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Sample records for microorganisms producing recombinant

  1. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD.

  2. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  3. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V.

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  4. Microorganisms for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  5. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  6. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  7. Engineered microorganisms capable of producing target compounds under anaerobic conditions

    DOEpatents

    Buelter, Thomas; Meinhold, Peter; Feldman, Reid M. Renny; Hawkins, Andrew C.; Urano, Jun; Bastian, Sabine; Arnold, Frances

    2012-01-17

    The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

  8. Biocorrosion produced by Thiobacillus-like microorganisms.

    PubMed

    López, A I; Marín, I; Amils, R

    1994-01-01

    Biocorrosion can be produced by many different microorganisms through diverse mechanisms. The biocorrosion produced by acidophilic microorganisms of the genus Thiobacillus is based on the production of sulfuric acid and ferric ion from pyrites or related mineral structures, as a result of the chemolithotrophic metabolism of these microorganisms. The products of this aerobic respiration are also powerful oxidant elements, which can produce chemical oxidations of other metallic structures. The Tinto River, a very unusual extremophilic habitat (pH around 2, and high concentration of ferric ion), product of the growth of strict chemolithotrophic microorganisms, is discussed as a model case. PMID:7946115

  9. Biocorrosion produced by Thiobacillus-like microorganisms.

    PubMed

    López, A I; Marín, I; Amils, R

    1994-01-01

    Biocorrosion can be produced by many different microorganisms through diverse mechanisms. The biocorrosion produced by acidophilic microorganisms of the genus Thiobacillus is based on the production of sulfuric acid and ferric ion from pyrites or related mineral structures, as a result of the chemolithotrophic metabolism of these microorganisms. The products of this aerobic respiration are also powerful oxidant elements, which can produce chemical oxidations of other metallic structures. The Tinto River, a very unusual extremophilic habitat (pH around 2, and high concentration of ferric ion), product of the growth of strict chemolithotrophic microorganisms, is discussed as a model case.

  10. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, J.L.; Clausen, E.C.

    1992-12-22

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H[sub 2]O and/or CO[sub 2] and H[sub 2] in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate. 3 figs.

  11. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, James L.; Clausen, Edgar C.

    1992-01-01

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H.sub.2 O and/or CO.sub.2 and H.sub.2 in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate.

  12. Biosurfactants Produced by Marine Microorganisms with Therapeutic Applications

    PubMed Central

    Gudiña, Eduardo J.; Teixeira, José A.; Rodrigues, Lígia R.

    2016-01-01

    Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments. PMID:26901207

  13. Biosurfactants Produced by Marine Microorganisms with Therapeutic Applications.

    PubMed

    Gudiña, Eduardo J; Teixeira, José A; Rodrigues, Lígia R

    2016-02-01

    Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments. PMID:26901207

  14. Biosurfactants Produced by Marine Microorganisms with Therapeutic Applications.

    PubMed

    Gudiña, Eduardo J; Teixeira, José A; Rodrigues, Lígia R

    2016-02-18

    Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

  15. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  16. Engineering biofuel tolerance in non-native producing microorganisms.

    PubMed

    Jin, Hu; Chen, Lei; Wang, Jiangxin; Zhang, Weiwen

    2014-01-01

    Large-scale production of renewable biofuels through microbiological processes has drawn significant attention in recent years, mostly due to the increasing concerns on the petroleum fuel shortages and the environmental consequences of the over-utilization of petroleum-based fuels. In addition to native biofuel-producing microbes that have been employed for biofuel production for decades, recent advances in metabolic engineering and synthetic biology have made it possible to produce biofuels in several non-native biofuel-producing microorganisms. Compared to native producers, these non-native systems carry the advantages of fast growth, simple nutrient requirements, readiness for genetic modifications, and even the capability to assimilate CO2 and solar energy, making them competitive alternative systems to further decrease the biofuel production cost. However, the tolerance of these non-native microorganisms to toxic biofuels is naturally low, which has restricted the potentials of their application for high-efficiency biofuel production. To address the issues, researches have been recently conducted to explore the biofuel tolerance mechanisms and to construct robust high-tolerance strains for non-native biofuel-producing microorganisms. In this review, we critically summarize the recent progress in this area, focusing on three popular non-native biofuel-producing systems, i.e. Escherichia coli, Lactobacillus and photosynthetic cyanobacteria.

