causes for concern. In addition to the radionuclides the TRU waste consists a variety of organic materials (cellulose, plastic, rubber, chelating agents) and inorganic compounds (nitrate and sulfate). Significant microbial activity is expected in the waste because of the presence of organic compounds and nitrate, which serve as carbon and nitrogen sources and in the absence of oxygen the microbes can use nitrate and sulfate as alternate electron acceptors. Biodegradation of the TRU waste can result in gas generation and pressurization of containment areas, and waste volume reduction and subsidence in the repository. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of actinides have been investigated, we have only limited information on the effects of microbial processes. Microbial activity could affect the chemical nature of the actinides by altering the speciation, solubility and sorption properties and thus could increase or decrease the concentrations of actinides in solution. Under appropriate conditions, dissolution or immobilization of actinides is brought about by direct enzymatic or indirect non-enzymatic actions of microorganisms. Dissolution of actinides by microorganisms is brought about by changes in the Eh and pH of the medium, by their production of organic acids, such as citric acid, siderophores and extracellular metabolites. Immobilization or precipitation of actinides is due to changes in the Eh of the environment, enzymatic reductive precipitation (reduction from higher to lower oxidation state), biosorption, bioaccumulation, biotransformation of actinides complexed

  11. Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria, bacteria-polymer mixtures and biofilms

    NASA Technical Reports Server (NTRS)

    Nichols, P. D.; Henson, J. M.; Guckert, J. B.; Nivens, D. E.; White, D. C.

    1985-01-01

    Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.

  12. Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria, bacteria-polymer mixtures and biofilms.

    PubMed

    Nichols, P D; Henson, J M; Guckert, J B; Nivens, D E; White, D C

    1985-01-01

    Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.

  13. From genes to genomes: universal scale-invariant properties of microbial chromosome organisation.

    PubMed

    Audit, Benjamin; Ouzounis, Christos A

    2003-09-19

    The availability of complete genome sequences for a large variety of organisms is a major advance in understanding genome structure and function. One attribute of genome structure is chromosome organisation in terms of gene localisation and orientation. For example, bacterial operons, i.e. clusters of co-oriented genes that form transcription units, enable functionally related genes to be expressed simultaneously. The description of genome organisation was pioneered with the study of the distribution of genes of the Escherichia coli partial genetic map before the full genome sequence was known. Deploying powerful techniques from circular statistics and signal processing, we revisit the issue of gene localisation and orientation using 89 complete microbial chromosomes from the eubacterial and archaeal domains. We demonstrate that there is no characteristic size pertinent to the description of chromosome structure, e.g. there does not exist any single length appropriate to describe gene clustering. Our results show that, for all 89 chromosomes, gene positions and gene orientations share a common form of scale-invariant correlations known as "long-range correlations" that we can reveal for distances from the gene length, up to the chromosome size. This observation indicates that genes tend to assemble and to co-orient over any scale of observation greater than a few kilobases. This unexpected property of chromosome structure can be portrayed as an operon-like organisation at all scales and implies that a complete scale range extending over more than three orders of magnitudes of chromosome segment lengths is necessary to properly describe prokaryotic genome organisation. We propose that this pattern results from the effects of the superhelical context on gene expression coupled with the structure and dynamics of the nucleoid, possibly accommodating the diverse gene expression profiles needed during the different stages of cellular life.

  14. 16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns

    PubMed Central

    Rundell, Ethan A.; Banta, Lois M.; Ward, Doyle V.; Watts, Corey D.; Birren, Bruce; Esteban, David J.

    2014-01-01

    A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities. PMID:25101630

  15. Fourier transform infrared spectroscopy as a tool for identification of crude microbial extracts with anti-malarial potential.

    PubMed

    Sankarganesh, P; Joseph, Baby

    2016-01-01

    Fourier transform infrared (FT-IR) spectroscopy is an indispensable tool for identifying biologically active functional groups in uncharacterized crude samples. Here, using FT-IR spectrum analysis, we identified crude extracts of Streptomyces that have anti-malarial activities and conducted a statistical analysis of their spectra. Among the three crude microbial extracts tested herein, an aromatic amine C-N stretching functional group was observed in the spectra of Streptomyces sp. BJSG1 and BJSG4 crude extracts. These extracts showed promising activity against Plasmodium falciparum in vitro cultures with IC50 values of 0.5 for BJSG1 and 0.4μg/mL for BJSG4. The present results showed that FT-IR analysis is necessary for the primary analysis of unknown samples in anti-malarial drug development.

  16. A novel one-step microbial transformation of betulin to betulinic acid catalysed by Cunninghamella blakesleeana.

    PubMed

    Feng, Yu; Li, Min; Liu, Jing; Xu, Teng-Yang; Fang, Ruo-Si; Chen, Qi-He; He, Guo-Qing

    2013-01-01

    Betulinic acid and its derivatives are potential bioactive compounds present in nature. This study investigated the biotransformation of betulin to betulinic acid by Cunninghamella blakesleeana cells. LC-MS analysis demonstrated that betulin could be transformed into at least five products from cultured C. blakesleeana cells, among which betulinic acid was the most important. The presented method provides an attractive alternative approach to chemical synthesis, because is less time-consuming and more environmentally friendly. C. blakesleeana can transform betulin into potent derivatives with high pharmacological activities.

  17. The Extent of Fermentative Transformation of Phenolic Compounds in the Bioanode Controls Exoelectrogenic Activity in a Microbial Electrolysis Cell

    DOE PAGES

    Zeng, Xiaofei; Collins, Maya; Borole, Abhijeet P.; ...

    2016-11-27

    Phenolic compounds in hydrolysate/pyrolysate and wastewater streams produced during the pretreatment of lignocellulosic biomass for biofuel production present a significant challenge in downstream processes. Bioelectrochemical systems are increasingly recognized as an alternative technology to handle biomass-derived streams and to promote water reuse in biofuel production. Thus, a thorough understanding of the fate of phenolic compounds in bioanodes is urgently needed. The present study investigated the biotransformation of three structurally similar phenolic compounds (syringic acid, SA; vanillic acid, VA; 4-hydroxybenzoic acid, HBA), and their individual contribution to exoelectrogenesis in a microbial electrolysis cell (MEC) bioanode. Fermentation of SA resulted in themore » highest exoelectrogenic activity among the three compounds tested, with 50% of the electron equivalents converted to current, compared to 12 and 9% for VA and HBA, respectively. The biotransformation of SA, VA and HBA was initiated by demethylation and decarboxylation reactions common to all three compounds, resulting in their corresponding hydroxylated analogs. SA was transformed to pyrogallol (1,2,3-trihydroxybenzene), whose aromatic ring was then cleaved via a phloroglucinol pathway, resulting in acetate production, which was then used in exoelectrogenesis. In contrast, more than 80% of VA and HBA was converted to catechol (1,2-dihydroxybenzene) and phenol (hydroxybenzene) as their respective dead-end products. The persistence of catechol and phenol is explained by the fact that the phloroglucinol pathway does not apply to di- or mono-hydroxylated benzenes. Previously reported, alternative ring-cleaving pathways were either absent in the bioanode microbial community or unfavorable due to high energy-demand reactions. With the exception of acetate oxidation, all biotransformation steps in the bioanode occurred via fermentation, independently of exoelectrogenesis. Therefore, the observed

  18. The extent of fermentative transformation of phenolic compounds in the bioanode controls exoelectrogenic activity in a microbial electrolysis cell.

    PubMed

    Zeng, Xiaofei; Collins, Maya A; Borole, Abhijeet P; Pavlostathis, Spyros G

    2017-02-01

    Phenolic compounds in hydrolysate/pyrolysate and wastewater streams produced during the pretreatment of lignocellulosic biomass for biofuel production present a significant challenge in downstream processes. Bioelectrochemical systems are increasingly recognized as an alternative technology to handle biomass-derived streams and to promote water reuse in biofuel production. Thus, a thorough understanding of the fate of phenolic compounds in bioanodes is urgently needed. The present study investigated the biotransformation of three structurally similar phenolic compounds (syringic acid, SA; vanillic acid, VA; 4-hydroxybenzoic acid, HBA), and their individual contribution to exoelectrogenesis in a microbial electrolysis cell (MEC) bioanode. Fermentation of SA resulted in the highest exoelectrogenic activity among the three compounds tested, with 50% of the electron equivalents converted to current, compared to 12 and 9% for VA and HBA, respectively. The biotransformation of SA, VA and HBA was initiated by demethylation and decarboxylation reactions common to all three compounds, resulting in their corresponding hydroxylated analogs. SA was transformed to pyrogallol (1,2,3-trihydroxybenzene), whose aromatic ring was then cleaved via a phloroglucinol pathway, resulting in acetate production, which was then used in exoelectrogenesis. In contrast, more than 80% of VA and HBA was converted to catechol (1,2-dihydroxybenzene) and phenol (hydroxybenzene) as their respective dead-end products. The persistence of catechol and phenol is explained by the fact that the phloroglucinol pathway does not apply to di- or mono-hydroxylated benzenes. Previously reported, alternative ring-cleaving pathways were either absent in the bioanode microbial community or unfavorable due to high energy-demand reactions. With the exception of acetate oxidation, all biotransformation steps in the bioanode occurred via fermentation, independently of exoelectrogenesis. Therefore, the observed

  19. The Extent of Fermentative Transformation of Phenolic Compounds in the Bioanode Controls Exoelectrogenic Activity in a Microbial Electrolysis Cell

    SciTech Connect

    Zeng, Xiaofei; Collins, Maya; Borole, Abhijeet P.; Pavlostathis, Spyros

    2016-11-27

    Phenolic compounds in hydrolysate/pyrolysate and wastewater streams produced during the pretreatment of lignocellulosic biomass for biofuel production present a significant challenge in downstream processes. Bioelectrochemical systems are increasingly recognized as an alternative technology to handle biomass-derived streams and to promote water reuse in biofuel production. Thus, a thorough understanding of the fate of phenolic compounds in bioanodes is urgently needed. The present study investigated the biotransformation of three structurally similar phenolic compounds (syringic acid, SA; vanillic acid, VA; 4-hydroxybenzoic acid, HBA), and their individual contribution to exoelectrogenesis in a microbial electrolysis cell (MEC) bioanode. Fermentation of SA resulted in the highest exoelectrogenic activity among the three compounds tested, with 50% of the electron equivalents converted to current, compared to 12 and 9% for VA and HBA, respectively. The biotransformation of SA, VA and HBA was initiated by demethylation and decarboxylation reactions common to all three compounds, resulting in their corresponding hydroxylated analogs. SA was transformed to pyrogallol (1,2,3-trihydroxybenzene), whose aromatic ring was then cleaved via a phloroglucinol pathway, resulting in acetate production, which was then used in exoelectrogenesis. In contrast, more than 80% of VA and HBA was converted to catechol (1,2-dihydroxybenzene) and phenol (hydroxybenzene) as their respective dead-end products. The persistence of catechol and phenol is explained by the fact that the phloroglucinol pathway does not apply to di- or mono-hydroxylated benzenes. Previously reported, alternative ring-cleaving pathways were either absent in the bioanode microbial community or unfavorable due to high energy-demand reactions. With the exception of acetate oxidation, all biotransformation steps in the bioanode occurred via fermentation, independently of exoelectrogenesis. Therefore, the observed

  20. Linking potential denitrification rates to microbial gene abundances in multiple boreal ecosystems

    NASA Astrophysics Data System (ADS)

    Petersen, D. G.; Blazewicz, S.; Herman, D. J.; Firestone, M. K.; Waldrop, M. P.

    2010-12-01

    The composition and functioning of boreal ecosystems are vulnerable to changes in climate, leading to changes in season length, fire regimes, and soil moisture status. To investigate the influence of vegetation and soil moisture on microbial nitrogen cycling several disparate boreal ecosystems was studied. The two primary objectives were to: (1) determine whether process rates could be predicted solely from soil physical and chemical characteristics and (2) determine if the abundance of functional genes could be an additional explanatory variable. Surface soils were sampled along an elevation-driven hydrologic gradient at the Bonanza Creek LTER that corresponds with five plant communities typical of interior Alaska. The plant communities included a black spruce stand, a deciduous stand, a tussock grassland, an emergent fen, and a rich fen. We examined the chemical composition of the surface organic moss and soil, measured gross N-mineralization, potential rates of nitrification and denitrification (DEA), and abundances of several functional groups of microorganisms from soil cores collected in mid summer. We used quantitative PCR to assess the gene abundances of ammonia oxidizers and denitrifiers based on a functional gene approach. Here, we focus on potential denitrification rates (PDR), and abundance of denitrifyers carrying NirS and NirK genes (nitrate reductase) and NosZ genes (nitrous oxide reductase). PDR increased dramatically with increasing soil moisture along the gradient, from 1 mg N/m2/h at the dry black spruce site to 300 mg N/m2/h in the rich fen, which is very high compared to other poorly drained soil environments. PDR were linearly related to the abundance of functional genes from the microorganisms responsible for this process. Abundances of NirS, NirK and NosZ genes correlated significantly to PDR (r2 = 0.61 p < 0.0001, r2 = 0.45 p < 0.0003, r2 = 0.81 p < 0.0001, respectively). In addition, PDR were better explained by functional gene abundances

  1. Novel enabling technologies of gene isolation and plant transformation for improved crop protection

    SciTech Connect

    Torok, Tamas

    2013-02-04

    Meeting the needs of agricultural producers requires the continued development of improved transgenic crop protection products. The completed project focused on developing novel enabling technologies of gene discovery and plant transformation to facilitate the generation of such products.

  2. The role of non-ras transforming genes in chemical carcinogenesis.

    PubMed Central

    Cooper, C S

    1991-01-01

    DNA transfection experiments using the NIH 3T3 mouse fibroblast cell line have demonstrated that chemically induced tumors and chemically transformed cell lines frequently contain dominant transforming genes. Although many of the genes detected using the NIH 3T3 transfection-transformation assay are activated versions of H-ras, K-ras, and N-ras, in some experimental systems activated forms of genes such as met and neu that are unrelated to ras have been observed. The activated met gene was originally detected in a human cell line that had been transformed by exposure to N-methyl-N'-nitro-N-nitrosoguanidine. Subsequent studies demonstrated that the met proto-oncogene encodes a novel growth factor receptor and that gene activation involves the production of a chimeric gene in which the regions of met encoding the extracellular and transmembrane domains of the receptor are replaced by the 5'-region of an unrelated gene called trp. The activated neu gene was detected in tumors of the nervous system that arose in mice following transplacental exposure to N-ethyl-N-nitrosourea. The neu gene also encodes a novel growth factor receptor but, in contrast to met, its activation involves a single T:A----A:T point mutation in the region of the neu gene encoding the receptor transmembrane domain. The presence of genetic alterations in chemically induced malignancies has also been assessed in cytogenetic studies and by Southern analysis of DNA from neoplastic cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1685444

  3. Analysis of microbial communities in the oil reservoir subjected to CO2-flooding by using functional genes as molecular biomarkers for microbial CO2 sequestration

    PubMed Central

    Liu, Jin-Feng; Sun, Xiao-Bo; Yang, Guang-Chao; Mbadinga, Serge M.; Gu, Ji-Dong; Mu, Bo-Zhong

    2015-01-01

    Sequestration of CO2 in oil reservoirs is considered to be one of the feasible options for mitigating atmospheric CO2 building up and also for the in situ potential bioconversion of stored CO2 to methane. However, the information on these functional microbial communities and the impact of CO2 storage on them is hardly available. In this paper a comprehensive molecular survey was performed on microbial communities in production water samples from oil reservoirs experienced CO2-flooding by analysis of functional genes involved in the process, including cbbM, cbbL, fthfs, [FeFe]-hydrogenase, and mcrA. As a comparison, these functional genes in the production water samples from oil reservoir only experienced water-flooding in areas of the same oil bearing bed were also analyzed. It showed that these functional genes were all of rich diversity in these samples, and the functional microbial communities and their diversity were strongly affected by a long-term exposure to injected CO2. More interestingly, microorganisms affiliated with members of the genera Methanothemobacter, Acetobacterium, and Halothiobacillus as well as hydrogen producers in CO2 injected area either increased or remained unchanged in relative abundance compared to that in water-flooded area, which implied that these microorganisms could adapt to CO2 injection and, if so, demonstrated the potential for microbial fixation and conversion of CO2 into methane in subsurface oil reservoirs. PMID:25873911

  4. Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

    PubMed Central

    Pimentel-Elardo, Sheila Marie; Grozdanov, Lubomir; Proksch, Sebastian; Hentschel, Ute

    2012-01-01

    Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis. PMID:22822366

  5. Overexpression of several Arabidopsis histone genes increases Agrobacterium-medicated transformation and transgene expression in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis histone H2A-1 is important for Agrobacterium-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, in the rat5 mutant results in decreased T-(transferred) DNA integration into the plant genome, whereas over-expression of HTA1 increases transformation freq...

  6. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

  7. Pituitary tumor transforming gene binding factor: a new gene in breast cancer.

    PubMed

    Watkins, Rachel J; Read, Martin L; Smith, Vicki E; Sharma, Neil; Reynolds, Gary M; Buckley, Laura; Doig, Craig; Campbell, Moray J; Lewy, Greg; Eggo, Margaret C; Loubiere, Laurence S; Franklyn, Jayne A; Boelaert, Kristien; McCabe, Christopher J

    2010-05-01

    Pituitary tumor transforming gene (PTTG) binding factor (PBF; PTTG1IP) is a relatively uncharacterized oncoprotein whose function remains obscure. Because of the presence of putative estrogen response elements (ERE) in its promoter, we assessed PBF regulation by estrogen. PBF mRNA and protein expression were induced by both diethylstilbestrol and 17beta-estradiol in estrogen receptor alpha (ERalpha)-positive MCF-7 cells. Detailed analysis of the PBF promoter showed that the region -399 to -291 relative to the translational start site contains variable repeats of an 18-bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 122 normal subjects revealed that subjects may be homozygous or heterozygous for between 1 and 6 repeats of the ERE. Chromatin immunoprecipitation and oligonucleotide pull-down assays revealed ERalpha binding to the PBF promoter. PBF expression was low or absent in normal breast tissue but was highly expressed in breast cancers. Subjects with greater numbers of ERE repeats showed higher PBF mRNA expression, and PBF protein expression positively correlated with ERalpha status. Cell invasion assays revealed that PBF induces invasion through Matrigel, an action that could be abrogated both by siRNA treatment and specific mutation. Furthermore, PBF is a secreted protein, and loss of secretion prevents PBF inducing cell invasion. Given that PBF is a potent transforming gene, we propose that estrogen treatment in postmenopausal women may upregulate PBF expression, leading to PBF secretion and increased cell invasion. Furthermore, the number of ERE half-sites in the PBF promoter may significantly alter the response to estrogen treatment in individual subjects.

  8. The use of microbial gene abundance in the development of fuel remediation guidelines in polar soils.

    PubMed

    Richardson, Elizabeth L; King, Catherine K; Powell, Shane M

    2015-04-01

    Terrestrial fuel spills in Antarctica commonly occur on ice-free land around research stations as the result of human activities. Successful spill clean-ups require appropriate targets that confirm contaminated sites are no longer likely to pose environmental risk following remediation. These targets are based on knowledge of the impacts of contaminants on the soil ecosystem and on the response of native biota to contamination. Our work examined the response of soil microbial communities to fuel contamination by measuring the abundance of genes involved in critical soil processes, and assessed the use of this approach as an indicator of soil health in the presence of weathered and fresh fuels. Uncontaminated and contaminated soils were collected from the site of remediation treatment of an aged diesel spill at Casey Station, East Antarctica in December 2012. Uncontaminated soil was spiked with fresh Special Antarctic Blend (SAB) diesel to determine the response of the genes to fresh fuel. Partly remediated soil containing weathered SAB diesel was diluted with uncontaminated soil to simulate a range of concentrations of weathered fuel and used to determine the response of the genes to aged fuel. Quantitative PCR (qPCR) was used to measure the abundance of rpoB, alkB, cat23, and nosZ in soils containing SAB diesel. Differences were observed between the abundance of genes in control soils versus soils containing weathered and fresh fuels. Typical dose-response curves were generated for genes in response to the presence of fresh fuel. In contrast, the response of these genes to the range of weathered fuel appeared to be due to dilution, rather than to the effect of the fuel on the microbial community. Changes in microbial genes in response to fresh contamination have potential as a sensitive measure of soil health and for assessments of the effect of fuel spills in polar soils. This will contribute to the development of remediation guidelines to assist in management

  9. Biodiversity of genes encoding anti-microbial traits within plant associated microbes

    PubMed Central

    Mousa, Walaa K.; Raizada, Manish N.

    2015-01-01

    The plant is an attractive versatile home for diverse associated microbes. A subset of these microbes produces a diversity of anti-microbial natural products including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins, and lytic enzymes. In recent years, detailed molecular analysis has led to a better understanding of the underlying genetic mechanisms. New genomic and bioinformatic tools have permitted comparisons of orthologous genes between species, leading to predictions of the associated evolutionary mechanisms responsible for diversification at the genetic and corresponding biochemical levels. The purpose of this review is to describe the biodiversity of biosynthetic genes of plant-associated bacteria and fungi that encode selected examples of antimicrobial natural products. For each compound, the target pathogen and biochemical mode of action are described, in order to draw attention to the complexity of these phenomena. We review recent information of the underlying molecular diversity and draw lessons through comparative genomic analysis of the orthologous coding sequences (CDS). We conclude by discussing emerging themes and gaps, discuss the metabolic pathways in the context of the phylogeny and ecology of their microbial hosts, and discuss potential evolutionary mechanisms that led to the diversification of biosynthetic gene clusters. PMID:25914708

  10. Similar Microbial Consortia and Genes Are Involved in the Biodegradation of Benzalkonium Chlorides in Different Environments.

    PubMed

    Ertekin, Emine; Hatt, Janet K; Konstantinidis, Konstantinos T; Tezel, Ulas

    2016-04-19

    Benzalkonium chlorides (BACs) are emerging pollutants. Identification of microorganisms and the genes involved in the biodegradation of BACs is crucial for better understanding the fate of BACs in the environment and developing treatment strategies. Four microbial communities degrading BACs were developed from sewage (SEW), activated sludge (AS), soil (SOIL) and sea sediment (SEA) samples. According to 16S rRNA pyrosequencing and shotgun metagenome sequencing analyses, the most abundant species represented uncharacterized members of the Pseudomonas and Achromobacter genera. BAC biotransformation rates of the enriched microbial communities were 2.8, 3.2, 17.8, and 24.3 μM hr(-1) for SEA, AS, SOIL, and SEW, respectively, and were positively correlated with the relative abundance of a particular Pseudomonas sp. strain, BIOMIG1. The strain BIOMIG1 mineralizes BACs at a rate up to 2.40 μmol hr(-1) 10(-11) cells. Genomes of four BAC degrading and nondegrading BIOMIG1 phenotypes were sequenced and differentially compared with each other. As a result, a gene cluster encoding for transporters, an integrase and a dioxygenase were involved in BAC biotransformation. Our results suggest that BIOMIG1 plays a key role on the fate of BACs in the environment and genes, other than those reported to date, are involved in BAC biotransformation in various habitats.

  11. Microbial transformations in Alkali Lake, Oregon. Final report, 1 Aug 88-31 Jul 91

    SciTech Connect

    Boone, D.R.

    1991-01-01

    An examination was made of the terminal metabolic processes in subsurface sediments near West Alkali Lake, Oregon, by performing microbial counts of methanogenic bacteria and isolating the predominant methanogenic culture. This methanogen was characterized and found to be physiologically and phylogenetically different from other described strains, so it represents a previously undescribed species of bacterium, which was named 'Methanohalophilus oregonensis'. In contrast to published descriptions of many other methanogens which have been isolated from hypersaline environments, this one is halotolerant rather than halophilic. Another important characteristic of this organism is that it is capable of catabolizing dimethylsulfide or methanethiol. This ability is important because these methylated sulfur compounds are major conduits by which sulfur moves between the atmosphere and terrestrial and aquatic ecosystems. Phylogenetic comparisons to known methanogens showed that this strain is closely related to another methanogen, 'Methanolobus siciliae' T4/M which was named but not described.

  12. Anaerobic Microbial Transformation of Aromatic Hydrocarbons and Mixtures of Aromatic Hydrocarbons and Halogenated Solvents.

    DTIC Science & Technology

    1992-08-25

    Final Technical I FROM 9/30/88 TO 3/31/92w August 25, 19921 149 16. SUPPLEMENTARY NOTATION Six peer- reviewed publications, acknowledging the sponsor...degradation, indicating that the presence of natural organic substrates may preclude anaerobic biodegradation of MAH in situ. Cyclohexane, CT, and...dechlorination rates with all the CB tested, except for the first step of PeCB transformation (reductive dechlorination to TTCB). Toluene, p- cresol , phenol, or

  13. Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA.

    PubMed

    Glassing, Angela; Dowd, Scot E; Galandiuk, Susan; Davis, Brian; Jorden, Jeffrey R; Chiodini, Rodrick J

    2015-12-01

    Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass.

  14. Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.

    PubMed

    Sandhu, S S; Kinghorn, J R; Rajak, R C; Unkles, S E

    2001-07-01

    An heterologous transformation system for entomopathogenic fungi B. bassiana and M. anisopliae was developed based on the use of A. nidulans nitrate reductase gene (niaD). B. bassiana and M. anisopliae niaD stable mutants were selected by treatment of protoplast with ethane methane sulphonate (EMS) and regenerated on chlorate medium. The cloned gene was capable of transforming B. bassiana and M. anisopliae at a frequency of 5.8 to 20 transformants per microg of DNA. Most of them were mitotically stable.

  15. Rhizome of life, catastrophes, sequence exchanges, gene creations, and giant viruses: how microbial genomics challenges Darwin.

    PubMed

    Merhej, Vicky; Raoult, Didier

    2012-01-01

    Darwin's theory about the evolution of species has been the object of considerable dispute. In this review, we have described seven key principles in Darwin's book The Origin of Species and tried to present how genomics challenge each of these concepts and improve our knowledge about evolution. Darwin believed that species evolution consists on a positive directional selection ensuring the "survival of the fittest." The most developed state of the species is characterized by increasing complexity. Darwin proposed the theory of "descent with modification" according to which all species evolve from a single common ancestor through a gradual process of small modification of their vertical inheritance. Finally, the process of evolution can be depicted in the form of a tree. However, microbial genomics showed that evolution is better described as the "biological changes over time." The mode of change is not unidirectional and does not necessarily favors advantageous mutations to increase fitness it is rather subject to random selection as a result of catastrophic stochastic processes. Complexity is not necessarily the completion of development: several complex organisms have gone extinct and many microbes including bacteria with intracellular lifestyle have streamlined highly effective genomes. Genomes evolve through large events of gene deletions, duplications, insertions, and genomes rearrangements rather than a gradual adaptative process. Genomes are dynamic and chimeric entities with gene repertoires that result from vertical and horizontal acquisitions as well as de novo gene creation. The chimeric character of microbial genomes excludes the possibility of finding a single common ancestor for all the genes recorded currently. Genomes are collections of genes with different evolutionary histories that cannot be represented by a single tree of life (TOL). A forest, a network or a rhizome of life may be more accurate to represent evolutionary relationships among

  16. Rhizome of life, catastrophes, sequence exchanges, gene creations, and giant viruses: how microbial genomics challenges Darwin

    PubMed Central

    Merhej, Vicky; Raoult, Didier

    2012-01-01

    Darwin's theory about the evolution of species has been the object of considerable dispute. In this review, we have described seven key principles in Darwin's book The Origin of Species and tried to present how genomics challenge each of these concepts and improve our knowledge about evolution. Darwin believed that species evolution consists on a positive directional selection ensuring the “survival of the fittest.” The most developed state of the species is characterized by increasing complexity. Darwin proposed the theory of “descent with modification” according to which all species evolve from a single common ancestor through a gradual process of small modification of their vertical inheritance. Finally, the process of evolution can be depicted in the form of a tree. However, microbial genomics showed that evolution is better described as the “biological changes over time.” The mode of change is not unidirectional and does not necessarily favors advantageous mutations to increase fitness it is rather subject to random selection as a result of catastrophic stochastic processes. Complexity is not necessarily the completion of development: several complex organisms have gone extinct and many microbes including bacteria with intracellular lifestyle have streamlined highly effective genomes. Genomes evolve through large events of gene deletions, duplications, insertions, and genomes rearrangements rather than a gradual adaptative process. Genomes are dynamic and chimeric entities with gene repertoires that result from vertical and horizontal acquisitions as well as de novo gene creation. The chimeric character of microbial genomes excludes the possibility of finding a single common ancestor for all the genes recorded currently. Genomes are collections of genes with different evolutionary histories that cannot be represented by a single tree of life (TOL). A forest, a network or a rhizome of life may be more accurate to represent evolutionary relationships

  17. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    NASA Astrophysics Data System (ADS)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  18. Functional comparison of three transformer gene introns regulating conditional female lethality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The trasformer gene plays a critical role in the sex determination pathways of many insects. We cloned two transformer gene introns from Anastrepha suspensa, the Caribbean fruit fly. These introns have sequences that putatively have a role in sex-specific splicing patterns that affect sex determinat...