  17. Biofuel and chemical production by recombinant microorganisms via fermentation of proteinaceous biomass

    DOEpatents

    Liao, James C.; Cho, Kwang Myung; Yan, Yajun; Huo, Yixin

    2016-03-15

    Provided herein are metabolically modified microorganisms characterized by having an increased keto-acid flux when compared with the wild-type organism and comprising at least one polynucleotide encoding an enzyme that when expressed results in the production of a greater quantity of a chemical product when compared with the wild-type organism. The recombinant microorganisms are useful for producing a large number of chemical compositions from various nitrogen containing biomass compositions and other carbon sources. More specifically, provided herein are methods of producing alcohols, acetaldehyde, acetate, isobutyraldehyde, isobutyric acid, n-butyraldehyde, n-butyric acid, 2-methyl-1-butyraldehyde, 2-methyl-1-butyric acid, 3-methyl-1-butyraldehyde, 3-methyl-1-butyric acid, ammonia, ammonium, amino acids, 2,3-butanediol, 1,4-butanediol, 2-methyl-1,4-butanediol, 2-methyl-1,4-butanediamine, isobutene, itaconate, acetoin, acetone, isobutene, 1,5-diaminopentane, L-lactic acid, D-lactic acid, shikimic acid, mevalonate, polyhydroxybutyrate (PHB), isoprenoids, fatty acids, homoalanine, 4-aminobutyric acid (GABA), succinic acid, malic acid, citric acid, adipic acid, p-hydroxy-cinnamic acid, tetrahydrofuran, 3-methyl-tetrahydrofuran, gamma-butyrolactone, pyrrolidinone, n-methylpyrrolidone, aspartic acid, lysine, cadeverine, 2-ketoadipic acid, and/or S-adenosyl-methionine (SAM) from a suitable nitrogen rich biomass.

  18. Recombinant protein vaccines produced in insect cells.

    PubMed

    Cox, Manon M J

    2012-02-27

    The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises. PMID:22265860

  19. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

  20. Six Siderophore-Producing Microorganisms Identified in Biological Soil Crusts

    NASA Astrophysics Data System (ADS)

    Noonan, K.; Anbar, A. D.; Garcia-Pichel, F.; Poret-peterson, A. T.; Hartnett, H. E.

    2011-12-01

    Biological soil crusts (BSCs) are diverse microbial communities that colonize soils in arid and semi-arid environments. Cyanobacteria in BSCs are pioneer organisms that increase ecosystem habitability by providing fixed carbon (C) and nitrogen (N) as well as by reducing water run-off and increasing infiltration. Photosynthesis and N fixation, in particular, require a variety of metals in large quantities, and yet, metals are predominantly insoluble in the environments where BSCs thrive. Therefore, BSC organisms must have efficient strategies for extracting metals from soil minerals. We hypothesized that BSC microbes, particularly the cyanobacteria, produce siderophores to serve their metal-acquisition needs. Siderophores are small organic compounds that bind Fe with high affinity and are produced by a variety of microorganisms, including cyanobacteria. Most siderophores bind Fe, primarily; however, some can also bind Mo, V, and Cu. Soil siderophores are released by microbes to increase the solubility of metals from minerals and to facilitate microbial uptake. Thus, siderophores serve as chemical weathering agents and provide a direct link between soil microbes and minerals. Studying siderophore production in BSCs provides insight into how BSCs tackle the challenge of acquiring insoluble metals, and may help conservationists determine useful fertilizers for BSC growth by facilitating metal acquisition. Biological soil crusts were collected near Moab, UT. Soil slurries were prepared in deionized water and transferred to modified BG-11 agar plates. The O-CAS agar plate assay was used to screen organisms for siderophore production. Siderophore producing microbes were isolated and identified by16S rRNA gene sequencing. Cultures were then grown in 3 L batch cultures under metal limitation, and siderophore presence was monitored using the traditional liquid CAS assay. After siderophore detection, cells were removed by centrifugation, organic compounds were separated using

  1. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  2. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  3. Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology.

    PubMed

    Liang, Ming-Hua; Jiang, Jian-Guo

    2013-10-01

    With the depletion of global petroleum and its increasing price, biodiesel has been becoming one of the most promising biofuels for global fuels market. Researchers exploit oleaginous microorganisms for biodiesel production due to their short life cycle, less labor required, less affection by venue, and easier to scale up. Many oleaginous microorganisms can accumulate lipids, especially triacylglycerols (TAGs), which are the main materials for biodiesel production. This review is covering the related researches on different oleaginous microorganisms, such as yeast, mold, bacteria and microalgae, which might become the potential oil feedstocks for biodiesel production in the future, showing that biodiesel from oleaginous microorganisms has a great prospect in the development of biomass energy. Microbial oils biosynthesis process includes fatty acid synthesis approach and TAG synthesis approach. In addition, the strategies to increase lipids accumulation via metabolic engineering technology, involving the enhancement of fatty acid synthesis approach, the enhancement of TAG synthesis approach, the regulation of related TAG biosynthesis bypass approaches, the blocking of competing pathways and the multi-gene approach, are discussed in detail. It is suggested that DGAT and ME are the most promising targets for gene transformation, and reducing PEPC activity is observed to be beneficial for lipid production.

  4. Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology.

    PubMed

    Liang, Ming-Hua; Jiang, Jian-Guo

    2013-10-01

    With the depletion of global petroleum and its increasing price, biodiesel has been becoming one of the most promising biofuels for global fuels market. Researchers exploit oleaginous microorganisms for biodiesel production due to their short life cycle, less labor required, less affection by venue, and easier to scale up. Many oleaginous microorganisms can accumulate lipids, especially triacylglycerols (TAGs), which are the main materials for biodiesel production. This review is covering the related researches on different oleaginous microorganisms, such as yeast, mold, bacteria and microalgae, which might become the potential oil feedstocks for biodiesel production in the future, showing that biodiesel from oleaginous microorganisms has a great prospect in the development of biomass energy. Microbial oils biosynthesis process includes fatty acid synthesis approach and TAG synthesis approach. In addition, the strategies to increase lipids accumulation via metabolic engineering technology, involving the enhancement of fatty acid synthesis approach, the enhancement of TAG synthesis approach, the regulation of related TAG biosynthesis bypass approaches, the blocking of competing pathways and the multi-gene approach, are discussed in detail. It is suggested that DGAT and ME are the most promising targets for gene transformation, and reducing PEPC activity is observed to be beneficial for lipid production. PMID:23685199

  5. Method of enhancing oil recovery by use of exopolymer producing microorganisms

    SciTech Connect

    Thompson, B.G.; Jack, T.R.

    1984-07-17

    A method of controlling and selectively reducing the permeability of zones in an oil-bearing underground formation which have higher permeabilities than the surrounding zones by injection of exopolymer producing microorganisms into the oil-bearing formation is described. The exopolymer production of the microorganisms used in the method is controlled by regulation of the availability to the microorganisms of a compound such as sucrose which triggers exopolymer production.

  6. Organic acid-tolerant microorganisms and uses thereof for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-05-06

    Organic acid-tolerant microorganisms and methods of using same. The organic acid-tolerant microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid. Further modifications to the microorganisms such as increasing expression of malonyl-CoA reductase and/or acetyl-CoA carboxylase provide or increase the ability of the microorganisms to produce 3HP. Methods of generating an organic acid with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers include replacing acsA or homologs thereof in cells with genes of interest and selecting for the cells comprising the genes of interest with amounts of organic acids effective to inhibit growth of cells harboring acsA or the homologs.

  7. Volatile dimethyl polonium produced by aerobic marine microorganisms.

    PubMed

    Bahrou, Andrew S; Ollivier, Patrick R L; Hanson, Thomas E; Tessier, Emmanuel; Amouroux, David; Church, Thomas M

    2012-10-16

    The production of volatile polonium (Po(v)), a naturally occurring radioactive element, by pure cultures of aerobic marine tellurite-resistant microorganisms was investigated. Rhodotorula mucilaginosa, a carotogenic yeast, and a Bacillus sp. strain, a Gram-positive bacterium, generated approximately one and 2 orders of magnitude, respectively, greater amounts of Po(v) compared to the other organisms tested. Gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS) analysis identified dimethyl polonide (DMPo) as the predominant volatile Po compound in culture headspace of the yeast. This species assignment is based on the exact relation between GC retention times and boiling points of this and other Group VI B analogues (S, Se, and Te). The extent of the biotic Po(v) production correlates exponentially with elevated particulate Po (Po(p)): dissolved Po (Po(aq)) ratios in the cultures, consistent with efficient Po bioaccumulation. Further experimentation demonstrated that some abiotic Po(v) generation is possible. However, high-level Po(v) generation in these cultures is predominantly biotic.

  8. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    PubMed

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

  9. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    PubMed

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses. PMID:27029122

  10. Genetic structure of a novel biofuel-producing microorganism community.

    PubMed

    de Felice, Bruna; Blasi, Vito Onofrio; de Castro, Olga; Cennamo, Paola; Martino, Laura; Trifuoggi, Marco; Condorelli, Valerio; di Onofrio, Valeria; Guida, Marco

    2012-08-01

    Biofuels are an important alternative, renewable source of energy in the face of the ongoing depletion of fossil fuels. Cheese whey is a dairy industry waste characterized by high lactose concentration, which represents a significant environmental problem. Bio-ethanol production by cheese whey could be an effective nonvegetable source for renewable energy production. Here, we report the isolation of a mixed microbial population, able to produce ethanol as main fermentation product from fermenting whey. The microbial consortium has been used to perform a batch fermentation of crude whey in both anoxic and hypoxic conditions. Maximum ethanol concentrations achieved in this study was obtained using the mixed culture in hypoxic conditions, grown at pH 4 and 30 °C, with ethanol production yield of 60 g/L. Our research has pointed out an alternative way to both dispose and valorize cheese whey, a dairy by-product that could cause water pollution and harm to the environment if not properly treated.

  11. Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids.

    PubMed

    Gong, Yangmin; Wan, Xia; Jiang, Mulan; Hu, Chuanjiong; Hu, Hanhua; Huang, Fenghong

    2014-10-01

    Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have received growing attention due to their significant roles in human health. Currently the main source of these nutritionally and medically important fatty acids is marine fish, which has not met ever-increasing global demand. Microorganisms are an important alternative source also being explored. Although many microorganisms accumulate omega-3 LC-PUFAs naturally, metabolic engineering might still be necessary for significantly improving their yields. Here, we review recent research involving the engineering of microorganisms for production of omega-3 LC-PUFAs, including eicospentaenoic acid and docosohexaenoic acid. Both reconstitution of omega-3 LC-PUFA biosynthetic pathways and modification of existing pathways in microorganisms have demonstrated the potential to produce high levels of omega-3 LC-PUFAs. However, the yields of omega-3 LC-PUFAs in host systems have been substantially limited by potential metabolic bottlenecks, which might be caused partly by inefficient flux of fatty acid intermediates between the acyl-CoA and different lipid class pools. Although fatty acid flux in both native and heterologous microbial hosts might be controlled by several acyltransferases, evidence has suggested that genetic manipulation of one acyltransferase alone could significantly increase the accumulation of LC-PUFAs. The number of oleaginous microorganisms that can be genetically transformed is increasing, which will advance engineering efforts to maximize LC-PUFA yields in microbial strains.