  19. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  20. Microbial Transformations of TRU and Mixed Wastes: Actinide Speciation and Waste Volume Reduction

    SciTech Connect

    Halada, Gary P.

    2005-06-01

    Cellosic samples were prepared 1/29/92 at BNL from various sources, including white and brown paper towel, and Kimwipes. The mixed cellulosics were cut into 1 cm x 1 cm squares and transferred to glass serum bottles and various treatments were conducted: unamended (U) samples were filled with nitrogen-purged brine from G-Seep (4.1 M Na+ and 5.1 Cl- with minor amounts of Mg, K, and Ca and 0.3 M sulfate (Brush, 1990)); unamended/inoculated (UI) samples were filled with bacteria-containing surface lake water, sediment, and halite from the underground at the WIPP site; amended/inoculated (AI) samples were inoculated in this fashion and amended with nutrients; and amended/inoculated/excess nitrate (AINO3) samples were inoculated with excess nitrate in the form of KNO3 (5 g L-1 (49.5 mM)). Further information on sample preparation is available. All samples were analyzed by Fourier transform infrared spectroscopy (FTIR) at SBU to identify any transformations in cellulosic material which may have occurred during treatment and storage.

  1. Microbial transformation of ginsenoside Rb1 to compound K by Lactobacillus paralimentarius.

    PubMed

    Quan, Lin-Hu; Kim, Yeon-Ju; Li, Guan Hao; Choi, Kwang-Tea; Yang, Deok-Chun

    2013-06-01

    In this study, the major ginsenoside Rb1 was transformed into the more pharmacologically active minor compound K by food grade Lactobacillus paralimentarius LH4, which was isolated from kimchi, a traditional Korean fermented food. The enzymatic reaction was analyzed by TLC, HPLC, and NMR. Using the cell-free enzyme of Lactobacillus paralimentarius LH4 at optimal conditions for 30 °C at pH 6.0, 1.0 mg ml(-1) ginsenoside Rb1 was transformed into 0.52 mg ml(-1) compound K within 72 h, with a corresponding molar conversion yield of 88 %. The cell-free enzyme hydrolyzed the two glucose moieties attached to the C-3 position and the outer glucose moiety attached to the C-20 position of the ginsenoside Rb1. The cell-free enzyme hydrolyzed the ginsenoside Rb1 along the following pathway: ginsenoside Rb1 → gypenoside XVII and ginsenoside Rd → ginsenoside F2 → compound K. Our results indicate that Lactobacillus paralimentarius LH4 has the potential to be applied for the preparation of compound K in the food industry.

  2. ANALYSIS OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) GENES REVEALS THE COMPLEX EVOLUTIONARY HISTORY OF A MICROBIAL EUKARYOTE1

    PubMed Central

    Chan, Cheong Xin; Soares, Marcelo B.; Bonaldo, Maria F.; Wisecaver, Jennifer H.; Hackett, Jeremiah D.; Anderson, Donald M.; Erdner, Deana L.; Bhattacharya, Debashish

    2012-01-01

    Microbial eukaryotes may extinguish much of their nuclear phylogenetic history due to endosymbiotic/horizontal gene transfer (E/HGT). We studied E/HGT in 32,110 contigs of expressed sequence tags (ESTs) from the dinoflagellate Alexandrium tamarense (Dinophyceae) using a conservative phylogenomic approach. The vast majority of predicted proteins (86.4%) in this alga are novel or dinoflagellate-specific. We searched for putative homologs of these predicted proteins against a taxonomically broadly sampled protein database that includes all currently available data from algae and protists and reconstructed a phylogeny from each of the putative homologous protein sets. Of the 2,523 resulting phylogenies, 14-17% are potentially impacted by E/HGT involving both prokaryote and eukaryote lineages, with 2-4% showing clear evidence of reticulate evolution. The complex evolutionary histories of the remaining proteins, many of which may also have been affected by E/HGT, cannot be interpreted using our approach with currently available gene data. We present empirical evidence of reticulate genome evolution that combined with inadequate or highly complex phylogenetic signal in many proteins may impede genome-wide approaches to infer the tree of microbial eukaryotes. PMID:23066170

  3. Soil-Atmosphere CO Exchanges and Microbial Biogeochemistry of CO Transformations in a Brazilian Agricultural Ecosystem†

    PubMed Central

    King, Gary M.; Hungria, M.

    2002-01-01

    Although anthropogenic land use has major impacts on the exchange of soil and atmosphere gas in general, relatively little is known about its impacts on carbon monoxide. We compared soil-atmosphere CO exchanges as a function of land use, crop type, and tillage treatment on an experimental farm in Parãna, Brazil, that is representative of regionally important agricultural ecosystems. Our results showed that cultivated soils consumed CO at rates between 3 and 6 mg of CO m−2 day−1, with no statistically significant effect of tillage method or crop. However, CO exchange for a pasture soil was near zero, and an unmanaged woodlot emitted CO at a rate of 9 mg of CO m−2 day−1. Neither nitrite, aluminum sulfate, nor methyl fluoride additions affected CO consumption by tilled or untilled soils from soybean plots, indicating that CO oxidation did not depend on ammonia oxidizers and that CO oxidation patterns differed in part from patterns reported for forest soils. The apparent Km for CO uptake, 5 to 11 ppm, was similar to values reported for temperate forest soils; Vmax values, approximately 1 μg of CO g (dry weight)−1 h−1, were comparable for woodlot and cultivated soils in spite of the fact that the latter consumed CO under ambient conditions. Short-term (24-h) exposure to elevated levels of CO (10% CO) partially inhibited uptake at lower concentrations (i.e., 100 ppm), suggesting that the sensitivity to CO of microbial populations that are active in situ differs from that of known carboxydotrophs. Soil-free soybean and corn roots consumed CO when they were incubated with 100-ppm concentrations and produced CO when they were incubated with ambient concentrations. These results document for the first time a role for cultivated plant roots in the dynamics of CO in an agricultural ecosystem. PMID:12200303

  4. Functional gene array-based analysis of microbial community structure in groundwaters with a gradient of contaminant levels

    SciTech Connect

    Waldron, P.J.; Wu, L.; Van Nostrand, J.D.; Schadt, C.W.; Watson, D.B.; Jardine, P.M.; Palumbo, A.V.; Hazen, T.C.; Zhou, J.

    2009-06-15

    To understand how contaminants affect microbial community diversity, heterogeneity, and functional structure, six groundwater monitoring wells from the Field Research Center of the U.S. Department of Energy Environmental Remediation Science Program (ERSP; Oak Ridge, TN), with a wide range of pH, nitrate, and heavy metal contamination were investigated. DNA from the groundwater community was analyzed with a functional gene array containing 2006 probes to detect genes involved in metal resistance, sulfate reduction, organic contaminant degradation, and carbon and nitrogen cycling. Microbial diversity decreased in relation to the contamination levels of the wells. Highly contaminated wells had lower gene diversity but greater signal intensity than the pristine well. The microbial composition was heterogeneous, with 17-70% overlap between different wells. Metal-resistant and metal-reducing microorganisms were detected in both contaminated and pristine wells, suggesting the potential for successful bioremediation of metal-contaminated groundwaters. In addition, results of Mantel tests and canonical correspondence analysis indicate that nitrate, sulfate, pH, uranium, and technetium have a significant (p < 0.05) effect on microbial community structure. This study provides an overall picture of microbial community structure in contaminated environments with functional gene arrays by showing that diversity and heterogeneity can vary greatly in relation to contamination.

  5. Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Münch, Steffen; Ludwig, Nancy; Floss, Daniela S; Sugui, Janyce A; Koszucka, Anna M; Voll, Lars M; Sonnewald, Uwe; Deising, Holger B

    2011-01-01

    A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

  6. SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

    PubMed Central

    Ma, Yan; Song, Jing; Chen, Bobin; Xu, Xiaoping; Lin, Guowei

    2016-01-01

    Objective Myelodysplastic syndromes (MDS) are a heterogenous group of clonal hematopoietic stem cell disorders characterized by increased risk of leukemic transformation. This study identifies microRNAs(miRNA) and miRNA targets that might represent leukemic transformation markers for MDS. Methods Based on our previously established nested case-control study cohort of MDS patients, we chose paired patients to undergo Angilent 8 × 15K human miRNA microarrays. Target prediction analysis was administrated using targetscan 5.1 software. We further investigated the function of target gene in MDS cell line using siRNA method, including cell proliferation, cell apoptosis, cell cycle and electron microscope. Results Finally we screened a subset of 7 miRNAs to be significantly differentially expressed between the case (at the end of follow up with leukemic transformation) and control group (at the end of follow up without leukemic transformation). Target prediction analysis revealed SLC7A5 was the common target gene of these 7 miRNAs. Further study on the function of SLC7A5 gene in SKM-1 cell line showed that downregulation of SLC7A5 inhibited SKM-1 cells proliferation, increased apoptosis and caused cell cycle arrest in the G0/G1 stage. Conclusion Our data indicate that SLC7A5 gene may act as a potential leukemic transformation target gene in MDS. PMID:26657287

  7. Expression of codon optimized genes in microbial systems: current industrial applications and perspectives.

    PubMed

    Elena, Claudia; Ravasi, Pablo; Castelli, María E; Peirú, Salvador; Menzella, Hugo G

    2014-01-01

    The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent "silent" nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.

  8. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

    PubMed Central

    Naveed, Muhammad; Mubeen, Samavia; khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

  9. A microbial transformation using Bacillus subtilis B7-S to produce natural vanillin from ferulic acid.

    PubMed

    Chen, Peng; Yan, Lei; Wu, Zhengrong; Li, Suyue; Bai, Zhongtian; Yan, Xiaojuan; Wang, Ningbo; Liang, Ning; Li, Hongyu

    2016-02-04

    Bacillus subtilis strain B7-S screened from18 strains is an aerobic, endospore-forming, model organism of Gram-positive bacteria which is capable to form vanillin during ferulic acid bioconversion. The bioconversion of ferulic acid to vanillin by Bacillus subtilis B7-S (B. subtilis B7-S) was investigated. Based on our results, the optimum bioconversion conditions for the production of vanillin by B. subtilis B7-S can be summarized as follows: temperature 35 °C; initial pH 9.0; inoculum volume 5%; ferulic acid concentration 0.6 g/L; volume of culture medium 20%; and shaking speed 200 r/min. Under these conditions, several repeated small-scale batch experiments showed that the maximum conversion efficiency was 63.30% after 3 h of bioconversion. The vanillin products were confirmed by spectral data achieved from UV-vis, inductively coupled plasma atomic emission spectroscope (ICP-AES) and Fourier transform infrared spectrometer (FT-IR) spectra. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) results confirmed that the cell surface of B. subtilis plays a role in the induction of ferulic acid tolerance. These results demonstrate that B. subtilis B7-S has the potential for use in vanillin production through bioconversion of ferulic acid.

  10. Microbial- and thiosulfate-mediated dissolution of mercury sulfide minerals and transformation to gaseous mercury

    PubMed Central

    Vázquez-Rodríguez, Adiari I.; Hansel, Colleen M.; Zhang, Tong; Lamborg, Carl H.; Santelli, Cara M.; Webb, Samuel M.; Brooks, Scott C.

    2015-01-01

    Mercury (Hg) is a toxic heavy metal that poses significant environmental and human health risks. Soils and sediments, where Hg can exist as the Hg sulfide mineral metacinnabar (β-HgS), represent major Hg reservoirs in aquatic environments. Metacinnabar has historically been considered a sink for Hg in all but severely acidic environments, and thus disregarded as a potential source of Hg back to aqueous or gaseous pools. Here, we conducted a combination of field and laboratory incubations to identify the potential for metacinnabar as a source of dissolved Hg within near neutral pH environments and the underpinning (a)biotic mechanisms at play. We show that the abundant and widespread sulfur-oxidizing bacteria of the genus Thiobacillus extensively colonized metacinnabar chips incubated within aerobic, near neutral pH creek sediments. Laboratory incubations of axenic Thiobacillus thioparus cultures led to the release of metacinnabar-hosted Hg(II) and subsequent volatilization to Hg(0). This dissolution and volatilization was greatly enhanced in the presence of thiosulfate, which served a dual role by enhancing HgS dissolution through Hg complexation and providing an additional metabolic substrate for Thiobacillus. These findings reveal a new coupled abiotic-biotic pathway for the transformation of metacinnabar-bound Hg(II) to Hg(0), while expanding the sulfide substrates available for neutrophilic chemosynthetic bacteria to Hg-laden sulfides. They also point to mineral-hosted Hg as an underappreciated source of gaseous elemental Hg to the environment. PMID:26157421

  11. A microbial transformation using Bacillus subtilis B7-S to produce natural vanillin from ferulic acid

    PubMed Central

    Chen, Peng; Yan, Lei; Wu, Zhengrong; Li, Suyue; Bai, Zhongtian; Yan, Xiaojuan; Wang, Ningbo; Liang, Ning; Li, Hongyu

    2016-01-01

    Bacillus subtilis strain B7-S screened from18 strains is an aerobic, endospore-forming, model organism of Gram-positive bacteria which is capable to form vanillin during ferulic acid bioconversion. The bioconversion of ferulic acid to vanillin by Bacillus subtilis B7-S (B. subtilis B7-S) was investigated. Based on our results, the optimum bioconversion conditions for the production of vanillin by B. subtilis B7-S can be summarized as follows: temperature 35 °C; initial pH 9.0; inoculum volume 5%; ferulic acid concentration 0.6 g/L; volume of culture medium 20%; and shaking speed 200 r/min. Under these conditions, several repeated small-scale batch experiments showed that the maximum conversion efficiency was 63.30% after 3 h of bioconversion. The vanillin products were confirmed by spectral data achieved from UV–vis, inductively coupled plasma atomic emission spectroscope (ICP-AES) and Fourier transform infrared spectrometer (FT-IR) spectra. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) results confirmed that the cell surface of B. subtilis plays a role in the induction of ferulic acid tolerance. These results demonstrate that B. subtilis B7-S has the potential for use in vanillin production through bioconversion of ferulic acid. PMID:26841717

  12. [Genetic transformation of OSISAP1 gene to onion (Allium cepa L.) mediated by amicroprojectile bombardment].

    PubMed

    Xu, Qi-Jiang; Cui, Cheng-Ri

    2007-06-01

    Microprojectile bombardment-mediated transformation method has been developed for onion (Allium cepa L.) using embryogenic calli, induced from stem discs, as target tissue. Zinc-finger protein gene OSISAP1 (Oryza sative subspecies indica stress-associated protein gene) was introduced into the open-pollinated onion cultivar (subs.) 'HG400B'. Bombardment parameters were optimized as: the pressure is 1,100 psi, the distance is 6 cm, two times, the ratio of mass between plasmid DNA and golden particles is 1:320. An efficient microprojectile bombardment-mediated transformation system of onion (Allium cepa L.) callus has been established. The binary vector used carried the nptII gene for kanamycin resistance and the GUS reporter gene. Transgenic cultures were screened for their ability to express the GUS reporter gene and to grow in the presence of kanamycin (150 mg/L). Transient expression of GUS reporter gene was observed through histochemical staining of embryogenic callus transformed by microprojectile bombardment. The putative transgenic plants were analysed at the molecular level using PCR, southern hybridization, and RT-PCR. The results confirmed that the OSISAP1 gene was integrated as one copy into the genome of onion and expression. Transgenic plants were produced efficiently with a transformation frequency of about 10%. Test of salinity-alkali stress showed that sodium chloride and sodium bicarbonate at 200 mmol/L effectively killed non-transgenic plants within 1 week of irrigation, while the transgenic plants were completely unaffected by salinity of 400 mmol/L. So transformation with the OSISAP1 gene raised the salinity-alkali-tolerance of the transgenic plants to a high level.

  13. Detection of methoxylated and hydroxylated polychlorinated biphenyls in sewage sludge in China with evidence for their microbial transformation

    PubMed Central

    Sun, Jianteng; Zhu, Lizhong; Pan, Lili; Wei, Zi; Song, Yao; Zhang, Yuduo; Qu, Liping; Zhan, Yu

    2016-01-01

    The concentrations of methoxylated polychlorinated biphenyls (MeO-PCBs) and hydroxylated polychlorinated biphenyls (OH-PCBs) were measured in the sewage sludge samples collected from twelve wastewater treatment plants in China. Two MeO-PCB congeners, including 3′-MeO-CB-65 and 4′-MeO-CB-101, were detected in three sludge with mean concentrations of 0.58 and 0.52 ng/g dry weight, respectively. OH-PCBs were detected in eight sludge samples, with an average total concentration of 4.2 ng/g dry weight. Furthermore, laboratory exposure was conducted to determine the possible source of OH-PCBs and MeO-PCBs in the sewage sludge, and their metabolism by the microbes. Both 4′-OH-CB-101 and 4′-MeO-CB-101 were detected as metabolites of CB-101 at a limited conversion rate after 5 days. Importantly, microbial interconversion between OH-PCBs and MeO-PCBs was observed in sewage sludge. Demethylation of MeO-PCBs was favored over methylation of OH-PCBs. The abundant and diverse microbes in sludge play a key role in the transformation processes of the PCB analogues. To our knowledge, this is the first report on MeO-PCBs in environmental matrices and on OH-PCBs in sewage sludge. The findings are important to understand the environmental fate of PCBs. PMID:27417462

  14. Detection of methoxylated and hydroxylated polychlorinated biphenyls in sewage sludge in China with evidence for their microbial transformation

    NASA Astrophysics Data System (ADS)

    Sun, Jianteng; Zhu, Lizhong; Pan, Lili; Wei, Zi; Song, Yao; Zhang, Yuduo; Qu, Liping; Zhan, Yu

    2016-07-01

    The concentrations of methoxylated polychlorinated biphenyls (MeO-PCBs) and hydroxylated polychlorinated biphenyls (OH-PCBs) were measured in the sewage sludge samples collected from twelve wastewater treatment plants in China. Two MeO-PCB congeners, including 3‧-MeO-CB-65 and 4‧-MeO-CB-101, were detected in three sludge with mean concentrations of 0.58 and 0.52 ng/g dry weight, respectively. OH-PCBs were detected in eight sludge samples, with an average total concentration of 4.2 ng/g dry weight. Furthermore, laboratory exposure was conducted to determine the possible source of OH-PCBs and MeO-PCBs in the sewage sludge, and their metabolism by the microbes. Both 4‧-OH-CB-101 and 4‧-MeO-CB-101 were detected as metabolites of CB-101 at a limited conversion rate after 5 days. Importantly, microbial interconversion between OH-PCBs and MeO-PCBs was observed in sewage sludge. Demethylation of MeO-PCBs was favored over methylation of OH-PCBs. The abundant and diverse microbes in sludge play a key role in the transformation processes of the PCB analogues. To our knowledge, this is the first report on MeO-PCBs in environmental matrices and on OH-PCBs in sewage sludge. The findings are important to understand the environmental fate of PCBs.

  15. Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

    SciTech Connect

    Skory, C.D.; Horng, J.S.; Pestka, J.J.; Linz, J.E. )

    1990-11-01

    The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per {mu}g of DNA was developed for A. parasiticus by using homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert ({sup 14}C)OMP to ({sup 14}C)UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert ({sup 14}C)OMP to ({sup 14}C)UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.

  16. Physical Factors Correlate to Microbial Community Structure and Nitrogen Cycling Gene Abundance in a Nitrate Fed Eutrophic Lagoon

    PubMed Central

    Highton, Matthew P.; Roosa, Stéphanie; Crawshaw, Josie; Schallenberg, Marc; Morales, Sergio E.

    2016-01-01

    Nitrogenous run-off from farmed pastures contributes to the eutrophication of Lake Ellesmere, a large shallow lagoon/lake on the east coast of New Zealand. Tributaries periodically deliver high loads of nitrate to the lake which likely affect microbial communities therein. We hypothesized that a nutrient gradient would form from the potential sources (tributaries) creating a disturbance resulting in changes in microbial community structure. To test this we first determined the existence of such a gradient but found only a weak nitrogen (TN) and phosphorous gradient (DRP). Changes in microbial communities were determined by measuring functional potential (quantification of nitrogen cycling genes via nifH, nirS, nosZI, and nosZII using qPCR), potential activity (via denitrification enzyme activity), as well as using changes in total community (via 16S rRNA gene amplicon sequencing). Our results demonstrated that changes in microbial communities at a phylogenetic (relative abundance) and functional level (proportion of the microbial community carrying nifH and nosZI genes) were most strongly associated with physical gradients (e.g., lake depth, sediment grain size, sediment porosity) and not nutrient concentrations. Low nitrate influx at the time of sampling is proposed as a factor contributing to the observed patterns. PMID:27826296

  17. Metagenomic analysis of stress genes in microbial mat communities from Antarctica and the High Arctic.

    PubMed

    Varin, Thibault; Lovejoy, Connie; Jungblut, Anne D; Vincent, Warwick F; Corbeil, Jacques

    2012-01-01

    Polar and alpine microbial communities experience a variety of environmental stresses, including perennial cold and freezing; however, knowledge of genomic responses to such conditions is still rudimentary. We analyzed the metagenomes of cyanobacterial mats from Arctic and Antarctic ice shelves, using high-throughput pyrosequencing to test the hypotheses that consortia from these extreme polar habitats were similar in terms of major phyla and subphyla and consequently in their potential responses to environmental stresses. Statistical comparisons of the protein-coding genes showed similarities between the mats from the two poles, with the majority of genes derived from Proteobacteria and Cyanobacteria; however, the relative proportions differed, with cyanobacterial genes more prevalent in the Antarctic mat metagenome. Other differences included a higher representation of Actinobacteria and Alphaproteobacteria in the Arctic metagenomes, which may reflect the greater access to diasporas from both adjacent ice-free lands and the open ocean. Genes coding for functional responses to environmental stress (exopolysaccharides, cold shock proteins, and membrane modifications) were found in all of the metagenomes. However, in keeping with the greater exposure of the Arctic to long-range pollutants, sequences assigned to copper homeostasis genes were statistically (30%) more abundant in the Arctic samples. In contrast, more reads matching the sigma B genes were identified in the Antarctic mat, likely reflecting the more severe osmotic stress during freeze-up of the Antarctic ponds. This study underscores the presence of diverse mechanisms of adaptation to cold and other stresses in polar mats, consistent with the proportional representation of major bacterial groups.

  18. Microbial- and thiosulfate-mediated dissolution of mercury sulfide minerals and transformation to gaseous mercury

    SciTech Connect

    Vázquez-Rodríguez, Adiari I.; Hansel, Colleen M.; Zhang, Tong; Lamborg, Carl H.; Santelli, Cara M.; Webb, Samuel M.; Brooks, Scott C.

    2015-06-23

    Mercury (Hg) is a toxic heavy metal that poses significant environmental and human health risks. Soils and sediments, where Hg can exist as the Hg sulfide mineral metacinnabar (β-HgS), represent major Hg reservoirs in aquatic environments. Metacinnabar has historically been considered a sink for Hg in all but severely acidic environments, and thus disregarded as a potential source of Hg back to aqueous or gaseous pools. In this study, we conducted a combination of field and laboratory incubations to identify the potential for metacinnabar as a source of dissolved Hg within near neutral pH environments and the underpinning (a)biotic mechanisms at play. We show that the abundant and widespread sulfur-oxidizing bacteria of the genus Thiobacillus extensively colonized metacinnabar chips incubated within aerobic, near neutral pH creek sediments. Laboratory incubations of axenic Thiobacillus thioparus cultures led to the release of metacinnabar-hosted Hg(II) and subsequent volatilization to Hg(0). This dissolution and volatilization was greatly enhanced in the presence of thiosulfate, which served a dual role by enhancing HgS dissolution through Hg complexation and providing an additional metabolic substrate for Thiobacillus. These findings reveal a new coupled abiotic-biotic pathway for the transformation of metacinnabar-bound Hg(II) to Hg(0), while expanding the sulfide substrates available for neutrophilic chemosynthetic bacteria to Hg-laden sulfides. Lastly, they also point to mineral-hosted Hg as an underappreciated source of gaseous elemental Hg to the environment.

  19. Microbial- and thiosulfate-mediated dissolution of mercury sulfide minerals and transformation to gaseous mercury

    DOE PAGES

    Vázquez-Rodríguez, Adiari I.; Hansel, Colleen M.; Zhang, Tong; ...

    2015-06-23

    Mercury (Hg) is a toxic heavy metal that poses significant environmental and human health risks. Soils and sediments, where Hg can exist as the Hg sulfide mineral metacinnabar (β-HgS), represent major Hg reservoirs in aquatic environments. Metacinnabar has historically been considered a sink for Hg in all but severely acidic environments, and thus disregarded as a potential source of Hg back to aqueous or gaseous pools. In this study, we conducted a combination of field and laboratory incubations to identify the potential for metacinnabar as a source of dissolved Hg within near neutral pH environments and the underpinning (a)biotic mechanismsmore » at play. We show that the abundant and widespread sulfur-oxidizing bacteria of the genus Thiobacillus extensively colonized metacinnabar chips incubated within aerobic, near neutral pH creek sediments. Laboratory incubations of axenic Thiobacillus thioparus cultures led to the release of metacinnabar-hosted Hg(II) and subsequent volatilization to Hg(0). This dissolution and volatilization was greatly enhanced in the presence of thiosulfate, which served a dual role by enhancing HgS dissolution through Hg complexation and providing an additional metabolic substrate for Thiobacillus. These findings reveal a new coupled abiotic-biotic pathway for the transformation of metacinnabar-bound Hg(II) to Hg(0), while expanding the sulfide substrates available for neutrophilic chemosynthetic bacteria to Hg-laden sulfides. Lastly, they also point to mineral-hosted Hg as an underappreciated source of gaseous elemental Hg to the environment.« less

  20. qPCR assays to quantify genes and gene expression associated with microbial perchlorate reduction.

    PubMed

    De Long, Susan K; Kinney, Kerry A; Kirisits, Mary Jo

    2010-11-01

    Quantitative PCR (qPCR) assays targeting cld (developed in this work) and pcrA (previously described) were used to quantify these perchlorate-related genes in a perchlorate-reducing enrichment culture. Transcript copies were quantified in perchlorate-reducing Rhodocyclaceae strain JDS4. Oxygen and nitrate inhibited expression of cld and pcrA.

  1. TRANSFORMER

    DOEpatents

    Baker, W.R.

    1959-08-25

    Transformers of a type adapted for use with extreme high power vacuum tubes where current requirements may be of the order of 2,000 to 200,000 amperes are described. The transformer casing has the form of a re-entrant section being extended through an opening in one end of the cylinder to form a coaxial terminal arrangement. A toroidal multi-turn primary winding is disposed within the casing in coaxial relationship therein. In a second embodiment, means are provided for forming the casing as a multi-turn secondary. The transformer is characterized by minimized resistance heating, minimized external magnetic flux, and an economical construction.

  2. Using Position-Specific 13C and 14C Labeling and 13C-PLFA Analysis to Assess Microbial Transformations of Free Versus Sorbed Alanine

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Herschbach, J.; Bore, E. K.; Kuzyakov, Y.; Dippold, M. A.