  12. Anti-Candida and anti-Cryptococcus antifungal produced by marine microorganisms.

    PubMed

    El Amraoui, B; El Amraoui, M; Cohen, N; Fassouane, A

    2014-12-01

    In order to search for antifungal from biological origin, we performed a screening of marine microorganisms isolated from seawater, seaweed, sediment and marine invertebrates collected from different coastal areas of the Moroccan Atlantic Ocean. The antifungal activities of these isolates were investigated against the pathogenic yeasts involved in medical mycology. Whole cultures of 34 marine microorganisms were screened for antifungal activities using the method of agar diffusion against four yeasts. The results showed that among the 34 isolates studied, 13 (38%) strains have antifungal activity against at least one out of four yeast species, 11 isolates have anti-Candida albicans CIP 48.72 activity, 12 isolates have anti-C. albicans CIP 884.65 activity, 13 isolates have anti-Cryptococcus neoformans activity and only 6 isolates are actives against Candida tropicalis R2 resistant to nystatin and amphotericin B. Nine isolates showed strong fungicidal activity. Fourteen microorganisms were identified and assigned to the genera Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Chromobacterium, Enterococcus, Pantoea, and Pseudomonas. Due to a competitive role for space and nutrient, the marine microorganisms could produce more antimicrobials; therefore these marine microorganisms were expected to be potential resources of natural products such as those we research: anti-Candida and anti-Cryptococcus fungicides.

  13. North Western Spain hot springs are a source of lipolytic enzyme-producing thermophilic microorganisms.

    PubMed

    Deive, Francisco J; Alvarez, María S; Sanromán, M Angeles; Longo, Maria A

    2013-02-01

    Several hot springs in Galicia (North Western Spain) have been investigated as potential sources of lipolytic enzyme-producing thermophilic microorganisms. After isolating 12 esterase producing strains, 9 of them were assured to be true lipase producers, and consequently grown in submerged cultures, obtaining high extracellular activities by two of them. Furthermore, a preliminary partial characterization of the crude lipase, obtained by ultrafiltration of the cell-free culture supernatant, was carried out at several pH and temperature values. It is outstanding that several enzymes turned out to be multiextremozymes, since they had their optimum temperature and pH at typical values from thermoalkalophiles. The thermal stability in aqueous solution of the crude enzymes was also assayed, and the influence of some potential enzyme stabilizing compounds was tested. Finally, the viability of the selected microorganisms has been demonstrated at bioreactor scale.

  14. North Western Spain hot springs are a source of lipolytic enzyme-producing thermophilic microorganisms.

    PubMed

    Deive, Francisco J; Alvarez, María S; Sanromán, M Angeles; Longo, Maria A

    2013-02-01

    Several hot springs in Galicia (North Western Spain) have been investigated as potential sources of lipolytic enzyme-producing thermophilic microorganisms. After isolating 12 esterase producing strains, 9 of them were assured to be true lipase producers, and consequently grown in submerged cultures, obtaining high extracellular activities by two of them. Furthermore, a preliminary partial characterization of the crude lipase, obtained by ultrafiltration of the cell-free culture supernatant, was carried out at several pH and temperature values. It is outstanding that several enzymes turned out to be multiextremozymes, since they had their optimum temperature and pH at typical values from thermoalkalophiles. The thermal stability in aqueous solution of the crude enzymes was also assayed, and the influence of some potential enzyme stabilizing compounds was tested. Finally, the viability of the selected microorganisms has been demonstrated at bioreactor scale. PMID:22763779

  15. Effect of enzyme producing microorganisms on the biomass of epigeic earthworms (eisenia fetida) in vermicompost.

    PubMed

    Hong, Sung Wook; Lee, Ju Sam; Chung, Kun Sub

    2011-05-01

    We analyzed the bacterial community structure of the intestines of earthworms and determined the effect of enzyme producing microorganisms on the biomass of earthworms in vermicompost. Fifty-seven bacterial 16S rDNA clones were identified in the intestines of earthworms by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Entomoplasma somnilux and Bacillus licheniformis were the dominant microorganisms; other strains included Aeromonas, Bacillus, Clostridium, Ferrimonas, and uncultured bacteria. Among these strains, Photobacterium ganghwense, Aeromonas hydrophila, and Paenibacillus motobuensis were enzyme-producing microorganisms. In the mixtures that were inoculated with pure cultures of A. hydrophila WA40 and P. motobuensis WN9, the highest survival rate was 100% and the average number of earthworms, young earthworms, and cocoons were 10, 4.00-4.33, and 3.00-3.33, respectively. In addition, P. motobuensis WN9 increased the growth of earthworms and production of casts in the vermicompost. These results show that earthworms and microorganisms have a symbiotic relationship.

  16. Milk production in lactating buffalo receiving recombinantly produced bovine somatotropin.

    PubMed

    Ludri, R S; Upadhyay, R C; Singh, M; Guneratne, J R; Basson, R P

    1989-09-01

    Thirty healthy Murrah buffalo (Bubalus bubalis) in their second to fourth lactations were selected from the herd at the National Dairy Research Institute, Karnal, Haryana, India, for use in a 35-d study to determine the effects of recombinantly produced bovine somatotropin on milk production, milk composition, and dry matter intake. Treatments were daily injections of 0, 25, or 50 mg somatotropin per animal for 14 d. All buffalo consumed green chopped fodder ad libitum plus a predetermined quantity of concentrate mixture to each animal, based on individual milk production during the 14-d pretreatment period. The quantity of concentrate mixture fed to each buffalo was not altered during the study. Net increase in milk volume for groups receiving 25 and 30 mg somatotropin was 16.8 and 29.5% over controls. Milk composition, DM intake, and body weights were not affected by treatment. PMID:2592642

  17. Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms.

    PubMed

    Ezeji, Thaddeus; Milne, Caroline; Price, Nathan D; Blaschek, Hans P

    2010-02-01

    Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.

  18. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  19. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  20. Identification of carotenoids with high antioxidant capacity produced by extremophile microorganisms.

    PubMed

    Mandelli, Fernanda; Miranda, Viviane S; Rodrigues, Eliseu; Mercadante, Adriana Z

    2012-04-01

    In this study, the carotenoids produced by the extremophile microorganisms Halococcus morrhuae, Halobacterium salinarium and Thermus filiformis were separated and identified by high-performance liquid chromatography connected to a diode array detector and a tandem mass spectrometer. The in vitro scavenging capacity of the carotenoid extracts against radical and non-radical species was evaluated. In halophilic microorganisms, the following carotenoids were identified: bacterioruberin, bisanhydrobacterioruberin, trisanhydrobacterioruberin and their derivatives. In the thermophilic bacterium, the carotenoids all-trans-zeaxanthin, zeaxanthin monoglucoside, thermozeaxanthins and thermobiszeaxanthins were identified. The antioxidant capacities of the carotenoid extracts of H. morrhuae (trolox equivalent antioxidant capacity = 5.07 and IC(50) = 0.85 μg mL(-1)) and H. salinarium (trolox equivalent antioxidant capacity = 5.28 and IC(50) = 0.84 μg mL(-1)) were similar and higher than those of the bacterium T. filiformis (trolox equivalent antioxidant capacity = 2.87 and IC(50) = 2.41 μg mL(-1)). This difference is related to the presence of acyclic carotenoids with both large numbers of conjugated double bounds and of hydroxyl groups in the major carotenoid of the halophilic microorganisms.

  1. A simple plate-assay for the screening of L-malic acid producing microorganisms.

    PubMed

    Peleg, Y; Rokem, J S; Goldberg, I

    1990-02-01

    A simple plate-assay has been developed to screen microorganisms for L-malic acid production. Acid producing organisms were identified, after microbial colony growth on media containing glucose or fumaric acid as sole carbons sources, by formation of a dark halo of formazan. The halo was observed when the plate was covered with a soft agar overlay containing NAD(+)-malate dehydrogenase, NAD+, phenazine methosulfate (PMS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay developed is simple, specific for L-malic acid and therefore can be used to identify L-malic acid producing filamentous fungi using glucose as carbon source (e.g. Aspergillus strains). The assay is also applicable for screening bacteria with high fumarase activity, able to convert fumaric acid to L-malic acid.

  2. Evaluation of terrestrial microcosms for detection, fate, and survival analysis of genetically engineered microorganisms and their recombinant genetic material

    SciTech Connect

    Fredrickson, J.K.; Seidler, R.J.

    1989-02-01

    The research included in this document represents the current scientific information available regarding the applicability of terrestrial microcosms and related methodologies for evaluating detection methods and the fate and survival of microorganisms in the environment. The three terrestrial microcosms described in this document were used to evaluate the survival and fate of recombinant bacteria in soils and in association with plant surfaces and insects and their transport through soil with percolating water and root systems, and to test new methods and procedures to improve detection and enumeration of bacteria in soil. Simple (potting soil composed of peat mix and perlite, lacking environmental control and monitoring) and complex microcosms (agricultural soil with partial control and monitoring of environmental conditions) were demonstrated to be useful tools for preliminary assessments of microbial viability in terrestrial ecosystems. These studies evaluated the survival patterns of Enterobacter cloacae (pBR322) in soil and on plant surfaces and the ingestion of this same microorganism by cutworms and survival in the foregut and frass. The Versacore microcosm design was used to monitor the fate and competitiveness of genetically engineered bacteria in soil. Both selective media and gene probes were used successfully to follow the fate of two recombinant Pseudomonas sp. introduced into Versacore microcosms. Intact soil-core microcosms were employed to evaluate the fate and transport of genetically altered Azospirillum sp. and Pseudomonas sp. in soil and the plant rhizosphere. The usefulness of these various microcosms as a tool for risk assessment is underscored by the ease in obtaining soil from a proposed field release site to evaluate subsequent GEM fate and survival.

  3. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    PubMed Central

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

  4. Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome.