    2015-12-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model substance for the pool of LMWOS. To assess transformations of sorbed alanine, we added position-specific and uniformly 13C and 14C labeled alanine tracer to soil that had previously been sterilized by γ-radiation. The labeled soil was added to non-sterilized soil from the same site and incubated. Soil labeled with the same tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time intervals. The incorporation of 14C into microbial biomass was determined by chloroform fumigation extraction (CFE), and utilization of individual C positions by distinct microbial groups was evaluated by 13C-phospholipid fatty acid analysis (PLFA). A dual peak in the respired CO2 revealed two sorption mechanisms. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we applied the divergence index (DI). The DI reveals the convergent or divergent behavior of C from individual molecule positions during microbial utilization. Alanine C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFA. This indicates that sorption by the COOH group does not protect this group from preferential oxidation. Microbial metabolism was determinative for the preferential oxidation of individual molecule positions. The use of position-specific labeling revealed mechanisms and kinetics of microbial utilization of sorbed and non

  3. Microbial trophic interactions and mcrA gene expression in monitoring of anaerobic digesters

    PubMed Central

    Alvarado, Alejandra; Montañez-Hernández, Lilia E.; Palacio-Molina, Sandra L.; Oropeza-Navarro, Ricardo; Luévanos-Escareño, Miriam P.; Balagurusamy, Nagamani

    2014-01-01

    Anaerobic digestion (AD) is a biological process where different trophic groups of microorganisms break down biodegradable organic materials in the absence of oxygen. A wide range of AD technologies is being used to convert livestock manure, municipal and industrial wastewaters, and solid organic wastes into biogas. AD gains importance not only because of its relevance in waste treatment but also because of the recovery of carbon in the form of methane, which is a renewable energy and is used to generate electricity and heat. Despite the advances on the engineering and design of new bioreactors for AD, the microbiology component always poses challenges. Microbiology of AD processes is complicated as the efficiency of the process depends on the interactions of various trophic groups involved. Due to the complex interdependence of microbial activities for the functionality of the anaerobic bioreactors, the genetic expression of mcrA, which encodes a key enzyme in methane formation, is proposed as a parameter to monitor the process performance in real time. This review evaluates the current knowledge on microbial groups, their interactions, and their relationship to the performance of anaerobic biodigesters with a focus on using mcrA gene expression as a tool to monitor the process. PMID:25429286

  4. Microbial trophic interactions and mcrA gene expression in monitoring of anaerobic digesters.

    PubMed

    Alvarado, Alejandra; Montañez-Hernández, Lilia E; Palacio-Molina, Sandra L; Oropeza-Navarro, Ricardo; Luévanos-Escareño, Miriam P; Balagurusamy, Nagamani

    2014-01-01

    Anaerobic digestion (AD) is a biological process where different trophic groups of microorganisms break down biodegradable organic materials in the absence of oxygen. A wide range of AD technologies is being used to convert livestock manure, municipal and industrial wastewaters, and solid organic wastes into biogas. AD gains importance not only because of its relevance in waste treatment but also because of the recovery of carbon in the form of methane, which is a renewable energy and is used to generate electricity and heat. Despite the advances on the engineering and design of new bioreactors for AD, the microbiology component always poses challenges. Microbiology of AD processes is complicated as the efficiency of the process depends on the interactions of various trophic groups involved. Due to the complex interdependence of microbial activities for the functionality of the anaerobic bioreactors, the genetic expression of mcrA, which encodes a key enzyme in methane formation, is proposed as a parameter to monitor the process performance in real time. This review evaluates the current knowledge on microbial groups, their interactions, and their relationship to the performance of anaerobic biodigesters with a focus on using mcrA gene expression as a tool to monitor the process.

  5. Effect of the gene transformer of Anastrepha on the somatic sexual development of Drosophila.

    PubMed

    Ruiz, María-Fernanda; Sánchez, Lucas

    2010-01-01

    The gene transformer (tra) is the key regulatory memory device for sex determination in tephritid insects. The present manuscript addressed the question about the functional conservation of the tephritid Anastrepha Transformer protein to direct somatic sexual development in Drosophila (Drosophilidae). The transformer cDNA of Anastrepha encoding the putative full-length Tra protein was cloned in pUAST and introduced into Drosophila melanogaster. To express this protein, the GAL4-UAS system was used. The Anastrepha Tra protein induced the female-specific splicing of both dsx and fru pre-mRNAs in Drosophila XY male flies, so that these became transformed into females, though this transformation was incomplete (the sexually dimorphic foreleg basitarsus and the external terminalia were monitored). It was found that the degree of female transformation directly depended on the dose of Anastrepha tra and Drosophila transformer-2 (tra-2) genes, and that the Anastrepha Tra-Drosophila Tra2 complex is not as efficient as the Drosophila Tra-Tra2 complex at inducing the female-specific splicing of Drosophila dsx pre-mRNA. This can explain why the Anastrepha Tra protein cannot fully substitute for the endogenous Drosophila Tra protein.

  6. Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes.

    PubMed

    Theilgaard, H; van Den Berg, M; Mulder, C; Bovenberg, R; Nielsen, J

    2001-02-20

    The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large

  7. The antiSMASH database, a comprehensive database of microbial secondary metabolite biosynthetic gene clusters

    PubMed Central

    Blin, Kai; Medema, Marnix H.; Kottmann, Renzo; Lee, Sang Yup; Weber, Tilmann

    2017-01-01

    Secondary metabolites produced by microorganisms are the main source of bioactive compounds that are in use as antimicrobial and anticancer drugs, fungicides, herbicides and pesticides. In the last decade, the increasing availability of microbial genomes has established genome mining as a very important method for the identification of their biosynthetic gene clusters (BGCs). One of the most popular tools for this task is antiSMASH. However, so far, antiSMASH is limited to de novo computing results for user-submitted genomes and only partially connects these with BGCs from other organisms. Therefore, we developed the antiSMASH database, a simple but highly useful new resource to browse antiSMASH-annotated BGCs in the currently 3907 bacterial genomes in the database and perform advanced search queries combining multiple search criteria. antiSMASH-DB is available at http://antismash-db.secondarymetabolites.org/. PMID:27924032

  8. The antiSMASH database, a comprehensive database of microbial secondary metabolite biosynthetic gene clusters.

    PubMed

    Blin, Kai; Medema, Marnix H; Kottmann, Renzo; Lee, Sang Yup; Weber, Tilmann

    2017-01-04

    Secondary metabolites produced by microorganisms are the main source of bioactive compounds that are in use as antimicrobial and anticancer drugs, fungicides, herbicides and pesticides. In the last decade, the increasing availability of microbial genomes has established genome mining as a very important method for the identification of their biosynthetic gene clusters (BGCs). One of the most popular tools for this task is antiSMASH. However, so far, antiSMASH is limited to de novo computing results for user-submitted genomes and only partially connects these with BGCs from other organisms. Therefore, we developed the antiSMASH database, a simple but highly useful new resource to browse antiSMASH-annotated BGCs in the currently 3907 bacterial genomes in the database and perform advanced search queries combining multiple search criteria. antiSMASH-DB is available at http://antismash-db.secondarymetabolites.org/.

  9. Improved ethanol production in the presence of cadmium ions by a Saccharomyces cerevisiae transformed with a novel cadmium-resistance gene DvCRP1.

    PubMed

    Hu, Jiajun; Xu, Qingyun; Wu, Mengnan; Meng, Xiangzong; Song, Rentao; Gao, Mintian

    2016-11-01

    The DvCRP1 gene obtained from Dunaliella viridis is a cadmium-resistance gene that induces cadmium accumulation in microbial and plant cells. In the present study, Saccharomyces cerevisiae was used as a model system to investigate the effect of DvCRP1 on both cadmium detoxification and ethanol production. Inhibitory effects of cadmium (50-300 µmol/L) on growth (29-92%), glucose consumption (23-89%), and ethanol production (17-92%) were observed at 24 h by S. cerevisiae. DvCRP1 alleviated the inhibitory effect of cadmium, with increase in the ethanol production. The established mathematical model showed that the initial inoculation concentration, cadmium concentration, and transformation of DvCRP1 were the most important factors for cell growth, glucose consumption, and ethanol production. Cadmium detoxification of yeast was also enhanced by increasing the initial concentration of yeast cells. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations (200 µmol/L). The present results evidenced the potential of the insertion of the DvCRP1 gene into yeast for use in bio-refineries during fermentation of heavy metals-contaminated substrates. In addition, this is a promising method for phytoremediation of agricultural soils highly contaminated by heavy metals.

  10. Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

    PubMed Central

    Takeya, T; Hanafusa, H; Junghans, R P; Ju, G; Skalka, A M

    1981-01-01

    Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes. Images PMID:6287213

  11. Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus.

    PubMed

    Yepes, L M; Mittak, V; Pang, S Z; Gonsalves, C; Slightom, J L; Gonsalves, D

    1995-08-01

    In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.

  12. Inhibition by interferon of biochemical transformation induced by cloned herpesvirus thymidine kinase genes.

    PubMed

    Otsuka, H; Qavi, H; Kit, S

    1982-10-01

    To learn whether interferon could prevent the biochemical transformations induced by cloned herpesvirus thymidine kinase (TK) genes, LM(TK-) mouse fibroblast cultures were pretreated for 24 h with 2.4-40 international units (I.U.)/ml mouse alpha + beta interferon, and subsequently transformed to the TK+ phenotype with recombinant plasmids containing the herpes simplex virus type 1 (HSV-1) TK gene (pAGO and pMH110) and the marmoset herpesvirus (MarHV) TK gene (pMAR035). Mouse alpha + beta interferon inhibited transformation and the inhibition was interferon dose-dependent. Transformation was also inhibited when LM(TK-) cells were pretreated for 2-5 h with 40 I.U./ml interferon. Maximal inhibitions of TK+ colony formation were observed following a 9-20 h pretreatment period with interferon. In contrast, 40 I.U./ml interferon treatment for 20 h did not reduce the rate or extent of LM(TK-) cell growth. Experiments in which cultures were first treated with plasmid pAGO and only afterwards treated with interferon also showed that, as the interferon concentration used, interferon did not inhibit the outgrowth of transformated colonies. Enzyme assays showed that pretreatment with interferon inhibited the induction of TK activity in cells that had been transfected with pAGO DNA.

  13. Generation of marker-free plastid transformants using a transiently cointegrated selection gene.

    PubMed

    Klaus, Sebastian M J; Huang, Fong-Chin; Golds, Timothy J; Koop, Hans-Ulrich

    2004-02-01

    Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.

  14. Transport and transformation of genetic information in the critical zone: The case of antibiotic resistance genes

    NASA Astrophysics Data System (ADS)

    Zhu, Y. G.

    2015-12-01

    In addition to material and energy flows, the dynamics and functions of the Earth's critical zone are intensively mediated by biological actions performed by diverse organisms. These biological actions are modulated by the expression of functional genes and their translation into enzymes that catalyze geochemical reactions, such as nutrient turnover and pollutant biodegradation. Although geobiology, as an interdisciplinary research area, is playing and vital role in linking biological and geochemical processes at different temporal and spatial scales, the distribution and transport of functional genes have rarely been investigated from the Earth's critical zone perspectives. To illustrate the framework of studies on the transport and transformation of genetic information in the critical zone, antibiotic resistance is taken as an example. Antibiotic resistance genes are considered as a group of emerging contaminants, and their emergence and spread within the critical zone on one hand are induced by anthropogenic activities, and on other hand are threatening human health worldwide. The transport and transformation of antibiotic resistance genes are controlled by both horizontal gene transfer between bacterial cells and the movement of bacteria harboring antibiotic resistance genes. In this paper, the fate and behavior of antibiotic resistance genes will be discussed in the following aspects: 1) general overview of environmental antibiotic resistance; 2) high through quantification of the resistome in various environmental media; 3) pathways of resistance gene flow within the critical zone; and 4) potential strategies in mitigating antibiotic resistance, particularly from the critical zone perspectives.

  15. Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene

    PubMed Central

    2012-01-01

    Background Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Results Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L-1 along with 250 mg L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Conclusion Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated. PMID:22691292

  16. Transformation of somatic embryos of Prunus incisa ‘February Pink’ with a visible reporter gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An Agrobacterium-mediated transformation system was developed for the ornamental cherry species Prunus incisa. This system uses both an antibiotic resistance gene (NPTII) and a visible selectable marker, the green fluorescent protein (GFP), to select plants. Cells from leaf and root explants were tr...

  17. A Novel Dominant Transformer Allele of the Sex-Determining Gene Her-1 of Caenorhabditis Elegans

    PubMed Central

    Trent, C.; Wood, W. B.; Horvitz, H. R.

    1988-01-01

    We have characterized a novel dominant allele of the sex-determining gene her-1 of Caenorhabditis elegans. This allele, called n695, results in the incomplete transformation of XX animals into phenotypic males. Previously characterized recessive her-1 alleles transform XO animals into phenotypic hermaphrodites. We have identified five new recessive her-1 mutations as intragenic suppressors of n695. Three of these suppressors are weak, temperature-sensitive alleles. We show that the recessive her-1 mutations are loss-of-function alleles, and that the her-1(n695) mutation results in a gain-of-function at the her-1 locus. The existence of dominant and recessive alleles that cause opposite phenotypic transformations demonstrates that the her-1 gene acts to control sexual identity in C. elegans. PMID:3220248

  18. GeoChip-based analysis of microbial functional gene diversity in a landfill leachate-contaminated aquifer

    USGS Publications Warehouse

    Lu, Zhenmei; He, Zhili; Parisi, Victoria A.; Kang, Sanghoon; Deng, Ye; Van Nostrand, Joy D.; Masoner, Jason R.; Cozzarelli, Isabelle M.; Suflita, Joseph M.; Zhou, Jizhong

    2012-01-01

    The functional gene diversity and structure of microbial communities in a shallow landfill leachate-contaminated aquifer were assessed using a comprehensive functional gene array (GeoChip 3.0). Water samples were obtained from eight wells at the same aquifer depth immediately below a municipal landfill or along the predominant downgradient groundwater flowpath. Functional gene richness and diversity immediately below the landfill and the closest well were considerably lower than those in downgradient wells. Mantel tests and canonical correspondence analysis (CCA) suggested that various geochemical parameters had a significant impact on the subsurface microbial community structure. That is, leachate from the unlined landfill impacted the diversity, composition, structure, and functional potential of groundwater microbial communities as a function of groundwater pH, and concentrations of sulfate, ammonia, and dissolved organic carbon (DOC). Historical geochemical records indicate that all sampled wells chronically received leachate, and the increase in microbial diversity as a function of distance from the landfill is consistent with mitigation of the impact of leachate on the groundwater system by natural attenuation mechanisms.

  19. A vast collection of microbial genes that are toxic to bacteria

    SciTech Connect

    Kimelman, Aya; Levy, Asaf; Sberro, Hila; Kidron, Shahar; Leavitt, Azita; Amitai, Gil; Yoder-Himes, Deborah; Wurtzel, Omri; Zhu, Yiwen; Rubin, Edward M; Sorek, Rotem

    2012-02-02

    In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes our assays revealed novel toxins and restriction enzymes, and new classes of small non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator dnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.

  20. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

    PubMed Central

    Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

    1990-01-01

    Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

  1. Simultaneous Transformation of Commingled Trichloroethylene, Tetrachloroethylene, and 1,4-Dioxane by a Microbially Driven Fenton Reaction in Batch Liquid Cultures.

    PubMed

    Sekar, Ramanan; Taillefert, Martial; DiChristina, Thomas J

    2016-11-01

    Improper disposal of 1,4-dioxane and the chlorinated organic solvents trichloroethylene (TCE) and tetrachloroethylene (also known as perchloroethylene [PCE]) has resulted in widespread contamination of soil and groundwater. In the present study, a previously designed microbially driven Fenton reaction system was reconfigured to generate hydroxyl (HO˙) radicals for simultaneous transformation of source zone levels of single, binary, and ternary mixtures of TCE, PCE, and 1,4-dioxane. The reconfigured Fenton reaction system was driven by fed batch cultures of the Fe(III)-reducing facultative anaerobe Shewanella oneidensis amended with lactate, Fe(III), and contaminants and exposed to alternating anaerobic and aerobic conditions. To avoid contaminant loss due to volatility, the Fe(II)-generating, hydrogen peroxide-generating, and contaminant transformation phases of the microbially driven Fenton reaction system were separated. The reconfigured Fenton reaction system transformed TCE, PCE, and 1,4-dioxane either as single contaminants or as binary and ternary mixtures. In the presence of equimolar concentrations of PCE and TCE, the ratio of the experimentally derived rates of PCE and TCE transformation was nearly identical to the ratio of the corresponding HO˙ radical reaction rate constants. The reconfigured Fenton reaction system may be applied as an ex situ platform for simultaneous degradation of commingled TCE, PCE, and 1,4-dioxane and provides valuable information for future development of in situ remediation technologies.

  2. Characterisation of microbial floras and functional gene levels in an anaerobic/aerobic bio-reactor for the degradation of carboxymethyl cellulose.

    PubMed

    Ji, Guodong; Wang, Chen; Guo, Feng

    2013-04-01

    The current study determined the carboxymethyl cellulose (CMC) degradation efficiency, dominant microbial flora, eubacteria and archaebacteria characteristics, and expression levels of genes cel5A, cel6B, and bglC in an anaerobic/aerobic bio-reactor consisting of two-stage UASB (U1 and U2) and two-stage BAF (B1 and B2). The results showed that under three CMC loads, the CMC degradation efficiency of the UASB-BAF system was 91.25%, 80.44%, and 78.73%, respectively. At higher CMC loads, the degradation of cellulose and transformation to cellobiose in U1 was higher, while the transformation to glucose was lower. The results of DGGE and real-time PCR indicated that cellulose degradation bacteria are dominant in U1, cellulose degradation bacteria and cellulose degradation symbiosis bacteria are dominant in B1, and non-cellulose degradation symbiosis bacteria are dominant in both U2 and B2. The rate-limiting enzyme gene of cellulose degradation in U1, B1, and B2 is cel6B, but it is cel5A in U2.

  3. Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker.

    PubMed

    Ogawa, Taiichi; Kawahigashi, Hiroyuki; Toki, Seiichi; Handa, Hirokazu

    2008-08-01

    Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.

  4. Microbial genes, brain & behaviour - epigenetic regulation of the gut-brain axis.

    PubMed

    Stilling, R M; Dinan, T G; Cryan, J F

    2014-01-01

    To date, there is rapidly increasing evidence for host-microbe interaction at virtually all levels of complexity, ranging from direct cell-to-cell communication to extensive systemic signalling, and involving various organs and organ systems, including the central nervous system. As such, the discovery that differential microbial composition is associated with alterations in behaviour and cognition has significantly contributed to establishing the microbiota-gut-brain axis as an extension of the well-accepted gut-brain axis concept. Many efforts have been focused on delineating a role for this axis in health and disease, ranging from stress-related disorders such as depression, anxiety and irritable bowel syndrome to neurodevelopmental disorders such as autism. There is also a growing appreciation of the role of epigenetic mechanisms in shaping brain and behaviour. However, the role of epigenetics in informing host-microbe interactions has received little attention to date. This is despite the fact that there are many plausible routes of interaction between epigenetic mechanisms and the host-microbiota dialogue. From this new perspective we put forward novel, yet testable, hypotheses. Firstly, we suggest that gut-microbial products can affect chromatin plasticity within their host's brain that in turn leads to changes in neuronal transcription and eventually alters host behaviour. Secondly, we argue that the microbiota is an important mediator of gene-environment interactions. Finally, we reason that the microbiota itself may be viewed as an epigenetic entity. In conclusion, the fields of (neuro)epigenetics and microbiology are converging at many levels and more interdisciplinary studies are necessary to unravel the full range of this interaction.

  5. Microbial transformations of free versus sorbed alanine analyzed by position-specific 13C and 14C labeling and 13C-PLFA analysis

    NASA Astrophysics Data System (ADS)

    Apostel, Carolin; Dippold, Michaela; Bore, Ezekiel; Kuzyakov, Yakov

    2015-04-01

    Sorption of charged or partially charged low molecular weight organic substances (LMWOS) to soil mineral surfaces delays microbial uptake and therefore mineralization of LMWOS to CO2, as well as all other biochemical transformations. We used position-specific labeling, a tool of isotope applications novel to soil sciences, to compare the transformation mechanisms of sorbed and non-sorbed alanine in soil. Alanine as an amino acid links C- and N-cycles in soil and therefore is a model representative for the pool of LMWOS. To assess transformations of sorbed alanine, we combined position-specifically and uniformly 13C and 14C labeled alanine tracer solution with a loamy haplic luvisol that had previously been sterilized by γ-radiation. After shaking the mixtures, the supernatant was removed, as was all non-sorbed alanine by repeated shaking with millipore water. The labeled soil was added to non-sterilized soil from the same site. To compare the effect of sorption, soil labeled with the same position-specifically labeled tracers without previous sorption was prepared and incubated as well. We captured the respired CO2 and determined its 14C-activity at increasing time steps. The incorporation of 14C into microbial biomass was determined by CFE, and utilization of individual C positions by distinct microbial groups was evaluated by 13C-PLFA analysis. A dual peak in the respired CO2 revealed the influence of two sorption mechanisms. Microbial uptake and transformation of the sorbed alanine was 3 times slower compared to non-sorbed alanine. To compare the fate of individual C atoms independent of their concentration and pool size in soil, we introduced the divergence index (DI). The DI reveals the convergent or divergent behaviour of C from individual molecule positions during microbial utilization. The DI revealed, that alanines C-1 position was mainly oxidized to CO2, while its C-2 and C-3 were preferentially incorporated in microbial biomass and PLFAs. This indicates

  6. Microbial transformation of nucleosides

    NASA Technical Reports Server (NTRS)

    Lamba, S. S.

    1979-01-01

    A study involving the use of coulter counter in studying the effects of neomycin on E. coli, S. aureus and A. aerogenes was completed. The purpose of this was to establish proper technique for enumeration of cells per ml. It was found that inhibitory effects on growth of E. coli and A. aerogenes, both gram negative organisms, were directly related to the concentration of neomycin used. However, in case S. aureus, a gram positive organism, a decreased inhibition was noted at higher concentrations. A paper entitled, Use of Coulter Counter in Studying Effect of Drugs on Cells in Culture 1 - Effects of Neomycin on E. coli, S. aureus and A. aerogenes, is attached in the appendix. Laboratory procedures were also established to study the effects of nucleoside antibiotic cordycepin on He La cell grown in suspension cultures.

  7. Screening Currency Notes for Microbial Pathogens and Antibiotic Resistance Genes Using a Shotgun Metagenomic Approach

    PubMed Central

    Jalali, Saakshi; Kohli, Samantha; Latka, Chitra; Bhatia, Sugandha; Vellarikal, Shamsudheen Karuthedath; Sivasubbu, Sridhar; Scaria, Vinod; Ramachandran, Srinivasan

    2015-01-01

    Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents. PMID:26035208

  8. Transformation of Neurospora crassa with the cloned am (glutamate dehydrogenase) gene.

    PubMed Central

    Kinsey, J A; Rambosek, J A

    1984-01-01

    We used DNA containing the am gene of Neurospora crassa, cloned in the lambda replacement vector lambdaL-47 (this clone is designated lambdaC-10), and plasmid vector subclones of this DNA to transform am deletion and point mutant strains. By means of subcloning, all sequences required for transformation to am prototrophy and expression of glutamate dehydrogenase have been shown to reside on a 2.5-kilobase BamHI fragment. We also characterized several am+ strains that were obtained after transformation with lambdaC-10. These strains showed Mendelian segregation of the am+ gene, although less than 50% of the transformed strains showed the normal linkage relationship of am with inl. In all cases tested, the strains had incorporated lambda DNA as well. The lambda DNA also showed a Mendelian segregation pattern. In one case, the incorporation of am DNA in a novel position was associated with a mutagenic event producing a strain with a very tight colonial morphology. In all cases in which the am+ gene had become the resident of a new chromosome, glutamate dehydrogenase was produced to only 10 to 20% of the wild-type levels. Images PMID:6230518

  9. Phylogenetic distribution and evolutionary dynamics of the sex determination genes doublesex and transformer in insects.

    PubMed

    Geuverink, E; Beukeboom, L W

    2014-01-01

    Sex determination in insects is characterized by a gene cascade that is conserved at the bottom but contains diverse primary signals at the top. The bottom master switch gene doublesex is found in all insects. Its upstream regulator transformer is present in the orders Hymenoptera, Coleoptera and Diptera, but has thus far not been found in Lepidoptera and in the basal lineages of Diptera. transformer is presumed to be ancestral to the holometabolous insects based on its shared domains and conserved features of autoregulation and sex-specific splicing. We interpret that its absence in basal lineages of Diptera and its order-specific conserved domains indicate multiple independent losses or recruitments into the sex determination cascade. Duplications of transformer are found in derived families within the Hymenoptera, characterized by their complementary sex determination mechanism. As duplications are not found in any other insect order, they appear linked to the haplodiploid reproduction of the Hymenoptera. Further phylogenetic analyses combined with functional studies are needed to understand the evolutionary history of the transformer gene among insects.

  10. Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology.

    PubMed

    Gestal, Alicia M; Liew, Elissa F; Coleman, Nicholas V

    2011-12-01

    Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (Gm(R) or Lac(+)) were obtained at frequencies ranging from 10(1) to 10(6) c.f.u. (µg DNA)(-1). Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter P(C) were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

  11. Transformation of tobacco plants by Yali PPO-GFP fusion gene and observation of subcellular localization.

    PubMed

    Qi, Jing; Li, Gui-Qin; Dong, Zhen; Zhou, Wei

    2016-01-01

    To explore the subcellular localization of Polyphenol oxidase (PPO) from Pyrus bretschneideri, the 1779 bp cDNA of PPO gene excluding the termination codon TAA was cloned and fused with GFP to construct a binary vector pBI121-PPO-GFP. Then, the binary vector was transformed into Nicotiana tabacum by the tumefanciens-mediated method. Using confocal laser scanning microscopy, green fluorescent signals were localized in chloroplasts of the transformed Nicotiana tabacum cell, suggesting that the Polyphenol oxidase from Pyrus bretschneideri was a chloroplast protein.

  12. Paradoxical performance of tryptophan synthase gene trp1 (+) in transformations of the basidiomycete Coprinopsis cinerea.

    PubMed

    Dörnte, Bastian; Kües, Ursula

    2016-10-01

    Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1 (+) wild-type gene. Regularly in C. cinerea, single-trp1 (+)-vector transformations yield about half the numbers of clones than cotransformations with a non-trp1 (+)-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB (+) gene shown here to function as a selection marker in C. cinerea. Parts of single-trp1 (+) - or single-trpB (+) -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1 (+)-vector transformants and also sets off loss of clones in cotransformation with a non-trp1 (+)-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 (+) copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in cotransformations due to preferred single trp1 (+) integration by homologous recombination at its native genomic site.

  13. Diversity and interactions of microbial functional genes under differing environmental conditions: insights from a membrane bioreactor and an oxidation ditch

    NASA Astrophysics Data System (ADS)

    Xia, Yu; Hu, Man; Wen, Xianghua; Wang, Xiaohui; Yang, Yunfeng; Zhou, Jizhong

    2016-01-01

    The effect of environmental conditions on the diversity and interactions of microbial communities has caused tremendous interest in microbial ecology. Here, we found that with identical influents but differing operational parameters (mainly mixed liquor suspended solid (MLSS) concentrations, solid retention time (SRT) and dissolved oxygen (DO) concentrations), two full-scale municipal wastewater treatment systems applying oxidation ditch (OD) and membrane bioreactor (MBR) processes harbored a majority of shared genes (87.2%) but had different overall functional gene structures as revealed by two datasets of 12-day time-series generated by a functional gene array-GeoChip 4.2. Association networks of core carbon, nitrogen and phosphorus cycling genes in each system based on random matrix theory (RMT) showed different topological properties and the MBR nodes showed an indication of higher connectivity. MLSS and DO were shown to be effective in shaping functional gene structures of the systems by statistical analyses. Higher MLSS concentrations resulting in decreased resource availability of the MBR system were thought to promote positive interactions of important functional genes. Together, these findings show the differences of functional potentials of some bioprocesses caused by differing environmental conditions and suggest that higher stress of resource limitation increased positive gene interactions in the MBR system.

  14. Diversity and interactions of microbial functional genes under differing environmental conditions: insights from a membrane bioreactor and an oxidation ditch

    PubMed Central

    Xia, Yu; Hu, Man; Wen, Xianghua; Wang, Xiaohui; Yang, Yunfeng; Zhou, Jizhong

    2016-01-01

    The effect of environmental conditions on the diversity and interactions of microbial communities has caused tremendous interest in microbial ecology. Here, we found that with identical influents but differing operational parameters (mainly mixed liquor suspended solid (MLSS) concentrations, solid retention time (SRT) and dissolved oxygen (DO) concentrations), two full-scale municipal wastewater treatment systems applying oxidation ditch (OD) and membrane bioreactor (MBR) processes harbored a majority of shared genes (87.2%) but had different overall functional gene structures as revealed by two datasets of 12-day time-series generated by a functional gene array-GeoChip 4.2. Association networks of core carbon, nitrogen and phosphorus cycling genes in each system based on random matrix theory (RMT) showed different topological properties and the MBR nodes showed an indication of higher connectivity. MLSS and DO were shown to be effective in shaping functional gene structures of the systems by statistical analyses. Higher MLSS concentrations resulting in decreased resource availability of the MBR system were thought to promote positive interactions of important functional genes. Together, these findings show the differences of functional potentials of some bioprocesses caused by differing environmental conditions and suggest that higher stress of resource limitation increased positive gene interactions in the MBR system. PMID:26743465

  15. Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation.