    PubMed

    Pozuelo, Marta; Panda, Suchita; Santiago, Alba; Mendez, Sara; Accarino, Anna; Santos, Javier; Guarner, Francisco; Azpiroz, Fernando; Manichanh, Chaysavanh

    2015-01-01

    The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity was associated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS.

  5. Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome

    PubMed Central

    Pozuelo, Marta; Panda, Suchita; Santiago, Alba; Mendez, Sara; Accarino, Anna; Santos, Javier; Guarner, Francisco; Azpiroz, Fernando; Manichanh, Chaysavanh

    2015-01-01

    The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity was associated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS. PMID:26239401

  6. Method for producing aldehyde from CO.sub.2

    DOEpatents

    Liao, James C.; Atsumi, Shota

    2015-09-29

    The invention provides recombinant microorganisms capable of producing isobutyraldehyde using CO.sub.2 as a carbon source. The invention further provides methods of preparing and using such microorganisms to produce isobutyraldehyde.

  7. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  8. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  9. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  10. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  11. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation...

  12. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  13. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus.

    PubMed

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  14. The Influence of Siderophores Produced by Alkaliphilic Microorganisms on Iron and Metal Contaminant Speciation and Solubility

    NASA Astrophysics Data System (ADS)

    Aiken, A. M.; Peyton, B. M.; Petersen, J. N.; Apel, W. A.; Camper, A. K.

    2003-12-01

    Halomonas campisalis strain 4A has been identified as capable of producing siderophores under halo-alkaliphilic growth conditions. Because of the scarcity of iron under the alkaline conditions in which Halomonas campisalis thrives, we hypothesize that the siderophores secreted by Halomonas campisalis and other alkaliphilic bacteria will have a stronger affinity for binding and solubilizing ferrous iron than siderophores produced by mesophilic bacteria. Siderophore production by Halomonas campisalis was confirmed through the use of the chrome azural S (CAS) agar plate method which showed a red orange halo around the bacterial colonies indicative of siderophore production. The siderophores were found to be produced under conditions of both high salinity and pH with a salt concentrations ranging from 0.4 - 1.8 M NaCl and pH ranging from 8 - 11. The siderophores produced have been determined to be of the hydroxamate class via the Csaky method. A negative response to the Arnow assay indicated that the siderophore produced does not contain any catechol moieties in its chemical structure. It was found that maximum siderophore production was equivalent to approximately 400 mM desferrioxamine and occurred during mid stationary phase. Similar results were found at pH 8, 10 and 11. A purification scheme was developed that involved an initial extraction of the siderophore from the growth medium into benzyl alcohol followed by precipitation with diethyl ether. Additional purification was achieved via ion exchange chromatography and size exclusion chromatography. Final purification was achieved via HPLC. The structure of the purified siderophore was analyzed via LC/MS/MS equipped with an ESI source. To date, few studies have included the siderophores produced by microorganisms capable of tolerating highly saline and alkaline environments. In addition to unique structure and high affinity for iron, it is further hypothesized that siderophores from alkaliphilic bacteria will also

  15. Selection and characterization of microorganisms utilizing thaxtomin A, a phytotoxin produced by streptomyces scabies

    PubMed

    Doumbou; Akimov; Beaulieu

    1998-11-01

    Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the gamma variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.

  16. Whole cell electrochemistry of electricity-producing microorganisms evidence an adaptation for optimal exocellular electron transport.

    PubMed

    Busalmen, Juan Pablo; Esteve-Nuñez, Abraham; Feliu, Juan Miguel

    2008-04-01

    The mechanism(s) by which electricity-producing microorganisms interact with an electrode is poorly understood. Outer membrane cytochromes and conductive pili are being considered as possible players, but the available information does not concur to a consensus mechanism yet. In this work we demonstrate that Geobacter sulfurreducens cells are able to change the way in which they exchange electrons with an electrode as a response to changes in the applied electrode potential. After several hours of polarization at 0.1 V Ag/AgCl-KCl (saturated), the voltammetric signature of the attached cells showed a single redox pair with a formal redox potential of about -0.08 V as calculated from chronopotentiometric analysis. A similar signal was obtained from cells adapted to 0.4 V. However, new redox couples were detected after conditioning at 0.6 V. A large oxidation process beyond 0.5 V transferring a higher current than that obtained at 0.1 V was found to be associated with two reduction waves at 0.23 and 0.50 V. The apparent equilibrium potential of these new processes was estimated to be at about 0.48 V from programmed current potentiometric results. Importantly, when polarization was lowered again to 0.1 V for 18 additional hours, the signals obtained at 0.6 V were found to greatly diminish in amplitude, whereas those previously found at the lower conditioning potential were recovered. Results clearly show the reversibility of cell adaptation to the electrode potential and pointto the polarization potential as a key variable to optimize energy production from an electricity producing population.

  17. Whole cell electrochemistry of electricity-producing microorganisms evidence an adaptation for optimal exocellular electron transport.

    PubMed

    Busalmen, Juan Pablo; Esteve-Nuñez, Abraham; Feliu, Juan Miguel

    2008-04-01

    The mechanism(s) by which electricity-producing microorganisms interact with an electrode is poorly understood. Outer membrane cytochromes and conductive pili are being considered as possible players, but the available information does not concur to a consensus mechanism yet. In this work we demonstrate that Geobacter sulfurreducens cells are able to change the way in which they exchange electrons with an electrode as a response to changes in the applied electrode potential. After several hours of polarization at 0.1 V Ag/AgCl-KCl (saturated), the voltammetric signature of the attached cells showed a single redox pair with a formal redox potential of about -0.08 V as calculated from chronopotentiometric analysis. A similar signal was obtained from cells adapted to 0.4 V. However, new redox couples were detected after conditioning at 0.6 V. A large oxidation process beyond 0.5 V transferring a higher current than that obtained at 0.1 V was found to be associated with two reduction waves at 0.23 and 0.50 V. The apparent equilibrium potential of these new processes was estimated to be at about 0.48 V from programmed current potentiometric results. Importantly, when polarization was lowered again to 0.1 V for 18 additional hours, the signals obtained at 0.6 V were found to greatly diminish in amplitude, whereas those previously found at the lower conditioning potential were recovered. Results clearly show the reversibility of cell adaptation to the electrode potential and pointto the polarization potential as a key variable to optimize energy production from an electricity producing population. PMID:18504979

  18. The role of microaerophilic Fe-oxidizing micro-organisms in producing banded iron formations.

    PubMed

    Chan, C S; Emerson, D; Luther, G W

    2016-09-01

    Despite the historical and economic significance of banded iron formations (BIFs), we have yet to resolve the formation mechanisms. On modern Earth, neutrophilic microaerophilic Fe-oxidizing micro-organisms (FeOM) produce copious amounts of Fe oxyhydroxides, leading us to wonder whether similar organisms played a role in producing BIFs. To evaluate this, we review the current knowledge of modern microaerophilic FeOM in the context of BIF paleoenvironmental studies. In modern environments wherever Fe(II) and O2 co-exist, microaerophilic FeOM proliferate. These organisms grow in a variety of environments, including the marine water column redoxcline, which is where BIF precursor minerals likely formed. FeOM can grow across a range of O2 concentrations, measured as low as 2 μm to date, although lower concentrations have not been tested. While some extant FeOM can tolerate high O2 concentrations, many FeOM appear to prefer and thrive at low O2 concentrations (~3-25 μm). These are similar to the estimated dissolved O2 concentrations in the few hundred million years prior to the 'Great Oxidation Event' (GOE). We compare biotic and abiotic Fe oxidation kinetics in the presence of varying levels of O2 and show that microaerophilic FeOM contribute substantially to Fe oxidation, at rates fast enough to account for BIF deposition. Based on this synthesis, we propose that microaerophilic FeOM were capable of playing a significant role in depositing the largest, most well-known BIFs associated with the GOE, as well as afterward when global O2 levels increased. PMID:27392195

  19. Allelochemicals produced by Caribbean macroalgae and cyanobacteria have species-specific effects on reef coral microorganisms

    NASA Astrophysics Data System (ADS)

    Morrow, K. M.; Paul, V. J.; Liles, M. R.; Chadwick, N. E.

    2011-06-01

    Coral populations have precipitously declined on Caribbean reefs while algal abundance has increased, leading to enhanced competitive damage to corals, which likely is mediated by the potent allelochemicals produced by both macroalgae and benthic cyanobacteria. Allelochemicals may affect the composition and abundance of coral-associated microorganisms that control host responses and adaptations to environmental change, including susceptibility to bacterial diseases. Here, we demonstrate that extracts of six Caribbean macroalgae and two benthic cyanobacteria have both inhibitory and stimulatory effects on bacterial taxa cultured from the surfaces of Caribbean corals, macroalgae, and corals exposed to macroalgal extracts. The growth of 54 bacterial isolates was monitored in the presence of lipophilic and hydrophilic crude extracts derived from Caribbean macroalgae and cyanobacteria using 96-well plate bioassays. All 54 bacterial cultures were identified by ribotyping. Lipophilic extracts from two species of Dictyota brown algae inhibited >50% of the reef coral bacteria assayed, and hydrophilic compounds from Dictyota menstrualis particularly inhibited Vibrio bacteria, a genus associated with several coral diseases. In contrast, both lipo- and hydrophilic extracts from 2 species of Lyngbya cyanobacteria strongly stimulated bacterial growth. The brown alga Lobophora variegata produced hydrophilic compounds with broad-spectrum antibacterial effects, which inhibited 93% of the bacterial cultures. Furthermore, bacteria cultured from different locations (corals vs. macroalgae vs. coral surfaces exposed to macroalgal extracts) responded differently to algal extracts. These results reveal that extracts from macroalgae and cyanobacteria have species-specific effects on the composition of coral-microbial assemblages, which in turn may increase coral host susceptibility to disease and result in coral mortality.

  20. Biologically produced bifunctional recombinant protein nanoparticles for immunoassays.

    PubMed

    Jääskeläinen, Anu; Harinen, Reija-Riitta; Soukka, Tero; Lamminmäki, Urpo; Korpimäki, Teemu; Virta, Marko

    2008-02-01

    Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and self-assembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles. PMID:18179181

  1. Synthesis of a tricyclic bisguanidine compound structurally related to saxitoxin and its identification in paralytic shellfish toxin-producing microorganisms.