    PubMed

    Zhou, H; Arrowsmith, J W; Fromm, M E; Hironaka, C M; Taylor, M L; Rodriguez, D; Pajeau, M E; Brown, S M; Santino, C G; Fry, J E

    1995-12-01

    The lack of alternative selectable markers in crop transformation has been a substantial barrier for commercial application of agricultural biotechnology. We have developed an efficient selection system for wheat transformation using glyphosate-tolerant CP4 and GOX genes as a selectable marker. Immature embryos of the wheat cultivar Bobwhite were bombarded with two separate plasmids harboring the CP4/GOX and GUS genes. After a 1 week delay, the bombarded embryos were transferred to a selection medium containing 2 mM glyphosate. Embryo-derived calli were subcultured onto the same selection medium every 3 weeks consecutively for 9-12 weeks, and were then regenerated and rooted on selection media with lower glyphosate concentrations. Transgenic plants tolerant to glyphosate were recovered. ELISA assay confirmed expression of the CP4 and GOX genes in R0 plants. Southern blot analysis demonstrated that the transgenes were integrated into the wheat genomes and transmitted to the following generation. The use of CP4 and GOX genes as a selectable marker provides an efficient, effective, and alternative transformation selection system for wheat.

  16. Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

    PubMed Central

    van Dillewijn, Pieter; Villadas, Pablo J.; Toro, Nicolás

    2002-01-01

    The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community. PMID:12200266

  17. Nitrogen transformations in a Vertisol under long-term tillage and no tillage management in dryland agricultual systems: key genes and potential rates

    NASA Astrophysics Data System (ADS)

    Melero, Sebastiana; Perez de Mora, Alfredo; Murillo, J. Manuel; Buegger, Franz; Kleinedam, Kristina; Kublik, Susanne; Vanderlinden, Karl; Moreno, Felix; Schloter, Michael

    2010-05-01

    The impact of tillage practices on microbial N transformations in semiarid regions is poorly understood and data from long-term field experiments are scarce. In this study, we evaluated the effects of traditional tillage (TT) vs no-tillage (NT) on key processes of the N cycle such as nitrification and denitrification in a long-term field experiment under a rainfed crop rotation system (cereal-sunflower-legumes) on a vertisol (SW Spain). Besides general soil chemical and biological parameters, we quantified the size of the ammonia oxidizing and denitrifying bacterial populations via real-time PCR (amoA, nirS and nosZ genes), and measured potential nitrification and denitrification rates. Soil was sampled at two depths (0-30, till layer; and 30-50 cm), once during the growing period of the crop (wheat) and another time after harvesting. Conservation tillage slightly increased total organic carbon and microbial biomass C content, whereas no effect on nutrient availability (C and N) was observed, likely due to the fertilization regime and the textural characteristics of the soil type (Vertisol). Gene abundance and potential rates were influenced by the interaction between tillage treatment and sampling period, mainly at 0-30 cm depth. In general, ammonia oxidizers and potential nitrification were enhanced under TT, particularly after harvesting. By contrast, higher abundance of denitrifiers, as reflected by both nirS and nosZ gene copy numbers and larger potential denitrification rates were found under NT during the growing period, but not after harvesting. Results also showed that the N2O/N2 ratio was constant throughout the experiment and thus was affected more significantly by environmental parameters such as the availability carbon than by changes in denitrifier abundance. Our results stress the importance of quantifying microbial populations to address the impact of agricultural practices on N transformations in soil. Furthermore, results suggest that the spatial

  18. Dissemination of Antimicrobial Resistance in Microbial Ecosystems through Horizontal Gene Transfer

    PubMed Central

    von Wintersdorff, Christian J. H.; Penders, John; van Niekerk, Julius M.; Mills, Nathan D.; Majumder, Snehali; van Alphen, Lieke B.; Savelkoul, Paul H. M.; Wolffs, Petra F. G.

    2016-01-01

    The emergence and spread of antibiotic resistance among pathogenic bacteria has been a rising problem for public health in recent decades. It is becoming increasingly recognized that not only antibiotic resistance genes (ARGs) encountered in clinical pathogens are of relevance, but rather, all pathogenic, commensal as well as environmental bacteria—and also mobile genetic elements and bacteriophages—form a reservoir of ARGs (the resistome) from which pathogenic bacteria can acquire resistance via horizontal gene transfer (HGT). HGT has caused antibiotic resistance to spread from commensal and environmental species to pathogenic ones, as has been shown for some clinically important ARGs. Of the three canonical mechanisms of HGT, conjugation is thought to have the greatest influence on the dissemination of ARGs. While transformation and transduction are deemed less important, recent discoveries suggest their role may be larger than previously thought. Understanding the extent of the resistome and how its mobilization to pathogenic bacteria takes place is essential for efforts to control the dissemination of these genes. Here, we will discuss the concept of the resistome, provide examples of HGT of clinically relevant ARGs and present an overview of the current knowledge of the contributions the various HGT mechanisms make to the spread of antibiotic resistance. PMID:26925045

  19. ESTIMATION OF MICROBIAL REDUCTIVE TRANSFORMATION RATES FOR CHLORINATED BENZENES AND PHENOLS USING A QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP APPROACH

    EPA Science Inventory

    A set of literature data was used to derive several quantitative structure-activity relationships (QSARs) to predict the rate constants for the microbial reductive dehalogenation of chlorinated aromatics. Dechlorination rate constants for 25 chloroaromatics were corrected for th...

  20. Characteristics and kinetic analysis of AQS transformation and microbial goethite reduction: Insight into “redox mediator-microbe-iron oxide” interaction process

    DOE PAGES

    Zhu, Weihuang; Shi, Mengran; Yu, Dan; ...

    2016-03-29

    Here, the characteristics and kinetics of redox transformation of a redox mediator, anthraquinone-2-sulfonate (AQS), during microbial goethite reduction by Shewanella decolorationis S12, a dissimilatory iron reduction bacterium (DIRB), were investigated to provide insights into “redox mediator-iron oxide” interaction in the presence of DIRB. Two pre-incubation reaction systems of the “strain S12-goethite” and the “strain S12-AQS” were used to investigate the dynamics of goethite reduction and AQS redox transformation. Results show that the concentrations of goethite and redox mediator, and the inoculation cell density all affect the characteristics of microbial goethite reduction, kinetic transformation between oxidized and reduced species of themore » redox mediator. Both abiotic and biotic reactions and their coupling regulate the kinetic process for “Quinone-Iron” interaction in the presence of DIRB. Our results provide some new insights into the characteristics and mechanisms of interaction among “quinone-DIRB- goethite” under biotic/abiotic driven.« less

  1. Characteristics and kinetic analysis of AQS transformation and microbial goethite reduction: Insight into “redox mediator-microbe-iron oxide” interaction process

    SciTech Connect

    Zhu, Weihuang; Shi, Mengran; Yu, Dan; Liu, Chongxuan; Huang, Tinglin; Wu, Fengchang

    2016-03-29

    Here, the characteristics and kinetics of redox transformation of a redox mediator, anthraquinone-2-sulfonate (AQS), during microbial goethite reduction by Shewanella decolorationis S12, a dissimilatory iron reduction bacterium (DIRB), were investigated to provide insights into “redox mediator-iron oxide” interaction in the presence of DIRB. Two pre-incubation reaction systems of the “strain S12-goethite” and the “strain S12-AQS” were used to investigate the dynamics of goethite reduction and AQS redox transformation. Results show that the concentrations of goethite and redox mediator, and the inoculation cell density all affect the characteristics of microbial goethite reduction, kinetic transformation between oxidized and reduced species of the redox mediator. Both abiotic and biotic reactions and their coupling regulate the kinetic process for “Quinone-Iron” interaction in the presence of DIRB. Our results provide some new insights into the characteristics and mechanisms of interaction among “quinone-DIRB- goethite” under biotic/abiotic driven.

  2. Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene.

    PubMed

    Azadi, Pejman; Otang, Ntui Valentaine; Supaporn, Hasthanasombut; Khan, Raham Sher; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2011-06-01

    Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

  3. Transformation by homeobox genes can be mediated by selective transcriptional repression.

    PubMed Central

    Qin, X F; Luo, Y; Suh, H; Wayne, J; Misulovin, Z; Roeder, R G; Nussenzweig, M C

    1994-01-01

    Altered transcription is a recurrent theme in the field of cancer biology. But despite the central role of transcription in transformation, little is known about the mechanism by which dominant nuclear oncogenes induce malignancies. Homeobox family proteins are prominent examples of transcriptional regulators which control development and can function as oncogenes. Here we explore the molecular basis for transformation by this class of regulators using Oct-2 and Oct-1. We show that the DNA binding POU domains of these proteins are selective and sequence-specific transcriptional repressors that produce malignant lymphomas when they are expressed in T cells of transgenic mice. Mutagenesis experiments identified a specific set of promoters, those containing octamer regulatory elements, as the targets for transformation by selective inhibition of gene expression. Images PMID:7813434

  4. Functional analysis of the white gene of Drosophila by P-factor-mediated transformation.

    PubMed

    Gehring, W J; Klemenz, R; Weber, U; Kloter, U

    1984-09-01

    A 12-kb DNA segment spanning the white (w) locus of Drosophila has been inserted into a P-transposon vector and used for P-factor-mediated germ-line transformation. Several red-eyed transformants were recovered which complement the white mutant phenotype. Analysis of the eye pigments and the interaction with the zeste mutation indicates that the w gene inserted at several new chromosomal sites is expressed normally. The tissue-specific accumulation of w transcripts, as studied by in situ hybridization to tissue sections, is the same in transformant and wild-type larvae. This indicates that all the genetic information specified by the w locus is contained within this 12-kb segment of DNA. By secondary mobilization it was shown that the w sequences have been inserted as a functional P(w) transposon which is capable of further transposition.

  5. Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.

    PubMed

    Koutinas, Michalis; Kiparissides, Alexandros; Silva-Rocha, Rafael; Lam, Ming-Chi; Martins Dos Santos, Vitor A P; de Lorenzo, Victor; Pistikopoulos, Efstratios N; Mantalaris, Athanasios

    2011-07-01

    The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses.

  6. Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships

    PubMed Central

    Lau, Maggie C. Y.; Cameron, Connor; Magnabosco, Cara; Brown, C. Titus; Schilkey, Faye; Grim, Sharon; Hendrickson, Sarah; Pullin, Michael; Sherwood Lollar, Barbara; van Heerden, Esta; Kieft, Thomas L.; Onstott, Tullis C.

    2014-01-01

    Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes. PMID:25400621

  7. Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships.

    PubMed

    Lau, Maggie C Y; Cameron, Connor; Magnabosco, Cara; Brown, C Titus; Schilkey, Faye; Grim, Sharon; Hendrickson, Sarah; Pullin, Michael; Sherwood Lollar, Barbara; van Heerden, Esta; Kieft, Thomas L; Onstott, Tullis C

    2014-01-01

    Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

  8. Delayed Leaf Senescence in Tobacco Plants Transformed with tmr, a Gene for Cytokinin Production in Agrobacterium.

    PubMed Central

    Smart, CM; Scofield, SR; Bevan, MW; Dyer, TA

    1991-01-01

    The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins. PMID:12324608

  9. Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

    PubMed Central

    Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

    1991-01-01

    Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

  10. High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

    PubMed Central

    Boast, S; La Mantia, G; Lania, L; Blasi, F

    1983-01-01

    Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells. Images Fig. 2. Figure 3. PMID:6321161

  11. Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection.

    PubMed

    Huang, F-C; Klaus, S M J; Herz, S; Zou, Z; Koop, H-U; Golds, T J

    2002-09-01

    Here we report on the development of a new dominant selection marker for plastid transformation in higher plants using the aminoglycoside phosphotransferase gene aphA-6 from Acinetobacter baumannii. Vectors containing chimeric aphA-6 gene constructs were introduced into the tobacco chloroplast using particle bombardment of alginate-embedded protoplast-derived micro colonies or polyethylene glycol (PEG)-mediated DNA uptake. Targeted insertion into the plastome was achieved via homologous recombination, and plastid transformants were recovered on the basis of their resistance to kanamycin. Variations in kanamycin resistance in transplastomic lines were observed depending on the 5' and 3' regulatory elements associated with the aphA-6 coding region. Transplastomic plants were fertile and showed maternal inheritance of the transplastome in the progeny.

  12. Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

    PubMed Central

    Tsai, Chia-Hong; Bullard, Blair; Cornish, Adam J.; Harvey, Christopher; Reca, Ida-Barbara; Thornburg, Chelsea; Achawanantakun, Rujira; Buehl, Christopher J.; Campbell, Michael S.; Cavalier, David; Childs, Kevin L.; Clark, Teresa J.; Deshpande, Rahul; Erickson, Erika; Armenia Ferguson, Ann; Handee, Witawas; Kong, Que; Li, Xiaobo; Liu, Bensheng; Lundback, Steven; Peng, Cheng; Roston, Rebecca L.; Sanjaya; Simpson, Jeffrey P.; TerBush, Allan; Warakanont, Jaruswan; Zäuner, Simone; Farre, Eva M.; Hegg, Eric L.; Jiang, Ning; Kuo, Min-Hao; Lu, Yan; Niyogi, Krishna K.; Ohlrogge, John; Osteryoung, Katherine W.; Shachar-Hill, Yair; Sears, Barbara B.; Sun, Yanni; Takahashi, Hideki; Yandell, Mark; Shiu, Shin-Han; Benning, Christoph

    2012-01-01

    Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing

  13. Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.

    PubMed

    Vieler, Astrid; Wu, Guangxi; Tsai, Chia-Hong; Bullard, Blair; Cornish, Adam J; Harvey, Christopher; Reca, Ida-Barbara; Thornburg, Chelsea; Achawanantakun, Rujira; Buehl, Christopher J; Campbell, Michael S; Cavalier, David; Childs, Kevin L; Clark, Teresa J; Deshpande, Rahul; Erickson, Erika; Armenia Ferguson, Ann; Handee, Witawas; Kong, Que; Li, Xiaobo; Liu, Bensheng; Lundback, Steven; Peng, Cheng; Roston, Rebecca L; Sanjaya; Simpson, Jeffrey P; Terbush, Allan; Warakanont, Jaruswan; Zäuner, Simone; Farre, Eva M; Hegg, Eric L; Jiang, Ning; Kuo, Min-Hao; Lu, Yan; Niyogi, Krishna K; Ohlrogge, John; Osteryoung, Katherine W; Shachar-Hill, Yair; Sears, Barbara B; Sun, Yanni; Takahashi, Hideki; Yandell, Mark; Shiu, Shin-Han; Benning, Christoph

    2012-01-01

    Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing

  14. Exogenous Gene Integration for Microalgal Cell Transformation Using a Nanowire-Incorporated Microdevice.

    PubMed

    Bae, Sunwoong; Park, Seunghye; Kim, Jung; Choi, Jong Seob; Kim, Kyung Hoon; Kwon, Donguk; Jin, EonSeon; Park, Inkyu; Kim, Do Hyun; Seo, Tae Seok

    2015-12-16

    Superior green algal cells showing high lipid production and rapid growth rate are considered as an alternative for the next generation green energy resources. To achieve the biomass based energy generation, transformed microalgae with superlative properties should be developed through genetic engineering. Contrary to the normal cells, microalgae have rigid cell walls, so that target gene delivery into cells is challengeable. In this study, we report a ZnO nanowire-incorporated microdevice for a high throughput microalgal transformation. The proposed microdevice was equipped with not only a ZnO nanowire in the microchannel for gene delivery into cells but also a pneumatic polydimethylsiloxane (PDMS) microvalve to modulate the cellular attachment and detachment from the nanowire. As a model, hygromycin B resistance gene cassette (Hyg3) was functionalized on the hydrothermally grown ZnO nanowires through a disulfide bond and released into green algal cells, Chlamydomonas reinhardtii, by reductive cleavage. During Hyg3 gene delivery, a monolithic PDMS membrane was bent down, so that algal cells were pushed down toward ZnO nanowires. The supply of vacuum in the pneumatic line made the PDMS membrane bend up, enabling the gene delivered algal cells to be recovered from the outlet of the microchannel. We successfully confirmed Hyg3 gene integrated in microalgae by amplifying the inserted gene through polymerase chain reaction (PCR) and DNA sequencing. The efficiency of the gene delivery to algal cells using the ZnO nanowire-incorporated microdevice was 6.52 × 10(4)- and 9.66 × 10(4)-fold higher than that of a traditional glass bead beating and electroporation.

  15. Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

    PubMed Central

    Zhao, Hongxi; Liu, Junlong; Li, Youquan; Yang, Congshan; Zhao, Shuaiyang; Liu, Juan; Liu, Aihong; Liu, Guangyuan; Yin, Hong; Guan, Guiquan; Luo, Jianxun

    2016-01-01

    Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata. PMID:26951977

  16. [Agrobacterium-mediated transformation of LJAMP2 gene into 'Red Sun' kiwifruit and its molecular identification].

    PubMed

    Zhou, Yue; Zhao, Xupeng; Wu, Xiuhua; Zhang, Yanling; Zhang, Lin; Luo, Keming; Tang, Shaohu

    2014-06-01

    Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for 'Red Sun' kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into 'Red Sun' kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of 'Red Sun' kiwifruit. Taken together, we established an efficient genetic transformation method for 'Red Sun' kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of 'Red Sun' kiwifruit for improvement of disease resistance.

  17. Effects of temperature on microbial transformation of organic matter - comparing stories told by purified enzyme assays, chemostat experiments and soils

    NASA Astrophysics Data System (ADS)

    Lehmeier, C.; Min, K.; Good, H. J.; Billings, S. A.

    2015-12-01

    Temperature (T) is a major determinant of microbial decomposition of soil organic matter (SOM). Quantifying T responses of microbial C fluxes is crucial to improve predictions of SOM dynamics and atmospheric CO2 concentrations, but interpretation of experimental data is complicated by many properties inherent to soils. Comparing such data with complementary, reductionist experiments can help to identify basic mechanisms and interpret soil measurements. We quantified T effects on activity levels (i.e., rates of substrate cleavage) of microbial extracellular enzymes β-glucosidase (BGase) and β-N-acetyl glucosaminidase (NAGase), and on rates of CO2 efflux in soil incubations. We compare the results to those derived from purified enzyme assays, and to measurements of microbial respiration rates in continuous-flow chemostat culture in which a population of the soil bacterium Pseudomonas fluorescens was grown on medium with similar C:N ratio as the incubated SOM (10:1). Activity levels of both BGase and NAGase decreased by 80% between 25 and 5 °C. These T responses were higher than predictions from intrinsic (i.e., maximum) T responses in purified assays of BGase (minus 50%) and NAGase (minus 67%). This suggests that factors like physical access to substrate or reduced microbial production of enzymes constrained substrate decomposition rates in the soils relatively more at low than at high T. In chemostats, (mass-)specific bacterial respiration rate at T 14.5 °C was 50% of the rate observed at 26.5 °C; in contrast, CO2 efflux from the soil incubations decreased by only ~25% from 25 to 15 °C. The reason for this discrepancy can be manifold, including changes in microbial community composition, but results from ongoing measurements of microbial biomass in the soil samples will allow a closer comparison of these respiration rate responses. Our efforts highlight the significance of experimenting across scales and complexity for a better understanding of SOM dynamics.

  18. Pellitorine, a potential anti-cancer lead compound against HL6 and MCT-7 cell lines and microbial transformation of piperine from Piper Nigrum.

    PubMed

    Ee, Gwendoline Cheng Lian; Lim, Chyi Meei; Rahmani, Mawardi; Shaari, Khozirah; Bong, Choon Fah Joseph

    2010-04-05

    Pellitorine (1), which was isolated from the roots of Piper nigrum, showed strong cytotoxic activities against HL60 and MCT-7 cell lines. Microbial transformation of piperine (2) gave a new compound 5-[3,4-(methylenedioxy)phenyl]-pent-2-ene piperidine (3). Two other alkaloids were also found from Piper nigrum. They are (E)-1-[3',4'-(methylenedioxy)cinnamoyl]piperidine (4) and 2,4-tetradecadienoic acid isobutyl amide (5). These compounds were isolated using chromatographic methods and their structures were elucidated using MS, IR and NMR techniques.

  19. Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans

    PubMed Central

    Nieto, Pamela A; Covarrubias, Paulo C; Jedlicki, Eugenia; Holmes, David S; Quatrini, Raquel

    2009-01-01

    Background Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data. Results Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans. Conclusion This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems. PMID:19555508

  20. Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Brenna, Andrea; Montanini, Barbara; Muggiano, Eleonora; Proietto, Marco; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.

  1. Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments.

    PubMed

    Hamamura, N; Macur, R E; Korf, S; Ackerman, G; Taylor, W P; Kozubal, M; Reysenbach, A-L; Inskeep, W P

    2009-02-01

    The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA-like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6-3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2-8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and beta-Proteobacteria. Modified primers designed around previously characterized and newly identified aroA-like genes successfully amplified new lineages of aroA-like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA-like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences

  2. Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.

    PubMed

    Zhang, Wan-Jun; Wang, Tao

    2015-05-01

    Generating salt tolerance forage plant is essential for use of the land affected by high salinity. A salt tolerance gene rstB was used as a selectable marker gene in Agrobacterium-mediated transformation of tobacco under a selective regime of 170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal phenotype was observed among the transgenic plants when compared with wild type (wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed improved germination rate and seedling growth under salt-stress condition. Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in transgenic plants was also verified by cytohistochemical localization of calcium. Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt plants had no flower formation. The results demonstrated that the expression of rstB gene improved salt tolerance in transgenic alfalfa.

  3. Blue Genes: An Integrative Laboratory to Differentiate Genetic Transformation from Gene Mutation for Underclassmen

    ERIC Educational Resources Information Center

    Militello, Kevin T.; Chang, Ming-Mei; Simon, Robert D.; Lazatin, Justine C.

    2016-01-01

    The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by…

  4. [Diversity of the Key Biphenyl Destruction Genes in the Microbial Community of the Anadyr Bay Coastal Sediments].

    PubMed

    Shumkova, E S; Voronina, A O; Kuznetsova, N V; Plotnikova, E G

    2015-07-01

    Biphenyl 2,3-dioxygenase is the key enzyme involved in the bacterial destruction of biphenyl and polychlo- rinated biphenyls (PCBs), which are highly stable toxic compounds. The diversity of bphA1 genes encoding the biphenyl 2,3-dioxygenase a subunit of biphenyl-decomposing bacteria from the microbial community of the Bering Sea coastal sediments (the Anadyr port area) was studied. The enrichment culture was obtained by the incubation of bottom sediments samples with biphenyl as the only carbon source. It was followed by total DNA extraction and PCR analysis with degenerate primers specific to the bacterial biphenyl 2,3-dioxygenase a subunit genes. Subsequent cloning of the PCR products led to the identification of three types of aromatic dioxygenase genes, which appeared to be phylogenetically close to the genes of the biphenyl/toluene dioxygenase and 3-phenylpropionate dioxygenase subfamilies of the Actinomycetales bacteria.

  5. Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene.

    PubMed

    Bowman, Jeff S; Rasmussen, Simon; Blom, Nikolaj; Deming, Jody W; Rysgaard, Søren; Sicheritz-Ponten, Thomas

    2012-01-01

    Dramatic decreases in the extent of Arctic multiyear ice (MYI) suggest this environment may disappear as early as 2100, replaced by ecologically different first-year ice. To better understand the implications of this loss on microbial biodiversity, we undertook a detailed census of the microbial community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than in underlying surface water, we found diversity to be comparable using the Simpson and Shannon's indices (for Simpson t=0.65, P=0.56; for Shannon t=0.25, P=0.84 for a Student's t-test of mean values). Cyanobacteria, comprising 6.8% of reads obtained from MYI, were observed for the first time in Arctic sea ice. In addition, several low-abundance clades not previously reported in sea ice were present, including the phylum TM7 and the classes Spartobacteria and Opitutae. Members of Coraliomargarita, a recently described genus of the class Opitutae, were present in sufficient numbers to suggest niche occupation within MYI.

  6. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

    SciTech Connect

    Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex; Waalkes, Michael P.

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular

  7. Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene.

    PubMed

    Cha, Mijeong; Shim, Sang Hee; Kim, Sung Hong; Kim, Ok Tae; Lee, Se-Weon; Kwon, Suk-Yoon; Baek, Kwang-Hyun

    2012-10-01

    Paclitaxel is produced by various species of yew trees and has been extensively used to treat tumors. In our research, a taxadiene synthase (TS) gene from Taxus brevifolia was used to transform the roots of cultured ginseng (Panax ginseng C.A. Meyer) to produce taxadiene, the unique skeletal precursor to taxol. The TS gene was successfully introduced into the ginseng genome, and the de novo formation of taxadiene was identified by mass spectroscopy profiling. Without any change in phenotypes or growth difference in a TS-transgenic ginseng line, the transgenic TSS3-2 line accumulated 9.1 μg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation was 14.6 and 15.9 μg per g of dry weight, respectively. This is the first report of the production of taxadiene by engineering ginseng roots with a taxadiene synthase gene.

  8. Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

    PubMed Central

    Cerutti, H; Johnson, A M; Gillham, N W; Boynton, J E

    1997-01-01

    The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation. PMID:9212467

  9. The efficiency of genetic transformation of mammalian cells by transfection and microinjection depends on the transferred gene.

    PubMed

    Strauss, M; Kiessling, U; Zavision, B A; Povitza, O N; Tikhonenko, T I; Geissler, E

    1983-01-01

    The efficiency of genetic transformation of mammalian cells was analysed with respect to the kind of the transferred gene and the selective system. Plasmids pAGO and pAG60 harboring the thymidine kinase gene of Herpes simplex virus type 1 and the bacterial neomycin resistance gene, respectively, were compared concerning their ability to transform mouse Ltk-aprt- cells. Using the calcium phosphate technique the neomycin resistance gene transformed at least ten times more efficiently than the thymidine kinase gene (3 X 10(-3) versus 2 X 10(-4] whereas the difference is even more impressive following microinjection of the plasmids into the nuclei (2 X 10(-1) versus 2.5 X 10(-3]. The neomycin system also proved to be more effective in secondary gene transfer experiments and, thus, seems to be the most convenient marker for cotransfer experiments.

  10. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    DOE PAGES

    Dupont, Chris L.; McCrow, John P.; Valas, Ruben; ...

    2014-10-21

    Here, transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enrichedmore » in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.« less

  11. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    SciTech Connect

    Dupont, Chris L.; McCrow, John P.; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L.; Bai, Jing; Johnson, Zackary I.; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A.; Venter, J. Craig; Allen, Andrew E.

    2014-10-21

    Here, transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

  12. Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

    PubMed Central

    Dupont, Chris L; McCrow, John P; Valas, Ruben; Moustafa, Ahmed; Walworth, Nathan; Goodenough, Ursula; Roth, Robyn; Hogle, Shane L; Bai, Jing; Johnson, Zackary I; Mann, Elizabeth; Palenik, Brian; Barbeau, Katherine A; Craig Venter, J; Allen, Andrew E

    2015-01-01

    Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM. PMID:25333462

  13. Excess of Organic Carbon in Mountain Spruce Forest Soils after Bark Beetle Outbreak Altered Microbial N Transformations and Mitigated N-Saturation.

    PubMed

    Kaňa, Jiří; Tahovská, Karolina; Kopáček, Jiří; Šantrůčková, Hana

    2015-01-01

    Mountain forests in National park Bohemian Forest (Czech Republic) were affected by bark beetle attack and windthrows in 2004-2008, followed by an extensive tree dieback. We evaluated changes in the biochemistry of the uppermost soil horizons with the emphasis on carbon (C) and nitrogen (N) cycling in a near-natural spruce (Picea abies) mountain forest after the forest dieback, and compared it with an undisturbed control plot of similar age, climate, elevation, deposition, N-saturation level, and land use history. We hypothesised that the high litter input after forest dieback at the disturbed plot and its consequent decomposition might influence the availability of C for microorganisms, and consequently, N transformations in the soil. The concentrations of dissolved organic C (DOC) and N (DON) in soil water extracts rapidly increased at the disturbed plot for 3 yeas and then continually decreased. Net ammonification exhibited a similar trend as DOC and DON, indicating elevated mineralization. Despite the high ammonium concentrations found after the forest dieback (an increase from 0.5 mmol kg-1 to 2-3 mmol kg-1), net nitrification was stable and low during these 3 years. After the DOC depletion and decrease in microbial biomass 5 years after the forest dieback, net nitrification started to rise, and nitrate concentrations increased from 0.2-1 mmol kg-1 to 2-3 mmol kg-1. Our results emphasize the key role of the availability of organic C in microbial N transformations, which probably promoted microbial heterotrophic activity at the expense of slow-growing nitrifiers.

  14. Genetic transformation of Nannochloropsis oculata with a bacterial phleomycin resistance gene as dominant selective marker

    NASA Astrophysics Data System (ADS)

    Ma, Xiaolei; Pan, Kehou; Zhang, Lin; Zhu, Baohua; Yang, Guanpin; Zhang, Xiangyang

    2016-04-01

    The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×107 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

  15. Pituitary tumor-transforming gene and its binding factor in endocrine cancer.

    PubMed

    Smith, Vicki E; Franklyn, Jayne A; McCabe, Christopher J

    2010-12-03

    The pituitary tumor-transforming gene (PTTG1) encodes a multifunctional protein (PTTG) that is overexpressed in numerous tumours, including pituitary, thyroid, breast and ovarian carcinomas. PTTG induces cellular transformation in vitro and tumourigenesis in vivo, and several mechanisms by which PTTG contributes to tumourigenesis have been investigated. Also known as the human securin, PTTG is involved in cell cycle regulation, controlling the segregation of sister chromatids during mitosis. This review outlines current information regarding PTTG structure, expression, regulation and function in the pathogenesis of neoplasia. Recent progress concerning the use of PTTG as a prognostic marker or therapeutic target will be considered. In addition, the PTTG binding factor (PBF), identified through its interaction with PTTG, has also been established as a proto-oncogene that is upregulated in several cancers. Current knowledge regarding PBF is outlined and its role both independently and alongside PTTG in endocrine and related cancers is discussed.

  16. Agrobacterium-mediated transformation of Malus robusta with tomato iron transporter gene.

    PubMed

    Qu, Shen-Chun; Huang, Xiao-De; Zhang, Zhen; Yao, Quan-Hong; Tao, Jian-Min; Qiao, Yu-Shan; Zhang, Jun-Yi

    2005-06-01

    The tomato iron transporter gene (LeIRT2) was introduced to Malus robusta Rehd. via Agrobacterium-mediated transformation to produce iron-deficiency tolerant apple rootstock. A total of 19 putative transformants were obtained, 11 of which were verified by PCR amplification to carry a fragment of the transgene. Among them, nine were confirmed to carry the transgene by Southern blot analysis with one to three copies of the transgene integrated into the plant genome. Two transgenic plants, one carrying one copy and the other three copies of the transgene, were hydroponically cultured to test their tolerance to iron-deficiency, which was found only in the transgenic plant with a single copy, which weighted 21%-4% greater than those of the control plants.

  17. Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot

    PubMed Central

    Armesto, Cecilia; Maia, Fernanda Gonçalves Martins; de Abreu, Mário Sobral; Figueira, Antonia dos Reis; da Silva, Bruno Marques; Monteiro, Fernando Pereira

    2012-01-01

    Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host × pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed. PMID:24031947

  18. [Rol of pituitary tumour-transforming gene (PTTG) in the pituitary adenomas].

    PubMed

    Sánchez-Ortiga, Ruth; Sánchez Tejada, Laura; Peiró Cabrera, Gloria; Moreno-Pérez, Oscar; Arias Mendoza, Nieves; Aranda López, F Ignacio; Picó Alfonso, Antonio

    2010-01-01

    The pathogenesis of pituitary tumours is far to be understood. Pituitary transforming tumour gene (PTTG), a gen that induces aneuploidy, genetic instability, cellular proliferation and to stimulate angiogenesis, has been involved in neoplasic transformation and shown overexpressed in many neoplasm as lung, breast, endometrium, thyroid and colon malignant tumours. On the other hand, PTTG has been inconsistently studied in pituitary tumours. The majority of studies have been performed in animals and there is a great variability in the methods used in its determination. The goal of this review is to resume the role of PTTG in tumourogenesis and critically to revise the studies published in humans in order to advance in the knowledge of the pathogenesis of pituitary adenomas and to find clinical useful predictors of the behavior of these tumours.

  19. Microbial diversity of mangrove sediment in Shenzhen Bay and gene cloning, characterization of an isolated phytase-producing strain of SPC09 B. cereus.

    PubMed

    Zhang, Shengpeng; Liao, Shao-An; Yu, Xiaoyuan; Lu, Hongwu; Xian, Jian-An; Guo, Hui; Wang, Anli; Xie, Jian

    2015-06-01

    Phytases hydrolyze phytate to release inorganic phosphate, which decreases the requirement for phosphorus in fertilizers for crops and thus reduces environmental pollutants. This study analyzed microbial communities in rhizosphere sediment, collected in September 2012 from Shenzhen Bay, Guangdong, China, using high-throughput pyrosequencing; the results showed that the dominant taxonomic phyla were Chloroflexi, Firmicutes, and Proteobacteria, and the proportion of the beneficial bacteria, Bacillus, was 4.95 %. Twenty-nine culturable, phytase-producing bacteria were isolated, their phosphorus solubilization capacity was analyzed, and they were taxonomically characterized. Their phylogenetic placement was determined using 16S ribosomal RNA (rRNA) gene sequence analysis. The result shows that most of the isolates are members of the order Bacillales, although seven strains of Enterobacteriales, two strains of Pseudomonadales, and one strain of Oceanospirillales were also identified. The phytase gene was cloned from SPC09, Bacillus cereus, which showed the highest phosphorus solubilizing ability among the isolated strains. The gene encoded a primary translation product of 335 amino acids. A construct including the 1005-nt ORF fragment, Bc-phy, was transformed into Escherichia coli. The recombinant phytase was produced and purified, which revealed the temperature optima at 60 °C and pH optima at 6.5. The assessment by quantitative PCR (qPCR) showed an abundance of bacteria containing the Bc-phy gene; the level was generally higher in the mangrove forest than in the tidal flats and in surface soil compared to bottom soil, and the highest value was obtained in June. Herein, we report on the cloning, characterization, and activity of a novel phytase isolated from a mangrove system.

  20. Neutral evolution of the sex-determining gene transformer in Drosophila.

    PubMed Central

    McAllister, B F; McVean, G A

    2000-01-01

    The amino acid sequence of the transformer (tra) gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana we find evidence for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function. PMID:10747064

  1. Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

    PubMed Central

    Leptak, C; Ramon y Cajal, S; Kulke, R; Horwitz, B H; Riese, D J; Dotto, G P; DiMaio, D

    1991-01-01

    To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells. Images PMID:1658398

  2. The Gene Transformer of Anastrepha Fruit Flies (Diptera, Tephritidae) and Its Evolution in Insects

    PubMed Central

    Salvemini, Marco; Eirín-López, José María; Perondini, André L. P.; Selivon, Denise; Polito, Catello; Saccone, Giuseppe; Sánchez, Lucas

    2007-01-01

    In the tephritids Ceratitis capitata and Bactrocera oleae, the gene transformer acts as the memory device for sex determination, via an auto-regulatory function; and functional Tra protein is produced only in females. This paper investigates the evolution of the gene tra, which was characterised in twelve tephritid species belonging to the less extensively analysed genus Anastrepha. Our study provided the following major conclusions. Firstly, the memory device mechanism used by this gene in sex determination in tephritids likely existed in the common ancestor of the Ceratitis, Bactrocera and Anastrepha phylogenetic lineages. This mechanism would represent the ancestral state with respect to the extant cascade seen in the more evolved Drosophila lineage. Secondly, Transformer2-specific binding intronic splicing silencer sites were found in the splicing regulatory region of transformer but not in doublesex pre-mRNAs in these tephritids. Thus, these sites probably provide the discriminating feature for the putative dual splicing activity of the Tra-Tra2 complex in tephritids. It acts as a splicing activator in dsx pre-mRNA splicing (its binding to the female-specific exon promotes the inclusion of this exon into the mature mRNA), and as a splicing inhibitor in tra pre-mRNA splicing (its binding to the male-specific exons prevents the inclusion of these exons into the mature mRNA). Further, a highly conserved region was found in the specific amino-terminal region of the tephritid Tra protein that might be involved in Tra auto-regulatory function and hence in its repressive splicing behaviour. Finally, the Tra proteins conserved the SR dipeptides, which are essential for Tra functionality. PMID:18043746

  3. Expression of copper-resistance genes in microbial communities under copper stress and oxic/anoxic conditions.

    PubMed

    Besaury, Ludovic; Pawlak, Barbara; Quillet, Laurent

    2016-03-01

    Microorganisms have developed copper-resistance mechanisms in order to survive in contaminated environments. The abundance and expression of the copper-resistance genes cusA and copA, encoding respectively for a Resistance Cell Nodulation protein and for a P-type ATP-ase pump, was assessed along a gradient of copper concentration in microcosms prepared from Seine estuary mudflat sediment. We demonstrated that the abundance of copA and cusA genes decreased with the increase of copper concentration and that cusA gene was up to ten times higher than the copA gene. Only the copA gene was expressed in both oxic and anoxic conditions. The abundance and activity of the microbial community remained constant whatever the concentrations of copper along the gradient. The molecular phylogeny of the two copper-resistance genes was studied and revealed that the increase of copper increased the diversity of copA and cusA gene sequences.

  4. Expression of immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation.

    PubMed

    Benatar, T; Iacampo, S; Tkalec, L; Ratcliffe, M J

    1991-10-01

    Analysis of the early stages of avian B lymphocyte differentiation has been hampered by the low frequency of extra-bursal B lineage cells in sites of hematopoiesis. Consequently, little is known about B lineage precursors prior to their migration into the bursa of Fabricius. Colonization of the bursa typically occurs between about days 8 and 14 of embryonic (e) development, although cells which can colonize the bursa, functionally defined as pre-bursal stem cells, can be demonstrated in embryo bone marrow up until about the time of hatch. As a novel approach to analyzing early stages of avian B lymphocyte development, we show here that transformed B lineage cells can be derived from chick embryo bone marrow after infection in vitro with the replication-defective retrovirus REV-T produced in the context of the non-cytopathic CSV helper virus. Thus, exposure of day 14e-15e chick embryo bone marrow cells to REV-T (CSV) results in the generation of transformed, polyclonal lines of cells. From these lines, cells expressing cell surface immunoglobulin were readily isolated by flow cytometric cell sorting and single cell cloning. Analysis of the phenotype of REV-T(CSV)-transformed clones with a panel of monoclonal antibody reagents demonstrated that transformation by v-rel likely leads to marked changes in cell surface antigen expression. Nonetheless, clones expressing cell surface immunoglobulin expressed apparently normal mRNA for immunoglobulin mu and light chain and contained apparently normal immunoglobulin heavy and light chain gene rearrangements. Furthermore, no evidence for chromosomal deletions or aberrations of the Ig loci was detected among either sIg+ or sIg- REV-T(CSV)-transformed clones.

  5. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

  6. Transformation of soybean Gy3 gene into Artemisaarenaria mediated by corona discharge

    NASA Astrophysics Data System (ADS)

    Chao, Lu-meng; Na, Ri; Xue, Dan; Xu, Yongze; Liu, Teng

    2013-03-01

    In order to improve the protein content of desert plant, a method of genetic transformation mediated by corona discharge was established. Artemisia seeds were processed in corona electric field for 120 min at 12 kV, and then soaked in 0.1 SSC media that contained Soybean Gy3 gene DNA to incubate for 12 h at 26 °C. Finally the seeds were inoculated on the differentiation medium. Polymerase Chain Reaction (PCR) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) detection showed that the Soybean Gy3 gene had been successfully introduced into genomic DNA of the regenerated plants of Artemisaarenaria. The study provided a new way for corona discharge in plant genetic modification.

  7. Quantification of Key Genes Steering the Microbial Nitrogen Cycle in the Rhizosphere of Sorghum Cultivars in Tropical Agroecosystems ▿

    PubMed Central

    Hai, Brigitte; Diallo, Ndeye Hélène; Sall, Saidou; Haesler, Felix; Schauss, Kristina; Bonzi, Moussa; Assigbetse, Komi; Chotte, Jean-Luc; Munch, Jean Charles; Schloter, Michael

    2009-01-01

    The effect of agricultural management practices on geochemical cycles in moderate ecosystems is by far better understood than in semiarid regions, where fertilizer availability and climatic conditions are less favorable. We studied the impact of different fertilizer regimens in an agricultural long-term observatory in Burkina Faso at three different plant development stages (early leaf development, flowering, and senescence) of sorghum cultivars. Using real-time PCR, we investigated functional microbial communities involved in key processes of the nitrogen cycle (nitrogen fixation, ammonia oxidation, and denitrification) in the rhizosphere. The results indicate that fertilizer treatments and plant development stages combined with environmental factors affected the abundance of the targeted functional genes in the rhizosphere. While nitrogen-fixing populations dominated the investigated communities when organic fertilizers (manure and straw) were applied, their numbers were comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing bacteria (AOB) increased not only in absolute numbers but also in relation to the other bacterial groups investigated in the urea-amended plots. Ammonia-oxidizing archaea exhibited higher numbers compared to AOB independent of fertilizer application. Similarly, denitrifiers were also more abundant in the urea-treated plots. Our data imply as well that, more than in moderate regions, water availability might shape microbial communities in the rhizosphere, since low gene abundance data were obtained for all tested genes at the flowering stage, when water availability was very limited. PMID:19502431

  8. [Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae].

    PubMed

    Zhang, Min; Jiang, Shaotong; Zheng, Juan; Zheng, Zhi; Li, Xingjiang; Pan, Lijun; Luo, Shuizhong

    2015-08-01

    To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.

  9. The role of the transformer gene in sex determination and reproduction in the tephritid fruit fly, Bactrocera dorsalis (Hendel)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transformer (tra) is a double-switch gene in the somatic sex-determination hierarchy that regulates sexual dimorphism based on RNA splicing in many insects. In tephritids, a Y-linked male determining gene (M) controls sex in the sex-determination pathway. Here, homologues of Drosophila tra and trans...

  10. Combining a regeneration-promoting ipt gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes.

    PubMed

    López-Noguera, Sonia; Petri, César; Burgos, Lorenzo

    2009-12-01

    The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted in the apricot cultivar 'Helena'. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants by site-specific recombination.

  11. [Expression analysis of a transformer gene in Daphnia pulex after RNAi].

    PubMed

    Guo, C Y; Chen, P; Zhang, M M; Ning, J J; Wang, С L; Wang, D L; Zhao, Y L

    2016-01-01

    In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

  12. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

    PubMed

    Weyda, István; Yang, Lei; Vang, Jesper; Ahring, Birgitte K; Lübeck, Mette; Lübeck, Peter S

    2017-04-01

    In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements.

  13. Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance

    PubMed Central

    Roehe, Rainer; Dewhurst, Richard J.; Duthie, Carol-Anne; Rooke, John A.; McKain, Nest; Ross, Dave W.; Hyslop, Jimmy J.; Waterhouse, Anthony; Freeman, Tom C.

    2016-01-01

    Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB) were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI) were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. human metabolism

  14. Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance.

    PubMed

    Roehe, Rainer; Dewhurst, Richard J; Duthie, Carol-Anne; Rooke, John A; McKain, Nest; Ross, Dave W; Hyslop, Jimmy J; Waterhouse, Anthony; Freeman, Tom C; Watson, Mick; Wallace, R John

    2016-02-01

    Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB) were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI) were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. human metabolism

  15. Biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen Cryptococcus neoformans.

    PubMed

    Taylor, Tonya; Bose, Indrani; Luckie, Taylor; Smith, Kerry

    2015-03-19

    The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide. Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only biolistic delivery provides an effective means to transform linear DNA that can be integrated into the genome by homologous recombination.  Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known. A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into C. neoformans. We then confirm integration of the ACK-mCherry fusion into the ACK locus.

  16. Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells

    SciTech Connect

    Yaswen, P.; Smoll, A.; Stampfer, M.R. ); Peehl, D.M. ); Trask, D.K.; Sager, R. )

    1990-10-01

    A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4 kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo({alpha})pyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to calmodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type {beta} increased its relative abundance. The protein encoded by NB-1 may have Ca{sup 2{sup plus}} binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined.

  17. Community structure and PAH ring-hydroxylating dioxygenase genes of a marine pyrene-degrading microbial consortium.

    PubMed

    Gallego, Sara; Vila, Joaquim; Tauler, Margalida; Nieto, José María; Breugelmans, Philip; Springael, Dirk; Grifoll, Magdalena

    2014-07-01

    Marine microbial consortium UBF, enriched from a beach polluted by the Prestige oil spill and highly efficient in degrading this heavy fuel, was subcultured in pyrene minimal medium. The pyrene-degrading subpopulation (UBF-Py) mineralized 31 % of pyrene without accumulation of partially oxidized intermediates indicating the cooperation of different microbial components in substrate mineralization. The microbial community composition was characterized by culture dependent and PCR based methods (PCR-DGGE and clone libraries). Molecular analyses showed a highly stable community composed by Alphaproteobacteria (84 %, Breoghania, Thalassospira, Paracoccus, and Martelella) and Actinobacteria (16 %, Gordonia). The members of Thalasosspira and Gordonia were not recovered as pure cultures, but five additional strains, not detected in the molecular analysis, that classified within the genera Novosphingobium, Sphingopyxis, Aurantimonas (Alphaproteobacteria), Alcanivorax (Gammaproteobacteria) and Micrococcus (Actinobacteria), were isolated. None of the isolates degraded pyrene or other PAHs in pure culture. PCR amplification of Gram-positive and Gram-negative dioxygenase genes did not produce results with any of the cultured strains. However, sequences related to the NidA3 pyrene dioxygenase present in mycobacterial strains were detected in UBF-Py consortium, suggesting the representative of Gordonia as the key pyrene degrader, which is consistent with a preeminent role of actinobacteria in pyrene removal in coastal environments affected by marine oil spills.

  18. Nitrification inhibition by hexavalent chromium Cr(VI)--Microbial ecology, gene expression and off-gas emissions.

    PubMed

    Kim, Young Mo; Park, Hongkeun; Chandran, Kartik

    2016-04-01

    The goal of this study was to investigate the responses in the physiology, microbial ecology and gene expression of nitrifying bacteria to imposition of and recovery from Cr(VI) loading in a lab-scale nitrification bioreactor. Exposure to Cr(VI) in the reactor strongly inhibited nitrification performance resulting in a parallel decrease in nitrate production and ammonia consumption. Cr(VI) exposure also led to an overall decrease in total bacterial concentrations in the reactor. However, the fraction of ammonia oxidizing bacteria (AOB) decreased to a greater extent than the fraction of nitrite oxidizing bacteria (NOB). In terms of functional gene expression, a rapid decrease in the transcript concentrations of amoA gene coding for ammonia oxidation in AOB was observed in response to the Cr(VI) shock. In contrast, transcript concentrations of the nxrA gene coding for nitrite oxidation in NOB were relatively unchanged compared to Cr(VI) pre-exposure levels. Therefore, Cr(VI) exposure selectively and directly inhibited activity of AOB, which indirectly resulted in substrate (nitrite) limitation to NOB. Significantly, trends in amoA expression preceded performance trends both during imposition of and recovery from inhibition. During recovery from the Cr(VI) shock, the high ammonia concentrations in the bioreactor resulted in an irreversible shift towards AOB populations, which are expected to be more competitive in high ammonia environments. An inadvertent impact during recovery was increased emission of nitrous oxide (N2O) and nitric oxide (NO), consistent with recent findings linking AOB activity and the production of these gases. Therefore, Cr(VI) exposure elicited multiple responses on the microbial ecology, gene expression and both aqueous and gaseous nitrogenous conversion in a nitrification process. A complementary interrogation of these multiple responses facilitated an understanding of both direct and indirect inhibitory impacts on nitrification.

  19. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior.

    PubMed

    Evans, Daniel S; Cline, Thomas W

    2013-11-19

    The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). Here we show instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. We inferred the existence of this unknown Sxl target from the observation that a constitutively feminizing tra transgene that restores fertility to tra(-) females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra(-). The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, we refer to this pathway as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, we find that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila.

  20. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    PubMed

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  1. Distribution of Microbial Arsenic Reduction, Oxidation and Extrusion Genes along a Wide Range of Environmental Arsenic Concentrations

    PubMed Central

    Escudero, Lorena V.; Casamayor, Emilio O.; Chong, Guillermo; Pedrós-Alió, Carles; Demergasso, Cecilia

    2013-01-01

    The presence of the arsenic oxidation, reduction, and extrusion genes arsC, arrA, aioA, and acr3 was explored in a range of natural environments in northern Chile, with arsenic concentrations spanning six orders of magnitude. A combination of primers from the literature and newly designed primers were used to explore the presence of the arsC gene, coding for the reduction of As (V) to As (III) in one of the most common detoxification mechanisms. Enterobacterial related arsC genes appeared only in the environments with the lowest As concentration, while Firmicutes-like genes were present throughout the range of As concentrations. The arrA gene, involved in anaerobic respiration using As (V) as electron acceptor, was found in all the systems studied. The As (III) oxidation gene aioA and the As (III) transport gene acr3 were tracked with two primer sets each and they were also found to be spread through the As concentration gradient. Sediment samples had a higher number of arsenic related genes than water samples. Considering the results of the bacterial community composition available for these samples, the higher microbial phylogenetic diversity of microbes inhabiting the sediments may explain the increased number of genetic resources found to cope with arsenic. Overall, the environmental distribution of arsenic related genes suggests that the occurrence of different ArsC families provides different degrees of protection against arsenic as previously described in laboratory strains, and that the glutaredoxin (Grx)-linked arsenate reductases related to Enterobacteria do not confer enough arsenic resistance to live above certain levels of As concentrations. PMID:24205341

  2. Distribution of microbial arsenic reduction, oxidation and extrusion genes along a wide range of environmental arsenic concentrations.

    PubMed

    Escudero, Lorena V; Casamayor, Emilio O; Chong, Guillermo; Pedrós-Alió, Carles; Demergasso, Cecilia

    2013-01-01

    The presence of the arsenic oxidation, reduction, and extrusion genes arsC, arrA, aioA, and acr3 was explored in a range of natural environments in northern Chile, with arsenic concentrations spanning six orders of magnitude. A combination of primers from the literature and newly designed primers were used to explore the presence of the arsC gene, coding for the reduction of As (V) to As (III) in one of the most common detoxification mechanisms. Enterobacterial related arsC genes appeared only in the environments with the lowest As concentration, while Firmicutes-like genes were present throughout the range of As concentrations. The arrA gene, involved in anaerobic respiration using As (V) as electron acceptor, was found in all the systems studied. The As (III) oxidation gene aioA and the As (III) transport gene acr3 were tracked with two primer sets each and they were also found to be spread through the As concentration gradient. Sediment samples had a higher number of arsenic related genes than water samples. Considering the results of the bacterial community composition available for these samples, the higher microbial phylogenetic diversity of microbes inhabiting the sediments may explain the increased number of genetic resources found to cope with arsenic. Overall, the environmental distribution of arsenic related genes suggests that the occurrence of different ArsC families provides different degrees of protection against arsenic as previously described in laboratory strains, and that the glutaredoxin (Grx)-linked arsenate reductases related to Enterobacteria do not confer enough arsenic resistance to live above certain levels of As concentrations.

  3. The structure of microbial evolutionary theory.

    PubMed

    Sapp, J

    2007-12-01

    The study of microbial phylogeny and evolution has emerged as an interdisciplinary synthesis, divergent in both methods and concepts from the classical evolutionary biology. The deployment of macromolecular sequencing in microbial classification has provided a deep evolutionary taxonomy hitherto deemed impossible. Microbial phylogenetics has greatly transformed the landscape of evolutionary biology, not only in revitalizing the field in the pursuit of life's history over billions of years, but also in transcending the structure of thought that has shaped evolutionary theory since the time of Darwin. A trio of primary phylogenetic lineages, along with the recognition of symbiosis and lateral gene transfer as fundamental processes of evolutionary innovation, are core principles of microbial evolutionary biology today. Their scope and significance remain contentious among evolutionists.

  4. Involvement and interaction of microbial communities in the transformation and stabilization of chromium during the composting of tannery effluent treated biomass of Vallisneria spiralis L.

    PubMed

    Shukla, O P; Rai, U N; Dubey, Smita

    2009-04-01

    Tannery effluent treated with aquatic macrophyte Vallisneria spiralis L. for 14 d showed significant improvement in physico-chemical properties and reduction in Cr concentration. Accumulation of Cr was found maximum in roots (358 microg g(-1)dw) as compared to shoot (62 microg g(-1)dw) of the plant. A laboratory scale composter was designed with the objectives to investigate the physico-chemical changes and role of microbes in stabilization and transformation of Cr in the composting material. Results revealed that the composting process was quick within 7-21 d as indicated by peak time for various physico-chemical parameters and drop in C/N ratio up to acceptable limit. The profile of microbial communities indicated that population of anaerobic, aerobic and nitrifying bacteria increased quickly at the initial phase, and reached a peak level of 4.2 x 10(6), 9.78 x 10(8) and 9.32 x 10(9) CFU g(-1), respectively at 21 d; while population of actinomycetes and fungi was found maximum i.e. 3.29 x 10(7) and 9.7 x 10(6) CFU g(-1), respectively, after 35 d of composting. Overall bacterial population dominated over the actinomycetes and fungi during the composting process. Cr((VI)) was transformed to Cr((III)) due to the microbial activity during the process. Sequential extraction of Cr fractionation showed its stabilization via changing into organic matter-bound and residual fractions during the composting.

  5. Microbial transformation of dissolved organic matter from different sources and its influence on disinfection byproduct formation potentials.

    PubMed

    Hur, Jin; Lee, Mi-Hee; Song, Hocheol; Schlatman, Mark A

    2013-06-01

    Biodegradation-induced changes in the characteristics of dissolved organic matter (DOM) and the subsequent effects on disinfection byproduct formation potentials (DBPFPs) were investigated using six different sources of DOM (algae, leaf litter, reed, compost, paddy water, and treated municipal sewage effluent). Microbial incubation of the DOM samples increased the specific ultraviolet absorbance and humic-like fluorescence but decreased the protein/tannin-like fluorescence and relative distribution of smaller-sized DOM components. Comparison of the original versus biodegraded DOM samples using resin fractionation and pyrolysis-gas chromatography/mass spectrometry revealed that the biodegradation-induced changes were highly dependent on DOM sources and exhibited no consistent trends among the different sources. Changes in DBPFPs also differed with DOM source. Vascular plant-derived DOM (leaf litter and reed) demonstrated an enhancement in specific DBPFP after biodegradation, whereas little change or even a slight decrease was observed for the other DOM sources. Correlations that were significant between specific DBPFPs and the aromatic content or humic-like fluorescence for the original DOM samples were no longer significant after microbial degradation. The relative abundance of hydrophobic to hydrophilic structures in DOM is suggested to be a general indicator for the formation potential of trihalomethanes irrespective of DOM source and the state of biodegradation.

  6. Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

    PubMed Central

    Vallone, D; Battista, S; Pierantoni, G M; Fedele, M; Casalino, L; Santoro, M; Viglietto, G; Fusco, A; Verde, P

    1997-01-01

    The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation. PMID:9311991

  7. Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil.

    PubMed

    Sawulski, Przemyslaw; Clipson, Nicholas; Doyle, Evelyn

    2014-11-01

    Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial

  8. Comparative Metagenomics of Gut and Ocean: Identification of Microbial Marker Genes for Complex Environmental Properties(2011 JGI User Meeting)

    ScienceCinema

    Bork, Peer

    2016-07-12

    The U.S. Department of Energy Joint Genome Institute (JGI) invited scientists interested in the application of genomics to bioenergy and environmental issues, as well as all current and prospective users and collaborators, to attend the annual DOE JGI Genomics of Energy & Environment Meeting held March 22-24, 2011 in Walnut Creek, Calif. The emphasis of this meeting was on the genomics of renewable energy strategies, carbon cycling, environmental gene discovery, and engineering of fuel-producing organisms. The meeting features presentations by leading scientists advancing these topics. Peer Bork of the European Molecular Biology Laboratory on "Comparative Metagenomics of Gut and Ocean: Identification of Microbial Marker Genes for Complex Environmental Properties" at the 6th annual Genomics of Energy & Environment Meeting on March 23, 2011

  9. Identification of genes associated with asexual reproduction in Phyllosticta citricarpa mutants obtained through Agrobacterium tumefaciens transformation.

    PubMed

    Goulin, Eduardo Henrique; Savi, Daiani Cristina; Petters, Desirrê Alexia Lourenço; Kava, Vanessa; Galli-Terasawa, Lygia; Silva, Geraldo José; Glienke, Chirlei

    2016-11-01

    Phyllosticta citricarpa is the epidemiological agent of Citrus Black Spot (CBS) disease, which is responsible for large economic losses worldwide. CBS is characterized by the presence of spores (pycnidiospores) in dark lesions of fruit, which are also responsible for short distance dispersal of the disease. The identification of genes involved in asexual reproduction of P. citricarpa can be an alternative for directional disease control. We analyzed a library of mutants obtained through Agrobacterium tumefaciens transformation system, looking for alterations in growth and reproductive structure formation. Two mutant strains were found to have lost the ability to form pycnidia. The flanking T-DNA insertion regions were identified on P. citricarpa genome by using blast analysis and further gene prediction. The predicted genes containing the T-DNA insertions were identified as Spindle Poison Sensitivity Scp3, Ion Transport protein, and Cullin Binding proteins. The Ion Transport and Cullin Binding proteins are known to be correlated with sexual and asexual reproduction in fungi; however, the exact mechanism by which these proteins act on spore formation in P. citricarpa needs to be better characterized. The Scp3 proteins are suggested here for the first time as being associated with asexual reproduction in fungus. This protein is associated with microtubule formation, and as microtubules play an essential role as spindle machinery for chromosome segregation and cytokinesis, insertions in this gene can lead to abnormal formations, such as that observed here in P. citricarpa. We suggest these genes as new targets for fungicide development and CBS disease control, by iRNA.

  10. Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation.

    PubMed

    Price, Alexander M; Luftig, Micah A

    2014-01-01

    Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses.

  11. Effect Of Spaceflight On Microbial Gene Expression And Virulence: Preliminary Results From Microbe Payload Flown On-Board STS-115

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.; HonerzuBentrup, K,; Schurr, M. J.; Buchanan, K.; Morici, L.; Hammond, T.; Allen, P.; Baker, C.; Ott, C. M.; Nelman-Gonzalez M.; Schurr, J. R.; Pierson, D. L.; Stodieck, L.; Hing, S.; Hammond, T.; Allen, P.; Baker, C.; Parra, M.; Dumars, P.; Stefanyshyn-Piper, H. M.; Nickerson, C. A.

    2007-01-01

    Human presence in space, whether permanent or temporary, is accompanied by the presence of microbes. However, the extent of microbial changes in response to spaceflight conditions and the corresponding changes to infectious disease risk is unclear. Previous studies have indicated that spaceflight weakens the immune system in humans and animals. In addition, preflight and in-flight monitoring of the International Space Station (ISS) and other spacecraft indicates the presence of opportunistic pathogens and the potential of obligate pathogens. Altered antibiotic resistance of microbes in flight has also been shown. As astronauts and cosmonauts live for longer periods in a closed environment, especially one using recycled water and air, there is an increased risk to crewmembers of infectious disease events occurring in-flight. Therefore, understanding how the space environment affects microorganisms and their disease potential is critically important for spaceflight missions and requires further study. The goal of this flight experiment, operationally called MICROBE, is to utilize three model microbial pathogens, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans to examine the global effects of spaceflight on microbial gene expression and virulence attributes. Specifically, the aims are (1) to perform microarray-mediated gene expression profiling of S. typhimurium, P. aeruginosa, and C. albicans, in response to spaceflight in comparison to ground controls and (2) to determine the effect of spaceflight on the virulence potential of these microorganisms immediately following their return from spaceflight using murine models. The model microorganisms were selected as they have been isolated from preflight or in-flight monitoring, represent different degrees of pathogenic behavior, are well characterized, and have sequenced genomes with available microarrays. In particular, extensive studies of S. typhimurium by the Principal Investigator, Dr. Nickerson

  12. Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation

    PubMed Central

    Dilshad, Erum; Cusido, Rosa Maria; Estrada, Karla Ramirez; Bonfill, Mercedes; Mirza, Bushra

    2015-01-01

    The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01–0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism. PMID:26444558

  13. Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-Gene Library

    DTIC Science & Technology

    2010-01-01

    tetracycline reistance ) gene to integrate into the antibiotic resistance gene present in the parent strain library for the 3rd and 4th rounds of DNA...is used as a generic desirable trait to provide a proof of the concept). An antibiotic gene marker was inserted into the E. coli chromosome to be...used as a target for homologous integration of a second antibiotic gene marker along with ~ 3 kb L. pl DNA fragments. The second antibiotic gene

  14. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

    PubMed Central

    Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

    2014-01-01

    Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important

  15. Psoriasis: new insight about pathogenesis, role of barrier organ integrity, NLR / CATERPILLER family genes and microbial flora.

    PubMed

    Mattozzi, Carlo; Richetta, Antonio G; Cantisani, Carmen; Macaluso, Laura; Calvieri, Stefano

    2012-09-01

    Psoriasis is a common, inflammatory, chronic, relapsing skin disease. New insight about the etiology of this disease shows the important role played by the epidermal barrier function, its integrity and pathogen responses in combination with microbial environmental factors. A pivotal role in the management of this balance is played by NLR genes, also known as NBD-LRR or CATERPILLER, that encode important mediators of innate immunity and provide the first line of defense against pathogens. The polymorphism of these genes is implicated in the pathogenesis of several immunological diseases and might be of importance in the pathogenesis of barrier organ disorders. Crohn's disease is considered archetypal of these kinds of disorders; similarities between Crohn's disease and psoriasis and their similar pathogenetic mechanisms may support the concept of psoriasis as a barrier organ disorder and common genetic ground lying behind these illnesses. Considering psoriasis as a "barrier organ disease" is not only a mere mental exercise; this consideration may, in fact, open new prospects in the treatment of these disorders just by preventing alterations of microbial flora or regulating the response of the host to infective diseases.

  16. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    PubMed

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  17. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    PubMed Central

    Fischer, Sandra; Kumar, Neeraj

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea. PMID:28097056

  18. Mechanisms and effects of arsanilic acid on antibiotic resistance genes and microbial communities during pig manure digestion.

    PubMed

    Sun, Wei; Qian, Xun; Gu, Jie; Wang, Xiao-Juan; Zhang, Li; Guo, Ai-Yun

    2017-03-08

    High concentrations of residual arsanilic acid occur in pig manure due to its use in feed to promote growth and control diseases. This study compared the effects of arsanilic acid at three concentrations (0, 325, and 650mg/kg dry pig manure) on the abundance of antibiotic resistance genes (ARGs) and the microbial community during anaerobic digestion. Addition of 650mg/kg arsanilic acid enhanced the absolute abundances of tetC, sul2, ermB, and gyrA more than twofold in the digestion product. Redundancy analysis indicated that the change in the microbial community structure was the main driver of variation in the ARGs profile. The As resistance gene arsC co-occurred with four ARGs and intI1, possibly causing the increase in ARGs under pressure by arsanilic acid. High arsanilic acid concentrations can increase the risk of ARGs occurring in anaerobic digestion products. The amount of arsanilic acid used as a feed additive should be controlled.

  19. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

    PubMed

    Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status.

  20. Dynamics of antibiotic resistance genes in microbial fuel cell-coupled constructed wetlands treating antibiotic-polluted water.

    PubMed

    Zhang, Shuai; Song, Hai-Liang; Yang, Xiao-Li; Huang, Shan; Dai, Zhe-Qin; Li, Hua; Zhang, Yu-Yue

    2017-07-01

    Microbial fuel cell-coupled constructed wetlands (CW-MFCs) use electrochemical, biological, and ecological functions to treat wastewater. However, few studies have investigated the risks of antibiotic resistance genes (ARGs) when using such systems to remove antibiotics. Therefore, three CW-MFCs were designed to assess the dynamics of ARGs in filler biofilm and effluent over 5000 h of operation. The experimental results indicated that relatively high steady voltages of 605.8 mV, 613.7 mV, and 541.4 mV were obtained at total influent antibiotic concentrations of 400, 1,000, and 1600 μg L(-1), respectively. The 16S rRNA gene level in the cathode layer was higher than those in the anode and two middle layers, but the opposite trend was observed for the sul and tet genes. The relative abundance of the three tested sul genes were in the order sulI > sulII > sulIII, and those of the five tet genes were in the order tetA > tetC > tetW > tetO > tetQ. The levels of sul and tet genes in the media biofilm showed an increase over the treatment period. The effluent water had relatively low abundances of sul and tet genes compared with the filler biofilm. No increases were observed for most ARGs over the treatment period, and no significant correlations were observed between the ARGs and 16S rRNA gene copy numbers, except for sulI and tetW in the effluent. However, significant correlations were observed among most of the ARG copy numbers.

  1. Chronic exposure to triclosan sustains microbial community shifts and alters antibiotic resistance gene levels in anaerobic digesters.

    PubMed

    Carey, Daniel E; Zitomer, Daniel H; Kappell, Anthony D; Choi, Melinda J; Hristova, Krassimira R; McNamara, Patrick J

    2016-08-10

    Triclosan, an antimicrobial chemical found in consumer personal care products, has been shown to stimulate antibiotic resistance in pathogenic bacteria. Although many studies focus on antibiotic resistance pertinent to medical scenarios, resistance developed in natural and engineered environments is less studied and has become an emerging concern for human health. In this study, the impacts of chronic triclosan (TCS) exposure on antibiotic resistance genes (ARGs) and microbial community structure were assessed in lab-scale anaerobic digesters. TCS concentrations from below detection to 2500 mg kg(-1) dry solids were amended into anaerobic digesters over 110 days and acclimated for >3 solid retention time values. Four steady state TCS concentrations were chosen (30-2500 mg kg(-1)). Relative abundance of mexB, a gene coding for a component of a multidrug efflux pump, was significantly higher in all TCS-amended digesters (30 mg kg(-1) or higher) relative to the control. TCS selected for bacteria carrying tet(L) and against those carrying erm(F) at concentrations which inhibited digester function; the pH decrease associated with digester failure was suspected to cause this selection. Little to no impact of TCS was observed on intI1 relative abundance. Microbial communities were also surveyed by high-throughput 16S rRNA gene sequencing. Compared to the control digesters, significant shifts in community structure towards clades containing commensal and pathogenic bacteria were observed in digesters containing TCS. Based on these results, TCS should be included in studies and risk assessments that attempt to elucidate relationships between chemical stressors (e.g. antibiotics), antibiotic resistance genes, and public health.

  2. A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass, precinct vegetation and the microbial community.

    PubMed

    Oguchi, Taichi; Kashimura, Yuko; Mimura, Makiko; Yu, Xiang; Matsunaga, Etsuko; Nanto, Kazuya; Shimada, Teruhisa; Kikuchi, Akira; Watanabe, Kazuo N

    2014-10-01

    A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial.

  3. CopyRighter: a rapid tool for improving the accuracy of microbial community profiles through lineage-specific gene copy number correction

    PubMed Central

    2014-01-01

    Background Culture-independent molecular surveys targeting conserved marker genes, most notably 16S rRNA, to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species. Results Based on 2,900 sequenced reference genomes, we show that 16S rRNA gene copy number (GCN) is strongly linked to microbial phylogenetic taxonomy, potentially under-representing Archaea in amplicon microbial profiles. Using this relationship, we inferred the GCN of all bacterial and archaeal lineages in the Greengenes database within a phylogenetic framework. We created CopyRighter, new software which uses these estimates to correct 16S rRNA amplicon microbial profiles and associated quantitative (q)PCR total abundance. CopyRighter parses microbial profiles and, because GCN estimates are pre-computed for all taxa in the reference taxonomy, rapidly corrects GCN bias. Software validation with in silico and in vitro mock communities indicated that GCN correction results in more accurate estimates of microbial relative abundance and improves the agreement between metagenomic and amplicon profiles. Analyses of human-associated and anaerobic digester microbiomes illustrate that correction makes tangible changes to estimates of qPCR total abundance, α and β diversity, and can significantly change biological interpretation. For example, human gut microbiomes from twins were reclassified into three rather than two enterotypes after GCN correction. Conclusions The CopyRighter bioinformatic tools permits rapid correction of GCN in microbial surveys, resulting in improved estimates of microbial abundance, α and β diversity. PMID:24708850

  4. Insights into functional genes and taxonomical/phylogenetic diversity of microbial communities in biological heap leaching system and their correlation with functions.

    PubMed

    Xiao, Yunhua; Liu, Xueduan; Liang, Yili; Niu, Jiaojiao; Zhang, Xian; Ma, Liyuan; Hao, Xiaodong; Gu, Yabin; Yin, Huaqun

    2016-11-01

    Although the taxonomical/phylogenetic diversity of microbial communities in biological heap leaching systems has been investigated, the diversity of functional genes was still unclear, and, especially, the differentiation and the relationships of diversity and functions of microbial communities in leaching heap (LH) and leaching solution (LS) were also still unclear. In our study, a functional gene array (GeoChip 5.0) was employed to investigate the functional gene diversity, and 16S rRNA gene sequencing was used to explore the taxonomical/phylogenetic diversity of microbial communities in LH and LS subsystems of Dexing copper mine (Jiangxi, China). Detrended correspondence analysis (DCA) showed that both functional gene structure and taxonomical/phylogenetic structure of microbial communities were significantly different between LH and LS. Signal intensities of genes, including genes for sulfur oxidation (e.g., soxB), metal homeostasis (e.g., arsm), carbon fixation (e.g., rubisco), polyphosphate degradation (e.g., ppk), and organic remediation (e.g., hydrocarbons) were significantly higher in LH, while signal intensities of genes for carbon degradation (e.g., amyA), polyphosphate synthesis (e.g., ppx), and sulfur reduction (e.g., dsrA) were significantly higher in LS. Further inspection revealed that microbial communities in LS and LH were dominated by Acidithiobacillus and Leptospirillum. However, rare species were relatively higher abundant in LH. Additionally, diversity index of functional genes was significantly different in LS (9.915 ± 0.074) and LH (9.781 ± 0.165), and the taxonomical/phylogenetic diversity index was also significantly different in LH (4.398 ± 0.508) and LS (3.014 ± 0.707). Functional tests, including sulfur-oxidizing ability, iron-oxidizing ability, and pyrite bioleaching ability, showed that all abilities of microbial communities were significantly stronger in LH than those in LS. Further studies found that most key genes (e

  5. Arsenic(V) Reduction in Relation to Iron(III) Transformation and Molecular Characterization of the Structural and Functional Microbial Community in Sediments of a Basin-Fill Aquifer in Northern Utah

    PubMed Central

    Mirza, Babur S.; Muruganandam, Subathra; Meng, Xianyu; Sorensen, Darwin L.; Dupont, R. Ryan

    2014-01-01

    Basin-fill aquifers of the Southwestern United States are associated with elevated concentrations of arsenic (As) in groundwater. Many private domestic wells in the Cache Valley Basin, UT, have As concentrations in excess of the U.S. EPA drinking water limit. Thirteen sediment cores were collected from the center of the valley at the depth of the shallow groundwater and were sectioned into layers based on redoxmorphic features. Three of the layers, two from redox transition zones and one from a depletion zone, were used to establish microcosms. Microcosms were treated with groundwater (GW) or groundwater plus glucose (GW+G) to investigate the extent of As reduction in relation to iron (Fe) transformation and characterize the microbial community structure and function by sequencing 16S rRNA and arsenate dissimilatory reductase (arrA) genes. Under the carbon-limited conditions of the GW treatment, As reduction was independent of Fe reduction, despite the abundance of sequences related to Geobacter and Shewanella, genera that include a variety of dissimilatory iron-reducing bacteria. The addition of glucose, an electron donor and carbon source, caused substantial shifts toward domination of the bacterial community by Clostridium-related organisms, and As reduction was correlated with Fe reduction for the sediments from the redox transition zone. The arrA gene sequencing from microcosms at day 54 of incubation showed the presence of 14 unique phylotypes, none of which were related to any previously described arrA gene sequence, suggesting a unique community of dissimilatory arsenate-respiring bacteria in the Cache Valley Basin. PMID:24632255

  6. Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice.

    PubMed

    Zhao, Feng; Li, Qiuying; Weng, Manli; Wang, Xiuliang; Guo, Baotai; Wang, Li; Wang, Wei; Duan, Delin; Wang, Bin

    2013-12-01

    The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice.

  7. Effects of microbial transformation on dissolved organic matter in the east Taiwan Strait and implications for carbon and nutrient cycling

    NASA Astrophysics Data System (ADS)

    Yang, Liyang; Chen, Chen-Tung Arthur; Lui, Hon-Kit; Zhuang, Wan-E.; Wang, Bing-Jye

    2016-10-01

    Dissolved inorganic and organic carbons (DIC and DOC) provide two of the largest pools of carbon in the ocean. However, limited information is available concerning the relationship between DIC and different constituents of dissolved organic matter (DOM), such as fluorescent compounds. This study investigates the dynamics of DOM and their implications for carbon and nutrient cycling in the east Taiwan Strait, using DOC, absorption spectroscopy, and fluorescence excitation-emission matrices-parallel factor analysis (EEM-PARAFAC). The study area was dominated by the waters from the South China Sea during the sampling period in summer 2013. The dynamics of DOM were influenced strongly by microbial activities, as indicated by the close correlations (the absolute value of r: 0.75-0.97, p < 0.001) between apparent oxygen utilization (AOU) and DOM parameters, including DOC, the absorption coefficient at 280 nm, the fluorescence intensity of protein-like component C3, and the humification index HIX. The contribution of DOC degradation to the net increase in DIC was approximately 15% and 21% in the north and the south of the east Taiwan Strait, respectively. The DIC was correlated negatively with protein-like fluorescence, revealing the production of DIC by the microbial degradation of labile components. The DIC was correlated positively with humic-like fluorescence and HIX, suggesting that the storage of carbon by produced refractory humic substances could not compensate for the release of DIC in the deeper waters. The correlations of nutrients with DOM parameters were similar to those of DIC, further indicating the profound impacts of the dynamics of labile DOM on nutrient cycling.

  8. [Construction of the plant expression vector with hepatitis a capsid protein fusion gene and genetic transformation of Citrus. Sinensis Osbeck].

    PubMed

    Hu, Rong; Wei, Hong; Chen, Shan-Chun; He, Yong-Rui

    2004-07-01

    The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments.13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.

  9. The Underlying Bases of Gene Expression Differences in Stable Transformants of the ROSY Locus in DROSOPHILA MELANOGASTER

    PubMed Central

    Daniels, Stephen B.; McCarron, Margaret; Love, Carol; Clark, Stephen H.; Chovnick, Arthur

    1986-01-01

    This report represents a continuation of our laboratory's effort to understand the major phenomena associated with P-M dysgenesis-mediated transformation in Drosophila. A group of stable transformants are characterized with respect to rosy gene expression. Stable, true-breeding, line-specific variants in gene expression are described. These are shown to be associated with single transposons present in each line, and the lines are free of functional P elements. The effects on expression are cis-acting, and there are no identifiable rosy DNA sequence lesions associated with these transposons. Evidence is presented that demonstrates that two features of the transformation experimental system are responsible for such variation. The first relates to the fact that the transposons insert at numerous genomic sites. Both heterochromatic and euchromatic position effects are characterized. The second relates to the fact that transformation involves dysgenic mobilization of a P-element transposon. This process is mutagenic, and such a mutation is characterized. PMID:3013723

  10. A Bayesian Framework for the Classification of Microbial Gene Activity States

    PubMed Central

    Disselkoen, Craig; Greco, Brian; Cook, Kaitlyn; Koch, Kristin; Lerebours, Reginald; Viss, Chase; Cape, Joshua; Held, Elizabeth; Ashenafi, Yonatan; Fischer, Karen; Acosta, Allyson; Cunningham, Mark; Best, Aaron A.; DeJongh, Matthew; Tintle, Nathan

    2016-01-01

    Numerous methods for classifying gene activity states based on gene expression data have been proposed for use in downstream applications, such as incorporating transcriptomics data into metabolic models in order to improve resulting flux predictions. These methods often attempt to classify gene activity for each gene in each experimental condition as belonging to one of two states: active (the gene product is part of an active cellular mechanism) or inactive (the cellular mechanism is not active). These existing methods of classifying gene activity states suffer from multiple limitations, including enforcing unrealistic constraints on the overall proportions of active and inactive genes, failing to leverage a priori knowledge of gene co-regulation, failing to account for differences between genes, and failing to provide statistically meaningful confidence estimates. We propose a flexible Bayesian approach to classifying gene activity states based on a Gaussian mixture model. The model integrates genome-wide transcriptomics data from multiple conditions and information about gene co-regulation to provide activity state confidence estimates for each gene in each condition. We compare the performance of our novel method to existing methods on both simulated data and real data from 907 E. coli gene expression arrays, as well as a comparison with experimentally measured flux values in 29 conditions, demonstrating that our method provides more consistent and accurate results than existing methods across a variety of metrics. PMID:27555837

  11. Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems.

    PubMed

    Li, Xiang; Harwood, Valerie J; Nayak, Bina; Weidhaas, Jennifer L

    2016-01-04

    Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.

  12. Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

    PubMed Central

    Li, Xiang; Harwood, Valerie J.; Nayak, Bina

    2016-01-01

    Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. PMID:26729716

  13. Association between shortage of energy supply and nuclear gene mutations leading to carcinomatous transformation.

    PubMed

    DU, Jianping

    2016-01-01

    Anaerobic bacteria use glycolysis, an oxygen-independent metabolic pathway, whereas energy metabolism in the evolved eukaryotic cell is performed via oxidative phosphorylation, with all eukaryotic cell activities depending upon high energy consumption. However, in cancer cells evolving from eukaryotic cells, the energy metabolism switches from oxidative phosphorylation to glycolysis. The shortage of energy supply induces cancer cells to acquire specific characteristics. Base pair renewal is the most energy-consuming process in the cell, and shortage of energy supply may lead to errors in this process; the more prominent the shortage in energy supply, the more errors are likely to occur in base pair renewal, resulting in gene mutations and expression of cancer cell characteristics. Thus, shortage of energy supply is associated with carcinomatous transformation.

  14. Gene Expression Changes during the Development of Acute Lung Injury Role of Transforming Growth Factor β

    PubMed Central

    Wesselkamper, Scott C.; Case, Lisa M.; Henning, Lisa N.; Borchers, Michael T.; Tichelaar, Jay W.; Mason, John M.; Dragin, Nadine; Medvedovic, Mario; Sartor, Maureen A.; Tomlinson, Craig R.; Leikauf, George D.

    2005-01-01

    Rationale: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain. Objectives: To integrate molecular pathways and investigate the role of transforming growth factor β (TGF-β) in acute lung injury in mice. Methods: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-β1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-β, was assessed in nickel-exposed mice. The effect of TGF-β on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells. Measurements and Main Results: Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-β signaling. MAPPFinder analysis further established TGF-β signaling to be significantly altered. TGF-β–inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-β1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-β attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-β1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-β–responsive region in the Sftpb promoter was identified. Conclusions: These data suggest that TGF-β acts as a central mediator of acute lung injury through the alteration of several different molecular pathways. PMID:16100012

  15. Discovery of the faithfulness gene: a model of transmission and transformation of scientific information.

    PubMed

    Green, Eva G T; Clémence, Alain

    2008-09-01

    The purpose of this paper is to study the diffusion and transformation of scientific information in everyday discussions. Based on rumour models and social representations theory, the impact of interpersonal communication and pre-existing beliefs on transmission of the content of a scientific discovery was analysed. In three experiments, a communication chain was simulated to investigate how laypeople make sense of a genetic discovery first published in a scientific outlet, then reported in a mainstream newspaper and finally discussed in groups. Study 1 (N=40) demonstrated a transformation of information when the scientific discovery moved along the communication chain. During successive narratives, scientific expert terminology disappeared while scientific information associated with lay terminology persisted. Moreover, the idea of a discovery of a faithfulness gene emerged. Study 2 (N=70) revealed that transmission of the scientific message varied as a function of attitudes towards genetic explanations of behaviour (pro-genetics vs. anti-genetics). Pro-genetics employed more scientific terminology than anti-genetics. Study 3 (N=75) showed that endorsement of genetic explanations was related to descriptive accounts of the scientific information, whereas rejection of genetic explanations was related to evaluative accounts of the information.

  16. Differential regulation of papilloma virus early gene expression in transformed fibroblasts and carcinoma cell lines.

    PubMed Central

    Kleiner, E; Dietrich, W; Pfister, H

    1986-01-01

    Treatment of bovine papilloma virus (BPV) 1-transformed mouse fibroblasts with cycloheximide led to a 10-fold increase in the amount of viral transcripts, after as little as 1 h of protein synthesis inhibition. Northern blots revealed no qualitative changes in the RNA pattern. Nuclear run-on experiments showed about a 7-fold increase in specific transcriptional activity after cycloheximide treatment. The half-life of BPV1 mRNA was twice as long as in untreated controls. These results indicate that both RNA synthesis and degradation of viral RNA are controlled by labile proteins. Cycloheximide stimulation turned out to be independent of the BPV1 E2 gene activity which enhances viral transcription. Cycloheximide treatment had no effect on the amount of human papilloma virus (HPV) 18 transcripts in cervical carcinoma derived HeLa and C4-1 cells. Transcription of HPV16 in the cervical carcinoma line SiHa was likewise unaffected. The differential regulation of transcription in transformed fibroblasts and cancer-derived cells, and the significance for malignant conversion are discussed. Images Fig. 1. Fig. 3. Fig. 4. Fig. 6. PMID:3019673

  17. Global analysis of gene expression dynamics within the marine microbial community during the VAHINE mesocosm experiment in the southwest Pacific

    NASA Astrophysics Data System (ADS)

    Pfreundt, Ulrike; Spungin, Dina; Bonnet, Sophie; Berman-Frank, Ilana; Hess, Wolfgang R.

    2016-07-01

    Microbial gene expression was followed for 23 days within a mesocosm (M1) isolating 50 m3 of seawater and in the surrounding waters in the Nouméa lagoon, New Caledonia, in the southwest Pacific as part of the VAriability of vertical and tropHIc transfer of diazotroph derived N in the south wEst Pacific (VAHINE) experiment. The aim of VAHINE was to examine the fate of diazotroph-derived nitrogen (DDN) in a low-nutrient, low-chlorophyll ecosystem. On day 4 of the experiment, the mesocosm was fertilized with phosphate. In the lagoon, gene expression was dominated by the cyanobacterium Synechococcus, closely followed by Alphaproteobacteria. In contrast, drastic changes in the microbial community composition and transcriptional activity were triggered within the mesocosm within the first 4 days, with transcription bursts from different heterotrophic bacteria in rapid succession. The microbial composition and activity of the surrounding lagoon ecosystem appeared more stable, although following similar temporal trends as in M1. We detected significant gene expression from Chromerida in M1, as well as the Nouméa lagoon, suggesting these photoautotrophic alveolates were present in substantial numbers in the open water. Other groups contributing substantially to the metatranscriptome were affiliated with marine Euryarchaeota Candidatus Thalassoarchaea (inside and outside) and Myoviridae bacteriophages likely infecting Synechococcus, specifically inside M1. High transcript abundances for ammonium transporters and glutamine synthetase in many different taxa (e.g., Pelagibacteraceae, Synechococcus, Prochlorococcus, and Rhodobacteraceae) was consistent with the known preference of most bacteria for this nitrogen source. In contrast, Alteromonadaceae highly expressed urease genes; Rhodobacteraceae and Prochlorococcus showed some urease expression, too. Nitrate reductase transcripts were detected on day 10 very prominently in Synechococcus and in Halomonadaceae. Alkaline

  18. Genetic Transformation and Analysis of Rice OsAPx2 Gene in Medicago sativa

    PubMed Central

    Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

    2012-01-01

    The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H2O2)concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H2O2 content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H2O2, sodium sulfite (Na2SO3), and sodium bicarbonate (NaHCO3). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H2O2 and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained. PMID:22848448

  19. Genetic transformation and analysis of rice OsAPx2 gene in Medicago sativa.

    PubMed

    Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

    2012-01-01

    The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H(2)O(2))concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H(2)O(2) content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H(2)O(2), sodium sulfite (Na(2)SO(3)), and sodium bicarbonate (NaHCO(3)). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H(2)O(2) and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained.

  20. Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit (Actinidia deliciosa cv. Hayward).

    PubMed

    Kim, Misun; Kim, Seong-Cheol; Song, Kwan Jeong; Kim, Ho Bang; Kim, In-Jung; Song, Eun-Young; Chun, Seung-Jong

    2010-12-01

    Genetic transformation using a micro-cross section (MCS) technique was conducted to improve the carotenoid content in kiwifruit (Actinidia deliciosa cv. Hayward). The introduced carotenoid biosynthetic genes include geranylgeranyl diphosphate synthase (GGPS), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), β-carotene hydroxylase (CHX), and phytoene synthase (PSY). The transformed explants were selected on half-strength MS medium containing 0.001 mg l(-1) of 2,4-D and 0.1 mg l(-1) of zeatin, either 5 mg l(-1) hygromycin or 25 mg l(-1) kanamycin, and 500 mg l(-1) cefotaxime. The genomic PCR, genomic Southern blot analysis, and RT-PCR were performed to confirm the integration and expression of the transgenes. The transformation efficiencies of either kanamycin- or hygromycin-resistant shoots ranged from 2.9 to 22.1% depending on the target genes, and from 2.9 to 24.2% depending on the reporter genes. The selection efficiencies ranged from 66.7 to 100% for the target genes and from 95.8 to 100% for the reporter genes. Changes of carotenoid content in the several PCR-positive plants were determined by UPLC analysis. As a result, transgenic plants expressing either GGPS or PSY increased about 1.2- to 1.3-fold in lutein or β-carotene content compared to non-transgenic plants. Our results suggest that the Agrobacterium-mediated transformation efficiency of kiwifruit can be greatly increased by this MCS method and that the carotenoid biosynthetic pathway can be modified in kiwifruit by genetic transformation. Our results further suggest that GGPS and PSY genes could be major target genes to increase carotenoid contents in kiwifruit.

  1. Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model

    PubMed Central

    Danielsson, Frida; Skogs, Marie; Huss, Mikael; Rexhepaj, Elton; O’Hurley, Gillian; Klevebring, Daniel; Pontén, Fredrik; Gad, Annica K. B.; Uhlén, Mathias; Lundberg, Emma

    2013-01-01

    The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that ∼6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation. PMID:23569271

  2. Cellular RNA homologous to the Abelson murine leukemia virus transforming gene: expression and relationship to the viral sequence.

    PubMed Central

    Wang, J Y; Baltimore, D

    1983-01-01

    To examine the expression of the cellular homolog of the Abelson murine leukemia virus transforming gene (the v-abl sequence), a DNA probe representing the v-abl sequence was prepared. The probe detected two cytoplasmic polyadenylic acid-containing c-abl RNAs of about 6.5 and 5.5 kilobases in a variety of rodent cells, and slightly larger RNAs were detected in human cells. These two RNA species were found in all normal tissues or cell lines examined, but at differing concentrations: liver cells had the least, fibroblastic cell lines had the most. By using a probe able to detect the cellular but not the viral gene, the two RNAs were shown to be present in Abelson murine leukemia virus-transformed cells at levels found either in their untransformed counterparts or in similar cell types transformed by other means. The target cells of the virus have a somewhat elevated level of the two RNAs although expression of the c-abl gene is not restricted to these cells. The v-abl sequence lacks 0.35 and 0.85 kilobases of the c-abl RNA on the 5' and 3' ends, respectively. Thus, the Abelson murine leukemia virus transforming gene is an internal fragment of the transcript of a normal cellular gene. Images PMID:6306446

  3. Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model.

    PubMed

    Danielsson, Frida; Skogs, Marie; Huss, Mikael; Rexhepaj, Elton; O'Hurley, Gillian; Klevebring, Daniel; Pontén, Fredrik; Gad, Annica K B; Uhlén, Mathias; Lundberg, Emma

    2013-04-23

    The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that ∼6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.

  4. Construction of efficient and effective transformation vectors for palmitoyl-acyl carrier protein thioesterase gene silencing in oil palm

    PubMed Central

    Bhore, Subhash Janardhan; Shah, Farida Habib

    2011-01-01

    Palm oil obtained from E. guineensis Jacq. Tenera is known to have about 44% of palmitic acid (C16:0). Palmitoyl-Acyl Carrier Protein Thioesterase (PATE) is one of the key enzymes involved in plastidial fatty acid biosynthesis; and it determines the level of the C16:0 assimilation in oilseeds. This enzyme's activity in oil palm is responsible for high (> 44 % in E. guineensis Jacq. Tenera and 25 % in E. oleifera) content of C16:0 in its oil. By post-transcriptional PATE gene silencing, C16:0 content can be minimized for nutritional value improvement of the palm oil. The objective of this study was the construction of novel transformation vectors for PATE gene silencing. Six different transformation vectors targeted against PATE gene were constructed using 619 bp long PATE gene (5' region) fragment (from GenBank AF507115). In one set of three transformation vectors, PATE gene fragment was fused with CaMV 35S promoter in antisense, intron-spliced inverted repeat (ISIR), and inverted repeat (IR) orientations to generate antisense mRNA and hair-pin RNAs (hpRNA). In another set of three transformation vectors with same design, CaMV 35S was replaced with Oil palm mesocarp tissue-specific promoter (MSP). The expression cassette of antisense, ISIR, and IR of PATE gene fragments were constructed in primary cloning vector, pHANNIBAL or its derivative/s. Finally, all 6 expression cassettes were sub-cloned into pCAMBIA 1301 which contains the Hygromycinr and the GUS reporter genes for transformant selection and transformation detection respectively. The results of the RE analyses of the constructs and sequence analyses of PATE and MSP shows and confirms the orientation, size and locations of all the components from constructs. We hypothesize that 4 (pISIRPATE-PC, pIRPATE-PC, pMISIRPATE-PC and pMIRPATE-PC) out of 6 transformation vectors constructed in this study will be efficient and effective in palmitoyl-ACP thioesterase gene silencing in oil palm. Abbreviations anti

  5. Evaluation of microbial population and functional genes during the bioremediation of petroleum-contaminated soil as an effective monitoring approach.

    PubMed

    Shahi, Aiyoub; Aydin, Sevcan; Ince, Bahar; Ince, Orhan

    2016-03-01

    This study investigated the abundance and diversity of soil n-alkane and polycyclic aromatic hydrocarbon (PAH)-degrading bacterial communities. It also investigated the quantity of the functional genes, the occurrence of horizontal gene transfer (HGT) in the identified bacterial communities and the effect that such HGT can have on biostimulation process. Illumina sequencing was used to detect the microbial diversity of petroleum-polluted soil prior to the biostimulation process, and quantitative real-time PCR was used to determine changes in the bacterial community and functional genes (alkB, phnAc and nah) expressions throughout the biostimulation of petroleum-contaminated soil. The illumine results revealed that γ-proteobacteria, Chloroflexi, Firmicutes, and δ-proteobacteria were the most dominant bacterial phyla in the contaminated site, and that most of the strains were Gram-negative. The results of the gene expression results revealed that gram-negative bacteria and alkB are critical to successful bioremediation. Failure to maintain the stability of hydrocarbon-degrading bacteria and functional gene will reduce the extend to which alkanes and PAHs are degraded. According to the results of the study, the application of a C:N:P ratio of was 100:15:1 in the biodegradation experiment resulted in the highest rate at which petroleum hydrocarbons were biodegraded. The diversity of pollutant-degrading bacteria and the effective transfer of degrading genes among resident microorganisms are essential factors for the successful biostimulation of petroleum hydrocarbons. As such, screening these factors throughout the biostimulation process represents an effective monitoring approach by which the success of the biostimulation can be assessed.

  6. Transformation of an edible crop with the pagA gene of Bacillus anthracis.

    PubMed

    Aziz, Mohammad Azhar; Sikriwal, Deepa; Singh, Samer; Jarugula, Sridhar; Kumar, P Anand; Bhatnagar, Rakesh

    2005-09-01

    Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here.

  7. Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

    NASA Astrophysics Data System (ADS)

    Versluis, Dennis; D'Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M.; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; Schaik, Willem Van; de Vos, Willem M.; Kleerebezem, Michiel; Smidt, Hauke; Passel, Mark W. J. Van

    2015-07-01

    Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

  8. Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes

    PubMed Central

    Azevedo, Analice C.; Bento, Cláudia B. P.; Ruiz, Jeronimo C.; Queiroz, Marisa V.

    2015-01-01

    Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides. PMID:26253660

  9. Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions.

    PubMed

    Versluis, Dennis; D'Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; van Schaik, Willem; de Vos, Willem M; Kleerebezem, Michiel; Smidt, Hauke; van Passel, Mark W J

    2015-07-08

    Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

  10. Rapid degradation of sulphamethoxazole and the further transformation of 3-amino-5-methylisoxazole in a microbial fuel cell.

    PubMed

    Wang, Lu; Liu, Yulei; Ma, Jun; Zhao, Feng

    2016-01-01

    Sulphamethoxazole (SMX) is extensively used in humans and livestock, but its appearance in natural water raises environmental concerns. This study demonstrated that SMX and its degradation product, 3-amino-5-methylisoxazole (3A5MI), could be effectively degraded in microbial fuel cell (MFC) reactors. Approximately 85% of 20 ppm SMX was degraded within 12 h, and this was a more rapid biodegradation rate than has been previously shown in the literature. In addition, 3A5MI, a toxic chemical that forms in the SMX degradation process, can be further mineralized. The degradation products of SMX were detected by mass spectrometry, and three speculated by-products were confirmed with chemical standards. It was observed that nitrogen atoms of SMX were progressively eliminated during the degradation process, which may relate with the degradation of SMX and 3A5MI. An antibacterial activity test showed that the biotoxicity of SMX towards Shewanella oneidensis MR-1 and Escherichia coli DH5α was greatly reduced after MFC treatment. Moreover, the ATP level of the MFC microbe was nearly threefold higher than that in open-circuit controls, which may be related to the rapid degradation of SMX in MFCs. This study can facilitate further investigations about the biodegradation of SMX.

  11. Antibiotic Resistance Genes and Correlations with Microbial Community and Metal Resistance Genes in Full-Scale Biogas Reactors As Revealed by Metagenomic Analysis.

    PubMed

    Luo, Gang; Li, Bing; Li, Li-Guan; Zhang, Tong; Angelidaki, Irini

    2017-04-04

    Digested residues from biogas plants are often used as biofertilizers for agricultural crops cultivation. The antibiotic resistance genes (ARGs) in digested residues pose a high risk to public health due to their potential spread to the disease-causing microorganisms and thus reduce the susceptibility of disease-causing microorganisms to antibiotics in medical treatment. A high-throughput sequencing (HTS)-based metagenomic approach was used in the present study to investigate the variations of ARGs in full-scale biogas reactors and the correlations of ARGs with microbial communities and metal resistance genes (MRGs). The total abundance of ARGs in all the samples varied from 7 × 10(-3) to 1.08 × 10(-1) copy of ARG/copy of 16S-rRNA gene, and the samples obtained from thermophilic biogas reactors had a lower total abundance of ARGs, indicating the superiority of thermophilic anaerobic digestion for ARGs removal. ARGs in all the samples were composed of 175 ARG subtypes; however, only 7 ARG subtypes were shared by all the samples. Principal component analysis and canonical correspondence analysis clustered the samples into three groups (samples from manure-based mesophilic reactors, manure-based thermophilic reactors, and sludge-based mesophilic reactors), and substrate, temperature, and hydraulic retention time (HRT) as well as volatile fatty acids (VFAs) were identified as crucial environmental variables affecting the ARGs compositions. Procrustes analysis revealed microbial community composition was the determinant of ARGs composition in biogas reactors, and there was also a significant correlation between ARGs composition and MRGs composition. Network analysis further revealed the co-occurrence of ARGs with specific microorganisms and MRGs.

  12. The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis

    PubMed Central

    Song, Wei; Li, Lingzhi; Huang, Hongliang; Jiang, Keji; Chen, Xuezhong

    2016-01-01

    Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics. PMID:27957494

  13. The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis.

    PubMed

    Song, Wei; Li, Lingzhi; Huang, Hongliang; Jiang, Keji; Zhang, Fengying; Chen, Xuezhong; Zhao, Ming; Ma, Lingbo

    2016-01-01

    Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics.

  14. Cloning and expression analysis of a transformer gene in Daphnia pulex during different reproduction stages.

    PubMed

    Chen, Ping; Xu, Shan-Liang; Zhou, Wei; Guo, Xiao-Ge; Wang, Chun-Lin; Wang, Dan-Li; Zhao, Yun-Long

    2014-05-01

    The full-length cDNA of a transformer gene (Dptra) was cloned from the cladoceran Daphnia pulex using RACE. Dptra expression was assessed by qPCR and whole-mount in situ hybridization in different reproductive stages. The Dptra cDNA, 1652bp in length, has a 1158-bp open reading frame that encodes a 385 amino acid polypeptide containing one Sex determination protein N terminal (SDP_N) superfamily, eight putative phosphorylation sites, and an arginine-serine (RS)-rich domain at the N-terminus. Dptra showed 81%, 53%, 51% and 45% identity to orthologous genes in Daphnia magna, Apis mellifera, Apis cerana and Bombus terrestris, respectively. Phylogenetic analysis based on deduced amino acid sequences revealed that Dptra clustered in the hymenopteran clade and was most closely related to D. magna and A. mellifera. qPCR showed that Dptra expression increased significantly (P<0.05) in different reproductive stages in the following order: male, ephippial female, parthenogenetic female, resting egg and juvenile female. Dptra expression is significantly different between males and females and it is significantly greater in ephippial females and males than in parthenogenetic D. pulex (with summer eggs). Whole-mount in situ hybridization revealed that Dptra was expressed at different levels between males and females. In males, hybridization signals were found in the first antennae, second antennae and thoracic limb, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. This suggests that the Dptra gene plays significant roles in switching modes of reproduction and in sexual differentiation.

  15. Relationships Between Nitrogen Transformation Rates and Gene Abundance in a Riparian Buffer Soil

    NASA Astrophysics Data System (ADS)

    Wu, Lin; Osmond, Deanna L.; Graves, Alexandria K.; Burchell, Michael R.; Duckworth, Owen W.

    2012-11-01

    Denitrification is a critical biogeochemical process that results in the conversion of nitrate to volatile products, and thus is a major route of nitrogen loss from terrestrial environments. Riparian buffers are an important management tool that is widely utilized to protect water from non-point source pollution. However, riparian buffers vary in their nitrate removal effectiveness, and thus there is a need for mechanistic studies to explore nitrate dynamics in buffer soils. The objectives of this study were to examine the influence of specific types of soluble organic matter on nitrate loss and nitrous oxide production rates, and to elucidate the relationships between these rates and the abundances of functional genes in a riparian buffer soil. Continuous-flow soil column experiments were performed to investigate the effect of three types of soluble organic matter (citric acid, alginic acid, and Suwannee River dissolved organic carbon) on rates of nitrate loss and nitrous oxide production. We found that nitrate loss rates increased as citric acid concentrations increased; however, rates of nitrate loss were weakly affected or not affected by the addition of the other types of organic matter. In all experiments, rates of nitrous oxide production mirrored nitrate loss rates. In addition, quantitative polymerase chain reaction (qPCR) was utilized to quantify the number of genes known to encode enzymes that catalyze nitrite reduction (i.e., nirS and nirK) in soil that was collected at the conclusion of column experiments. Nitrate loss and nitrous oxide production rates trended with copy numbers of both nir and 16s rDNA genes. The results suggest that low-molecular mass organic species are more effective at promoting nitrogen transformations than large biopolymers or humic substances, and also help to link genetic potential to chemical reactivity.

  16. Transforming growth factor-beta receptor requirements for the induction of the endothelin-1 gene.

    PubMed

    Castañares, Cristina; Redondo-Horcajo, Mariano; Magan-Marchal, Noemi; Lamas, Santiago; Rodriguez-Pascual, Fernando

    2006-06-01

    Expression of the endothelin (ET)-1 gene is subject to complex regulation by numerous factors, among which the cytokine transforming growth factor-beta (TGF-beta) is one of the most important. TGF-beta action is based on the activation of the Smad signaling pathway. Smad proteins activate transcription of the gene by cooperation with activator protein-1 (AP-1) at specific sites on the ET-1 promoter. Smad signaling pathway is initiated by binding of the cytokine to a heteromeric complex of type I and type II receptors. Signal is then propagated to the nucleus by specific members of the Smad family. Most cell types contain a type I receptor known as ALK5. However, endothelial cells are unique because they coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they actually activate different Smad-mediated expression programs that lead to specific endothelial phenotypes. TGF-beta/ALK5/Smad3 pathway is associated to a mature endothelium because it leads to inhibition of cell migration/proliferation. Conversely, TGF-beta/ALK1/Smad5 activates both processes and is more related to the angiogenic state. We have analyzed the TGF-beta receptor subtype requirements for the activation of the ET-1 gene. For that purpose, we have overexpressed type I receptor and Smad isoforms in endothelial cells and analyzed the effect on ET-1 expression. Our experiments indicate that TGF-beta induces ET-1 expression preferentially through the activation of the ALK5/Smad3 pathway and, therefore, the expression of the vaso-constrictor may be associated to a quiescent and mature endothelial phenotype.

  17. Genome-Resolved Metagenomic Analysis Reveals Roles for Candidate Phyla and Other Microbial Community Members in Biogeochemical Transformations in Oil Reservoirs

    PubMed Central

    Hu, Ping; Tom, Lauren; Singh, Andrea; Thomas, Brian C.; Baker, Brett J.; Piceno, Yvette M.; Andersen, Gary L.

    2016-01-01

    ABSTRACT Oil reservoirs are major sites of methane production and carbon turnover, processes with significant impacts on energy resources and global biogeochemical cycles. We applied a cultivation-independent genomic approach to define microbial community membership and predict roles for specific organisms in biogeochemical transformations in Alaska North Slope oil fields. Produced water samples were collected from six locations between 1,128 m (24 to 27°C) and 2,743 m (80 to 83°C) below the surface. Microbial community complexity decreased with increasing temperature, and the potential to degrade hydrocarbon compounds was most prevalent in the lower-temperature reservoirs. Sulfate availability, rather than sulfate reduction potential, seems to be the limiting factor for sulfide production in some of the reservoirs under investigation. Most microorganisms in the intermediate- and higher-temperature samples were related to previously studied methanogenic and nonmethanogenic archaea and thermophilic bacteria, but one candidate phylum bacterium, a member of the Acetothermia (OP1), was present in Kuparuk sample K3. The greatest numbers of candidate phyla were recovered from the mesothermic reservoir samples SB1 and SB2. We reconstructed a nearly complete genome for an organism from the candidate phylum Parcubacteria (OD1) that was abundant in sample SB1. Consistent with prior findings for members of this lineage, the OD1 genome is small, and metabolic predictions support an obligately anaerobic, fermentation-based lifestyle. At moderate abundance in samples SB1 and SB2 were members of bacteria from other candidate phyla, including Microgenomates (OP11), Atribacteria (OP9), candidate phyla TA06 and WS6, and Marinimicrobia (SAR406). The results presented here elucidate potential roles of organisms in oil reservoir biological processes. PMID:26787827

  18. Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

    PubMed

    Delforno, Tiago Palladino; Lacerda Júnior, Gileno Vieira; Noronha, Melline F; Sakamoto, Isabel K; Varesche, Maria Bernadete A; Oliveira, Valéria M

    2017-02-23

    The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

  19. High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae.

    PubMed

    Sheng, Ping; Li, Yushan; Marshall, Sean D G; Zhang, Hongyu

    2015-07-21

    In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

  20. High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae

    PubMed Central

    Sheng, Ping; Li, Yushan; Marshall, Sean D. G.; Zhang, Hongyu

    2015-01-01

    In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect. PMID:26197317

  1. Why Microbial Communities?

    ScienceCinema

    Fredrickson, Jim (PNNL)

    2016-07-12

    The Microbial Communities Initiative is a 5-year investment by Pacific Northwest National Laboratory that integrates biological/ecological experimentation, analytical chemistry, and simulation modeling. The objective is to create transforming technologies, elucidate mechanistic forces, and develop theoretical frameworks for the analysis and predictive understanding of microbial communities. Dr. Fredrickson introduces the symposium by defining microbial communities and describing their scientific relevance as they relate to solving problems in energy, climate, and sustainability.

  2. Production of androgens by microbial transformation of progesterone in vitro: a model for androgen production in rivers.

    PubMed

    Jenkins, Ronald L; Wilson, Elizabeth M; Angus, Robert A; Howell, W Mike; Kirk, Marion; Moore, Ray; Nance, Marione; Brown, Amber

    2004-11-01

    We have previously documented the presence of progesterone and androstenedione in the water column and bottom sediments of the Fenholloway River, Taylor County, Florida. This river receives paper mill effluent and contains masculinized female mosquitofish. We hypothesized that plant sterols (e.g., ss-sitosterol) derived from the pulping of pine trees are transformed by bacteria into progesterone and subsequently into 17alpha-hydroxyprogesterone, androstenedione, and other androgens. In this study, we demonstrate that these same androgens can be produced in vitro from the bacterium Mycobacterium smegmatis. In a second part to this study, we reextracted and reanalyzed the sediment from the Fenholloway River and verified the presence of androstadienedione, a delta1 steroid with androgen activity.

  3. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression.

    PubMed

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X; Kemp, Sadie E; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2014-04-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells.

  4. Microbial Evolution Is in the Cards: Horizontal Gene Transfer in the Classroom

    ERIC Educational Resources Information Center

    Kagle, Jeanne; Hay, Anthony G.

    2007-01-01

    Horizontal gene transfer, the exchange of genetic material between bacteria, is a potentially important factor in the degradation of synthetic compounds introduced to the environment and in the acquisition of other characteristics including antibiotic resistance. This game-based activity illustrates the role of horizontal gene transfer in the…

  5. Responses in colonic microbial community and gene expression of pigs to a long-term high resistant starch diet

    PubMed Central

    Sun, Yue; Zhou, Liping; Fang, Lingdong; Su, Yong; Zhu, Weiyun

    2015-01-01

    Intake of raw potato starch (RPS) has been associated with various intestinal health benefits, but knowledge of its mechanism in a long-term is limited. The aim of this study was to investigate the effects of long-term intake of RPS on microbial composition, genes expression profiles in the colon of pigs. Thirty-six Duroc × Landrace × Large White growing barrows were randomly allocated to corn starch (CS) and RPS groups with a randomized block design. Each group consisted of six replicates (pens), with three pigs per pen. Pigs in the CS group were offered a corn/soybean-based diet, while pigs in the RPS group were put on a diet in which 230 g/kg (growing period) or 280 g/kg (finishing period) purified CS was replaced with purified RPS during a 100-day trial. Real-time PCR assay showed that RPS significantly decreased the number of total bacteria in the colonic digesta. MiSeq sequencing of the V3-V4 region of the 16S rRNA genes showed that RPS significantly decreased the relative abundance of Clostridium, Treponema, Oscillospira, Phascolarctobacterium, RC9 gut group, and S24-7-related operational taxonomic units (OTUs), and increased the relative abundance of Turicibacter, Blautia, Ruminococcus, Coprococcus, Marvinbryantia, and Ruminococcus bromii-related OTUs in colonic digesta and mucosa. Analysis of the colonic transcriptome profiles revealed that the RPS diet changed the colonic expression profile of the host genes mainly involved in immune response pathways. RPS significantly increased proinflammartory cytokine IL-1β gene expression and suppressed genes involved in lysosome. Our findings suggest that long-term intake of high resistant starch (RS) diet may result in both positive and negative roles in gut health. PMID:26379652

  6. A Culture-Independent Approach to Unravel Uncultured Bacteria and Functional Genes in a Complex Microbial Community

    PubMed Central

    Wang, Yun; Chen, Yin; Zhou, Qian; Huang, Shi; Ning, Kang; Xu, Jian; Kalin, Robert M.; Rolfe, Stephen; Huang, Wei E.

    2012-01-01

    Most microorganisms in nature are uncultured with unknown functionality. Sequence-based metagenomics alone answers ‘who/what are there?’ but not ‘what are they doing and who is doing it and how?’. Function-based metagenomics reveals gene function but is usually limited by the specificity and sensitivity of screening strategies, especially the identification of clones whose functional gene expression has no distinguishable activity or phenotypes. A ‘biosensor-based genetic transducer’ (BGT) technique, which employs a whole-cell biosensor to quantitatively detect expression of inserted genes encoding designated functions, is able to screen for functionality of unknown genes from uncultured microorganisms. In this study, BGT was integrated with Stable isotope probing (SIP)-enabled Metagenomics to form a culture-independent SMB toolbox. The utility of this approach was demonstrated in the discovery of a novel functional gene cluster in naphthalene contaminated groundwater. Specifically, metagenomic sequencing of the 13C-DNA fraction obtained by SIP indicated that an uncultured Acidovorax sp. was the dominant key naphthalene degrader in-situ, although three culturable Pseudomonas sp. degraders were also present in the same groundwater. BGT verified the functionality of a new nag2 operon which co-existed with two other nag and two nah operons for naphthalene biodegradation in the same microbial community. Pyrosequencing analysis showed that the nag2 operon was the key functional operon in naphthalene degradation in-situ, and shared homology with both nag operons in Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2. The SMB toolbox will be useful in providing deep insights into uncultured microorganisms and unravelling their ecological roles in natural environments. PMID:23082176

  7. A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone releas...

  8. Are microbial N transformation rates in a permanent grassland soil after 17 years of elevated atmospheric CO2 sensitive to soil temperature?

    NASA Astrophysics Data System (ADS)

    Moser, Gerald; Gorenflo, André; Brenzinger, Kristof; Clough, Tim; Braker, Gesche; Müller, Christoph

    2016-04-01

    Long-term observations (17 years) within the Giessen Free Air Carbon dioxide Enrichment (Giessen FACE) study on permanent grassland showed that the carbon fertilization caused significant changes in the ecosystem nitrogen cycle. These changes are responsible for a doubling of annual N2O emissions under elevated atmospheric CO2 (eCO2) caused by increased emissions during the plant growing season. The goal of this lab study was to understand how soil temperature influences the long-term effects of eCO2 and plant carbon input on microbial N transformations in the Giessen FACE. Therefore, a pulse labelling study with 15N tracing of 15NH4+ and 15NO3- was carried out with incubated soil samples from elevated and ambient CO2 FACE rings in climate chambers at two different temperatures (10°C and 19°C), while water filled pore space of the samples was adjusted to the same level. The various N pools in the soil (NH4+, NO3-, NO2-, soil organic matter), N2O emissions and simultaneous gross N transformation rates were quantified. The quantification of the gross N transformations are based on the turnover of 15NH4+, 15NO3-, 15NO2- and shall illuminate the interaction between carbon fertilization, temperature and changes in nitrogen cycle in this grassland soil. While the soil respiration after labelling was significantly increased at 19°C compared to 10°C, N2O emissions showed no significant differences. There were also no significant differences of N2O emissions between soil samples from control and elevated CO2 rings within each temperature level. As the soil temperature (within the range of 10-19°C) had no significant effects on N transformations responsible for the observed doubling of N2O emissions under eCO2, it seems most likely that other factors like direct carbon input by plants and/or soil moisture differences between ambient and elevated rings in the field are responsible for the observed increase in N2O emissions under eCO2.

  9. Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster

    SciTech Connect

    Maschat, F.; Dubertret, M.L.; Lepesant, J.A. )

    1991-05-01

    The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between {minus}138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from {minus}138 to {minus}68 are essential for this expression.

  10. Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

    PubMed

    Lewy, Gregory D; Ryan, Gavin A; Read, Martin L; Fong, Jim C W; Poole, Vikki; Seed, Robert I; Sharma, Neil; Smith, Vicki E; Kwan, Perkin P K; Stewart, Sarah L; Bacon, Andrea; Warfield, Adrian; Franklyn, Jayne A; McCabe, Christopher J; Boelaert, Kristien

    2013-11-01

    Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

  11. Microbial community structure and denitrification in a wetland mitigation bank.

    PubMed

    Peralta, Ariane L; Matthews, Jeffrey W; Kent, Angela D

    2010-07-01

    Wetland mitigation is implemented to replace ecosystem functions provided by wetlands; however, restoration efforts frequently fail to establish equivalent levels of ecosystem services. Delivery of microbially mediated ecosystem functions, such as denitrification, is influenced by both the structure and activity of the microbial community. The objective of this study was to compare the relationship between soil and vegetation factors and microbial community structure and function in restored and reference wetlands within a mitigation bank. Microbial community composition was assessed using terminal restriction fragment length polymorphism targeting the 16S rRNA gene (total bacteria) and the nosZ gene (denitrifiers). Comparisons of microbial function were based on potential denitrification rates. Bacterial community structures differed significantly between restored and reference wetlands; denitrifier community assemblages were similar among reference sites but highly variable among restored sites throughout the mitigation bank. Potential denitrification was highest in the reference wetland sites. These data demonstrate that wetland restoration efforts in this mitigation bank have not successfully restored denitrification and that differences in potential denitrification rates may be due to distinct microbial assemblages observed in restored and reference (natural) wetlands. Further, we have identified gradients in soil moisture and soil fertility that were associated with differences in microbial community structure. Microbial function was influenced by bacterial community composition and soil fertility. Identifying soil factors that are primary ecological drivers of soil bacterial communities, especially denitrifying populations, can potentially aid the development of predictive models for restoration of biogeochemical transformations and enhance the success of wetland restoration efforts.

  12. Microbial Electricity Generation Enhances Decabromodiphenyl Ether (BDE-209) Degradation

    PubMed Central

    Yang, Yonggang; Xu, Meiying; He, Zhili; Guo, Jun; Sun, Guoping; Zhou, Jizhong

    2013-01-01

    Due to environmental persistence and biotoxicity of polybrominated diphenyl ethers (PBDEs), it is urgent to develop potential technologies to remediate PBDEs. Introducing electrodes for microbial electricity generation to stimulate the anaerobic degradation of organic pollutants is highly promising for bioremediation. However, it is still not clear whether the degradation of PBDEs could be promoted by this strategy. In this study, we hypothesized that the degradation of PBDEs (e.g., BDE-209) would be enhanced under microbial electricity generation condition. The functional compositions and structures of microbial communities in closed-circuit microbial fuel cell (c-MFC) and open-circuit microbial fuel cell (o-MFC) systems for BDE-209 degradation were detected by a comprehensive functional gene array, GeoChip 4.0, and linked with PBDE degradations. The results indicated that distinctly different microbial community structures were formed between c-MFCs and o-MFCs, and that lower concentrations of BDE-209 and the resulting lower brominated PBDE products were detected in c-MFCs after 70-day performance. The diversity and abundance of a variety of functional genes in c-MFCs were significantly higher than those in o-MFCs. Most genes involved in chlorinated solvent reductive dechlorination, hydroxylation, methoxylation and aromatic hydrocarbon degradation were highly enriched in c-MFCs and significantly positively correlated with the removal of PBDEs. Various other microbial functional genes for carbon, nitrogen, phosphorus and sulfur cycling, as well as energy transformation process, were also significantly increased in c-MFCs. Together, these results suggest that PBDE degradation could be enhanced by introducing the electrodes for microbial electricity generation and by specifically stimulating microbial functional genes. PMID:23940625

  13. Microbial electricity generation enhances decabromodiphenyl ether (BDE-209) degradation.

    PubMed

    Yang, Yonggang; Xu, Meiying; He, Zhili; Guo, Jun; Sun, Guoping; Zhou, Jizhong

    2013-01-01

    Due to environmental persistence and biotoxicity of polybrominated diphenyl ethers (PBDEs), it is urgent to develop potential technologies to remediate PBDEs. Introducing electrodes for microbial electricity generation to stimulate the anaerobic degradation of organic pollutants is highly promising for bioremediation. However, it is still not clear whether the degradation of PBDEs could be promoted by this strategy. In this study, we hypothesized that the degradation of PBDEs (e.g., BDE-209) would be enhanced under microbial electricity generation condition. The functional compositions and structures of microbial communities in closed-circuit microbial fuel cell (c-MFC) and open-circuit microbial fuel cell (o-MFC) systems for BDE-209 degradation were detected by a comprehensive functional gene array, GeoChip 4.0, and linked with PBDE degradations. The results indicated that distinctly different microbial community structures were formed between c-MFCs and o-MFCs, and that lower concentrations of BDE-209 and the resulting lower brominated PBDE products were detected in c-MFCs after 70-day performance. The diversity and abundance of a variety of functional genes in c-MFCs were significantly higher than those in o-MFCs. Most genes involved in chlorinated solvent reductive dechlorination, hydroxylation, methoxylation and aromatic hydrocarbon degradation were highly enriched in c-MFCs and significantly positively correlated with the removal of PBDEs. Various other microbial functional genes for carbon, nitrogen, phosphorus and sulfur cycling, as well as energy transformation process, were also significantly increased in c-MFCs. Together, these results suggest that PBDE degradation could be enhanced by introducing the electrodes for microbial electricity generation and by specifically stimulating microbial functional genes.

  14. Bioreactor performance and functional gene analysis of microbial community in a limited-oxygen fed bioreactor for co-reduction of sulfate and nitrate with high organic input.

    PubMed

    Xu, Xi-jun; Chen, Chuan; Wang, Ai-jie; Yu, Hao; Zhou, Xu; Guo, Hong-liang; Yuan, Ye; Lee, Duu-jong; Zhou, Jizhong; Ren, Nan-qi

    2014-08-15

    Limited-oxygen mediated synergistic relationships between sulfate-reducing bacteria (SRB), nitrate-reducing bacteria (NRB) and sulfide-oxidizing bacteria (SOB, including nitrate-reducing, sulfide-oxidizing bacteria NR-SOB) were predicted to simultaneously remove contaminants of nitrate, sulfate and high COD, and eliminate sulfide generation. A lab-scale experiment was conducted to examine the impact of limited oxygen on these oxy-anions degradation, sulfide oxidation and associated microbial functional responses. In all scenarios tested, the reduction of both nitrate and sulfate was almost complete. When limited-oxygen was fed into bioreactors, S(0) formation was significantly improved up to ∼ 70%. GeoChip 4.0, a functional gene microarray, was used to determine the microbial gene diversity and functional potential for nitrate and sulfate reduction, and sulfide oxidation. The diversity of the microbial community in bioreactors was increased with the feeding of limited oxygen. Whereas the intensities of the functional genes involved in sulfate reduction did not show a significant difference, the abundance of the detected denitrification genes decreased in limited oxygen samples. More importantly, sulfide-oxidizing bacteria may alter their populations/genes in response to limited oxygen potentially to function more effectively in sulfide oxidation, especially to elemental sulfur. The genes fccA/fccB from nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB), such as Paracoccus denitrificans, Thiobacillus denitrificans, Beggiatoa sp., Thiomicrospira sp., and Thioalkalivibrio sp., were more abundant under limited-oxygen condition.

  15. Microfluidics and microbial engineering.

    PubMed

    Kou, Songzi; Cheng, Danhui; Sun, Fei; Hsing, I-Ming

    2016-02-07

    The combination of microbial engineering and microfluidics is synergistic in nature. For example, microfluidics is benefiting from the outcome of microbial engineering and many reported point-of-care microfluidic devices employ engineered microbes as functional parts for the microsystems. In addition, microbial engineering is facilitated by various microfluidic techniques, due to their inherent strength in high-throughput screening and miniaturization. In this review article, we firstly examine the applications of engineered microbes for toxicity detection, biosensing, and motion generation in microfluidic platforms. Secondly, we look into how microfluidic technologies facilitate the upstream and downstream processes of microbial engineering, including DNA recombination, transformation, target microbe selection, mutant characterization, and microbial function analysis. Thirdly, we highlight an emerging concept in microbial engineering, namely, microbial consortium engineering, where the behavior of a multicultural microbial community rather than that of a single cell/species is delineated. Integrating the disciplines of microfluidics and microbial engineering opens up many new opportunities, for example in diagnostics, engineering of microbial motors, development of portable devices for genetics, high throughput characterization of genetic mutants, isolation and identification of rare/unculturable microbial species, single-cell analysis with high spatio-temporal resolution, and exploration of natural microbial communities.

  16. Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses.

    PubMed

    Fang, Jiasong; Shizuka, Arakawa; Kato, Chiaki; Schouten, Stefan

    2006-09-01

    Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.

  17. Gene Editing by Co-Transformation of TALEN and Chimeric RNA/DNA Oligonucleotides on the Rice OsEPSPS Gene and the Inheritance of Mutations

    PubMed Central

    Wang, Mugui; Liu, Yujun; Zhang, Cuicui; Liu, Jianping; Liu, Xin; Wang, Liangchao; Wang, Wenyi; Chen, Hao; Wei, Chuchu; Ye, Xiufen; Li, Xinyuan; Tu, Jumin

    2015-01-01

    Although several site-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT) in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs), including different strand composition such as RNA/DNA (C1) or DNA/RNA (C2) but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP), we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19) as well as co-transformation of TELAN with either HRP (5/30) or C1 (2/25) or C2 (5/31). Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type. PMID:25856577

  18. The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

    PubMed

    Rakosy-Tican, Elena; Aurori, Cristian M; Dijkstra, Camelia; Thieme, Ramona; Aurori, Adriana; Davey, Michael R

    2007-05-01

    Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

  19. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    PubMed

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  20. Single gene locus changes perturb complex microbial communities as much as apex predator loss.

    PubMed

    McClean, Deirdre; McNally, Luke; Salzberg, Letal I; Devine, Kevin M; Brown, Sam P; Donohue, Ian

    2015-09-10

    Many bacterial species are highly social, adaptively shaping their local environment through the production of secreted molecules. This can, in turn, alter interaction strengths among species and modify community composition. However, the relative importance of such behaviours in determining the structure of complex communities is unknown. Here we show that single-locus changes affecting biofilm formation phenotypes in Bacillus subtilis modify community structure to the same extent as loss of an apex predator and even to a greater extent than loss of B. subtilis itself. These results, from experimentally manipulated multitrophic microcosm assemblages, demonstrate that bacterial social traits are key modulators of the structure of their communities. Moreover, they show that intraspecific genetic variability can be as important as strong trophic interactions in determining community dynamics. Microevolution may therefore be as important as species extinctions in shaping the response of microbial communities to environmental change.

  1. Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants

    SciTech Connect

    Shaw, G.M.; Wasserman, C.R.; O`Malley, C.D.

    1996-03-01

    Results of studies determine whether women who smoke during early pregnancy are at increased risk of delivering infants with orofacial clefts have been mixed, and recently a gene-environment interaction between maternal smoking, transforming growth factor-alpha (TGFa), and clefting has been reported. Using a large population-based case-control study, we investigated whether parental periconceptional cigarette smoking was associated with an increased risk for having offspring with orofacial clefts. We also investigated the influence of genetic variation of the TGFa locus on the relation between smoking and clefting. Parental smoking information was obtained from telephone interviews with mothers of 731 (84.7% of eligible) orofacial cleft case infants and with mothers of 734 (78.2%) nonmalformed control infants. DNA was obtained from newborn screening blood spots and genotyped for the allelic variants of TGFa. We found that risks associated with maternal smoking were most elevated for isolated cleft lip with or without cleft palate, (odds ratio 2.1 [95% confidence interval 1.3-3.6]) and for isolated cleft palate (odds ratio 2.2 [1.1-4.5]) when mothers smoked {ge} 20 cigarrettes/d. These risks for white infants ranged from 3-fold to 11-fold across phenotypic groups. Paternal smoking was not associated with clefting among the offspring of nonsmoking mothers, and passive smoke exposures were associated with at most slightly increased risks. This study offers evidence that the risk for orofacial clefting in infants may be influenced by maternal smoke exposures alone as well as in combination (gene-environment interaction) with the presence of the uncommon TGFa allele. 56 refs., 5 tabs.

  2. The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes.

    PubMed

    Ross, T M; Pettiford, S M; Green, P L

    1996-08-01

    The mechanism of human T-cell leukemia virus (HTLV)-mediated transformation and induction of malignancy is unknown; however, several studies have implicated the viral gene product, Tax. Conclusive evidence for the role of Tax in the HTLV malignant process has been impeded by the inability to mutate tax in the context of an infectious virus and dissociate viral replication from cellular transformation. To circumvent this problem we constructed a mutant of HTLV type 2 (HTLV-2) that replicates by a Tax-independent mechanism. For these studies, the Tax response element in the viral long terminal repeat was replaced with the cytomegalovirus immediate-early promoter enhancer (C-enh). Transcription of the chimeric HTLV-2 (HTLVC-enh) was efficiently directed by this heterologous promoter. Also, the chimeric virus transformed primary human T lymphocytes with an efficiency similar to that of wild-type HTLV-2. A tax-knockout virus, termed HTLVC-enhDeltaTax, was constructed to directly assess the importance of Tax in cellular transformation. Transfection and infection studies indicated that HTLVC-enhDeltaTax was replication competent; however, HTLVC-enhDeltaTax failed to transform primary human T lymphocytes. We conclude that Tax is essential for HTLV-mediated transformation of human T lymphocytes. Furthermore, this chimeric HTLV, that replicates in the absence of Tax, should facilitate studies to determine the precise mechanism of T-lymphocyte transformation by HTLV.

  3. Anti‐microbial peptide gene expression during oral vaccination: analysis of a randomized controlled trial

    PubMed Central

    Simuyandi, M.; Kapulu, M.

    2016-01-01

    Summary We have observed previously that micronutrient supplementation ameliorated suppression of α‐defensin expression during diarrhoea. However, how interactions between anti‐microbial peptide (AMP) expression and diarrhoeal disease are altered by micronutrient supplementation remain unclear. Using oral vaccination as a model of intestinal infection, we measured changes in AMP expression during multiple micronutrient supplementation. In the first part, volunteers underwent duodenal jejunal biopsy before and at 1, 2, 4 or 7 days after administration of one of three live, attenuated oral vaccines against rotavirus, typhoid and enterotoxigenic Escherichia coli. In the second part, participants were randomized to receive a multiple micronutrient supplement or placebo for 6 weeks before undergoing intestinal biopsy, vaccination against typhoid and rebiopsy after 14 days. Expression of human alpha‐defensin (HD)5, HD6, hBD1, hBD2 and LL‐37 was measured by quantitative reverse transcription–polymerase chain reaction. Taken together, the bacterial vaccines, but not rotavirus vaccine, reduced HD5 expression (P = 0·02, signed‐rank test) and reduced LL‐37 expression in seven of the eight individuals whose biopsies had expression prevaccination (P = 0·03). hBD2 was not detected. In the controlled trial, HD5 and HD6 expression after vaccination was lower [median ratio 0·5, interquartile range (IQR) = 0·07–2·2 and 0·58, IQR = 0·13–2·3, respectively] than before vaccination. There was no significant effect detected of micronutrient supplementation on expression of HD5, HD6, hBD1 or LL‐37. We conclude that live attenuated bacterial vaccines, but not rotavirus vaccine, can reduce intestinal α‐defensins, and typhoid vaccine reduced LL‐37 expression. We found no evidence that micronutrient supplementation in the short term had any impact on anti‐microbial peptide expression. PMID:27465597

  4. The diversity changes of soil microbial communities stimulated by climate, soil type and vegetation type analyzed via a functional gene array.

    PubMed

    Chen, Fu; Tan, Min; Yang, Yongjun; Ma, Jing; Zhang, Shaoliang; Li, Gang

    2015-11-01

    The aim of this study was to analyze the changes of soil microbial communities stimulated by climate, soil type and vegetation type using a functional gene array. The dataset GSE51592 was obtained from Gene Expression Omnibus, including 54 soil samples. Genetic diversity variation of samples under different sites, soil and vegetation types was examined by calculating the percentage of specific gene number in each sample. Furthermore, gene functional categories and microorganism species in samples under different environmental factors were respectively divided. Gene number in samples with cropping was higher than in samples without cropping. When site, soil type and vegetation type were as the sole variable, respectively, the percentage of specific genes in samples from Yingtan, in phaeozem samples and in samples with cropping was higher. Furthermore, the percentage of gene number in organic remediation for phaeozem and cambisol samples was significant at p < 0.05, comparing with that for acrisol samples. At superkingdom level of microorganisms, for the same category, there was no significant difference (p < 0.05) between the samples. At phylum level, for the categories of Bacteroidetes and Cyanobacteria, the percentage of gene number in cambisol samples was significantly higher (p < 0.05). Conversely, in the category of Proteobacteria, the percentage of gene number in phaeozem and acrisol samples was markedly higher (p < 0.05). Microbial diversity of soil was cooperatively driven by climate, soil type and vegetation type.

  5. Structural genomics of highly conserved microbial genes of unknown function in search of new antibacterial targets.

    PubMed

    Abergel, Chantal; Coutard, Bruno; Byrne, Deborah; Chenivesse, Sabine; Claude, Jean-Baptiste; Deregnaucourt, Céline; Fricaux, Thierry; Gianesini-Boutreux, Celine; Jeudy, Sandra; Lebrun, Régine; Maza, Caroline; Notredame, Cédric; Poirot, Olivier; Suhre, Karsten; Varagnol, Majorie; Claverie, Jean-Michel

    2003-01-01

    With more than 100 antibacterial drugs at our disposal in the 1980's, the problem of bacterial infection was considered solved. Today, however, most hospital infections are insensitive to several classes of antibacterial drugs, and deadly strains of Staphylococcus aureus resistant to vancomycin--the last resort antibiotic--have recently begin to appear. Other life-threatening microbes, such as Enterococcus faecalis and Mycobacterium tuberculosis are already able to resist every available antibiotic. There is thus an urgent, and continuous need for new, preferably large-spectrum, antibacterial molecules, ideally targeting new biochemical pathways. Here we report on the progress of our structural genomics program aiming at the discovery of new antibacterial gene targets among evolutionary conserved genes of uncharacterized function. A series of bioinformatic and comparative genomics analyses were used to identify a set of 221 candidate genes common to Gram-positive and Gram-negative bacteria. These genes were split between two laboratories. They are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallization, X-ray diffraction, structure interpretation, and function prediction. We describe here our strategies for the 111 genes processed in our laboratory. Bioinformatics is used at most stages of the production process and out of 111 genes processed--and 17 months into the project--108 have been successfully cloned, 103 have exhibited detectable expression, 84 have led to the production of soluble protein, 46 have been purified, 12 have led to usable crystals, and 7 structures have been determined.

  6. Process of nitrogen transformation and microbial community structure in the Fe(0)-carbon-based bio-carrier filled in biological aerated filter.

    PubMed

    Deng, Shihai; Li, Desheng; Yang, Xue; Zhu, Shanbin; Li, Jinlong

    2016-04-01

    Nitrogen pollutants in low-organic carbon wastewater are difficult to biodegrade. Therefore, the Fe(0)-carbon-based bio-carrier (FCBC) was firstly used as hydrogen producer in a biological-aerated filter (BAF) to make up for the lack of organic carbon in biological nitrogen removal. Physical and chemical properties of FCBC were detected and compared in this study. The nitrogen removal rate for low COD/TN ratio wastewater, nitrogen transformation process, and microbial communities in the FCBC filled in BAF were investigated. Results showed that the nitrogen removal rates was 0.38-0.41 kg N m(-3) day(-1) in the FCBC filled BAF and reached 0.62 kg N m(-3) day(-1) within the filter depth of 60-80 cm, under the conditions of the dissolved oxygen 3.5 ± 0.2 mg L(-1) and the inlet pH 7.2 ± 0.1. Hydrogenophaga (using hydrogen as electron donor), Sphaerotilus (absorbing [Fe(3+)]), Nitrospira (nitrificaion), and Nitrosomonas (ammonia oxidation) were found to be the predominant genera in the reactor. The reaction schemes in the FCBC filled in BAF was calculated: hydrogen and [Fe(3+)] were produced by Fe(0)-C galvanic cells in the FCBC, ammonia was oxidized into nitrate by Nitrosomonas and Nitrospira genera, hydrogen was used as electron donors by Hydrogenophaga genus to reduce nitrate into N2, and [Fe(3+)] was partly absorbed by Sphaerotilus and diverted via sludge discharging.

  7. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    PubMed

    Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

  8. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance

    PubMed Central

    Pandey, Sonika; Patel, Manish Kumar; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas. PMID:27411057

  9. The Sex Determination Gene transformer Regulates Male-Female Differences in Drosophila Body Size

    PubMed Central

    Rideout, Elizabeth J.; Narsaiya, Marcus S.; Grewal, Savraj S.

    2015-01-01

    Almost all animals show sex differences in body size. For example, in Drosophila, females are larger than males. Although Drosophila is widely used as a model to study growth, the mechanisms underlying this male-female difference in size remain unclear. Here, we describe a novel role for the sex determination gene transformer (tra) in promoting female body growth. Normally, Tra is expressed only in females. We find that loss of Tra in female larvae decreases body size, while ectopic Tra expression in males increases body size. Although we find that Tra exerts autonomous effects on cell size, we also discovered that Tra expression in the fat body augments female body size in a non cell-autonomous manner. These effects of Tra do not require its only known targets doublesex and fruitless. Instead, Tra expression in the female fat body promotes growth by stimulating the secretion of insulin-like peptides from insulin producing cells in the brain. Our data suggest a model of sex-specific growth in which body size is regulated by a previously unrecognized branch of the sex determination pathway, and identify Tra as a novel link between sex and the conserved insulin signaling pathway. PMID:26710087

  10. The Sex Determination Gene transformer Regulates Male-Female Differences in Drosophila Body Size.

    PubMed

    Rideout, Elizabeth J; Narsaiya, Marcus S; Grewal, Savraj S

    2015-12-01

    Almost all animals show sex differences in body size. For example, in Drosophila, females are larger than males. Although Drosophila is widely used as a model to study growth, the mechanisms underlying this male-female difference in size remain unclear. Here, we describe a novel role for the sex determination gene transformer (tra) in promoting female body growth. Normally, Tra is expressed only in females. We find that loss of Tra in female larvae decreases body size, while ectopic Tra expression in males increases body size. Although we find that Tra exerts autonomous effects on cell size, we also discovered that Tra expression in the fat body augments female body size in a non cell-autonomous manner. These effects of Tra do not require its only known targets doublesex and fruitless. Instead, Tra expression in the female fat body promotes growth by stimulating the secretion of insulin-like peptides from insulin producing cells in the brain. Our data suggest a model of sex-specific growth in which body size is regulated by a previously unrecognized branch of the sex determination pathway, and identify Tra as a novel link between sex and the conserved insulin signaling pathway.

  11. Harnessing microbial gene pools to remediate persistent organic pollutants using genetically modified plants--a viable technology?

    PubMed

    Rylott, Elizabeth L; Johnston, Emily J; Bruce, Neil C

    2015-11-01

    It has been 14 years since the international community came together to legislate the Stockholm Convention on Persistent Organic Pollutants (POPs), restricting the production and use of specific chemicals that were found to be environmentally stable, often bioaccumulating, with long-term toxic effects. Efforts are continuing to remove these pollutants from the environment. While incineration and chemical treatment can be successful, these methods require the removal of tonnes of soil, at high cost, and are damaging to soil structure and microbial communities. The engineering of plants for in situ POP remediation has had highly promising results, and could be a more environmentally-friendly alternative. This review discusses the characterization of POP-degrading bacterial pathways, and how the genes responsible have been harnessed using genetic modification (GM) to introduce these same abilities into plants. Recent advances in multi-gene cloning, genome editing technologies and expression in monocot species are accelerating progress with remediation-applicable species. Examples include plants developed to degrade 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), trichloroethylene (TCE), and polychlorinated biphenyls (PCBs). However, the costs and timescales needed to gain regulatory approval, along with continued public opposition, are considerable. The benefits and challenges in this rapidly developing and promising field are discussed.

  12. Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

    PubMed Central

    Suzuki, Marcelino T.; Taylor, Lance T.; DeLong, Edward F.

    2000-01-01

    Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. PMID:11055900

  13. Altered biochemical profile and gene expression in aflatoxin B-1-transformed C3H10T1/2 cells.

    PubMed

    Nadadur, S; Lisciandro, K; Mudipalli, A; Maccubbin, A; Faletto, M; Gurtoo, H

    1997-06-01

    A transformed cell line 7SA, obtained by transformation of C3H10T1/2 cells with irt vitro activated aflatoxin B-1 (AFB(1)), was used to investigate biochemical and molecular alterations associated with transformation by AFB(1). 7SA cells demonstrate an altered biochemical phenotype characterized by alterations in phase I and phase II enzymes in a manner that would allow these cells to survive in a hostile chemical environment. Investigations of the molecular basis of transformation revealed no mutations in codons 12/13 and 61 of ras genes (Ha-, Ki- and N-ras) and in exons 5, 6, 7 and 8 of p53 tumor suppressor gene. However, subtractive hybridization led to the isolation of seven novel cDNA clones that demonstrated 2 to 10-fold overexpression of the mRNAs corresponding to the five cDNAs (SK1, SK2, SK3, SK4 and SK5) and >400 fold overexpression of the mRNAs corresponding to the other two cDNAs (SK67 and SK153). In addition, part of the sequence of the cDNA clone SK5 demonstrated >88% identity with L1-like mobile genetic element and Southern analysis of the DNA with SK5 cDNA as a probe revealed gene rearrangement in 7SA DNA, compared to DNA from C3H10T1/2 cells.

  14. Photochemical and microbial transformation of terrestrial dissolved organic matter - Lena River vs. rivers in mid and low latitudes

    NASA Astrophysics Data System (ADS)

    Vähätalo, A. V.; Aarnos, H.; Paolucci, E. M.; Musibono, D. E.; Khan, S. R.; Gelinas, Y.; Shantz, A.; Huang, Q.; Schneider, W.; Rezende, C. E.; Petrescu, E.; Reader, H. E.

    2012-04-01

    The aim of this study was to assess the fate of riverine dissolved organic matter (DOM) in coastal ocean. In that environment after the sedimentation of terrestrial particulate matter and the advective mixing of river water to optically clear marine water, the photochemical transformation of riverine DOM has a large potential for decomposing riverine DOM. For this study, we collected water samples from ten large rivers and carried out laboratory experiments with the river water samples. The potential for the photochemical decomposition of riverine dissolved organic matter was assessed by exposing sterile-filtered river waters to simulated solar radiation. Dark control samples we treated similarly but were not irradiated. The exposures were designed to decompose photochemically chromophoric dissolved organic matter, the major absorber of UV-vis radiation and the primary initiator of the direct photoreactions of DOM. In the end of irradiation, the difference in the concentration of dissolved organic carbon between the irradiated and the dark control sample corresponded to that portion of dissolved organic carbon (DOC) decomposable through direct photoreactions to carbon dioxide. In order to assess the amount of DOC phototransformed into biologically available forms, the irradiated and the dark control water samples received indigenous inoculums of riverine microbes and were incubated in the darkness. After a month, the difference in the concentration of DOC between the irradiated and dark control sample was considered to represent the photoreactive portion of DOC, while the residual DOC made up the DOC resistant to photochemical reactions. The photoreactive portion of DOC varied among rivers and was related to the amount of chromophoric dissolved organic matter in the water samples. The biological decomposition experiments were extended to last up to one year and predictions about the biodegradability of irradiated and dark control DOM were made by fitting a

  15. Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and Gradual Control of Microbial Gene Expression.

    PubMed

    Binder, Dennis; Bier, Claus; Grünberger, Alexander; Drobietz, Dagmar; Hage-Hülsmann, Jennifer; Wandrey, Georg; Büchs, Jochen; Kohlheyer, Dietrich; Loeschcke, Anita; Wiechert, Wolfgang; Jaeger, Karl-Erich; Pietruszka, Jörg; Drepper, Thomas

    2016-02-15

    Controlling cellular functions by light allows simple triggering of biological processes in a non-invasive fashion with high spatiotemporal resolution. In this context, light-regulated gene expression has enormous potential for achieving optogenetic control over almost any cellular process. Here, we report on two novel one-step cleavable photocaged arabinose compounds, which were applied as light-sensitive inducers of transcription in bacteria. Exposure of caged arabinose to UV-A light resulted in rapid activation of protein production, as demonstrated for GFP and the complete violacein biosynthetic pathway. Moreover, single-cell analysis revealed that intrinsic heterogeneity of arabinose-mediated induction of gene expression was overcome when using photocaged arabinose. We have thus established a novel phototrigger for synthetic bio(techno)logy applications that enables precise and homogeneous control of bacterial target gene expression.

  16. Post-Weaning Diet Affects Faecal Microbial Composition but Not Selected Adipose Gene Expression in the Cat (Felis catus)

    PubMed Central

    Bermingham, Emma N.; Kittelmann, Sandra; Young, Wayne; Kerr, Katherine R.; Swanson, Kelly S.; Roy, Nicole C.; Thomas, David G.

    2013-01-01

    The effects of pre- (i.e., gestation and during lactation) and post-weaning diet on the composition of faecal bacterial communities and adipose expression of key genes in the glucose and insulin pathways were investigated in the cat. Queens were maintained on a moderate protein:fat:carbohydrate kibbled (“Diet A”; 35:20:28% DM; n  =  4) or high protein:fat:carbohydrate canned (“Diet B”; 45:37:2% DM; n = 3) diet throughout pregnancy and lactation. Offspring were weaned onto these diets in a nested design (n  =  5 per treatment). Faecal samples were collected at wk 8 and 17 of age. DNA was isolated from faeces and bacterial 16S rRNA gene amplicons were analysed by pyrosequencing. RNA was extracted from blood (wk 18) and adipose tissue and ovarian/testicular tissues (wk 24) and gene expression levels determined using RT-qPCR. Differences (P<0.05) in composition of faecal bacteria were observed between pregnant queens fed Diet A or B. However, pre-weaning diet had little effect on faecal bacterial composition in weaned kittens. In contrast, post-weaning diet altered bacterial population profiles in the kittens. Increased (P<0.05) abundance of Firmicutes (77% vs 52% of total reads) and Actinobacteria (0.8% vs 0.2% of total reads), and decreased (P<0.05) abundance of Fusobacteria (1.6% vs 18.4% of total reads) were observed for kittens fed the Diet A compared to those fed Diet B post-weaning. Feeding Diet B pre-weaning increased (P<0.05) the expression levels of INRS, LEPT, PAI-1 and tended to increase GLUT1, while the expression levels of IRS-1 in blood increased in kittens fed Diet A pre-weaning. Post-weaning diet had no effect on expression levels of target genes. Correlations between the expression levels of genes involved in glucose and insulin pathways and faecal Bacteriodetes and Firmicutes phyla were identified. The reasons for why post-weaning diet affects microbial populations and not gene expression levels are of interest. PMID:24312255