    PubMed

    Tsuchiya, Shigeki; Cho, Yuko; Konoki, Keiichi; Nagasawa, Kazuo; Oshima, Yasukatsu; Yotsu-Yamashita, Mari

    2015-05-18

    We recently reported the chemical synthesis and identification of the genetically predicted biosynthetic intermediates of saxitoxin (STX), including a 2-aminoimidazole-bearing monoguanidine compound (Int-C'2) in two paralytic shellfish toxin (PST)-producing microorganisms. In this study, we achieved the direct conversion of Int-C'2 into a tricyclic bisguanidine compound (called Cyclic-C'), which is structurally related to STX, through oxidative intramolecular guanidine transfer to 2-aminoimidazole catalyzed by Pd/C under basic conditions in air. By using HPLC-MS analysis, Cyclic-C' was also identified in the PST-producing microorganisms, suggesting that Cyclic-C' is either another biosynthetic intermediate or a shunt product of PSTs. In addition, a weak inhibitory activity of Cyclic-C' to the voltage-gated sodium channels was detected by using a cell-based assay.

  2. Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

    PubMed

    Perveen, Zakia; Ando, Hitomi; Ueno, Akio; Ito, Yukiya; Yamamoto, Yusuke; Yamada, Yohko; Takagi, Tomoko; Kaneko, Takako; Kogame, Kazuhiro; Okuyama, Hidetoshi

    2006-02-01

    A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism.

  3. Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

    PubMed

    Perveen, Zakia; Ando, Hitomi; Ueno, Akio; Ito, Yukiya; Yamamoto, Yusuke; Yamada, Yohko; Takagi, Tomoko; Kaneko, Takako; Kogame, Kazuhiro; Okuyama, Hidetoshi

    2006-02-01

    A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism. PMID:16489498

  4. Vaccination against Anthrax with Attenuated Recombinant Strains of Bacillus anthracis That Produce Protective Antigen

    PubMed Central

    Barnard, John P.; Friedlander, Arthur M.

    1999-01-01

    The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro. PMID:9916059

  5. A rapid, efficient and sensitive plate assay for detection and screening of l-asparaginase-producing microorganisms.

    PubMed

    Mahajan, Richi V; Saran, Saurabh; Saxena, Rajendra K; Srivastava, Ayush K

    2013-04-01

    l-Asparaginase-producing microbes are conventionally screened on phenol red l-asparagine-containing plates. However, sometimes the contrast of the zone obtained (between yellow and pink) is not very sharp and distinct. In the present investigation, an improved method for screening of the microorganisms producing extracellular l-asparaginase is reported wherein bromothymol blue (BTB) is incorporated as pH indicator in l-asparagine-containing medium instead of phenol red. Plates containing BTB at acidic pH are yellow and turn dark blue at alkaline pH. Thus, a dense dark blue zone is formed around microbial colonies producing l-asparaginase, differentiating between enzyme producers and non-producers. The present method is more sensitive and accurate than the conventional method for screening of both fungi and bacteria producing extracellular l-asparaginase. Furthermore, BTB gives a transient green colour at neutral pH (7.0) and dark blue colour at higher pH 8.0-9.0, indicating the potency of the microorganism for l-asparaginase production. PMID:23398626

  6. Antibiotic producing microorganisms from River Wiwi, Lake Bosomtwe and the Gulf of Guinea at Doakor Sea Beach, Ghana

    PubMed Central

    2012-01-01

    Background Microorganisms have provided a wealth of metabolites with interesting activities such as antimicrobial, antiviral and anticancer. In this study, a total of 119 aquatic microbial isolates from 30 samples (taken from water bodies in Ghana) were screened by the agar-well diffusion method for ability to produce antibacterial-metabolites. Results Antibacterial activity was exhibited by 27 of the isolates (14 bacteria, 9 actinomycetes and 4 fungi) against at least one of the indicator microorganisms: Enterococcus faecalis (ATCC 29212), Bacillus thuringiensis (ATCC 13838), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Proteus vulgaris (NCTC 4635) and Bacillus Subtilis (NCTC 10073). A sea isolate MAI2 (identified as a strain of Pseudomonas aeruginosa) exhibited the highest antibacterial activity (lowest zone of inhibition = 22 mm). The metabolites of MAI2 extracted with chloroform were stable to heat and gave minimum inhibitory concentrations ranging between 250 and 2000 μg/ml. Bioautography of the extract revealed seven active components. Conclusion This study has therefore uncovered the potential of water bodies in the West African sub-region as reservoirs of potent bioactive metabolite producing microorganisms. PMID:23072432

  7. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  8. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H.C.

    1998-07-14

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  9. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

    PubMed Central

    Gasser, Brigitte; Saloheimo, Markku; Rinas, Ursula; Dragosits, Martin; Rodríguez-Carmona, Escarlata; Baumann, Kristin; Giuliani, Maria; Parrilli, Ermenegilda; Branduardi, Paola; Lang, Christine; Porro, Danilo; Ferrer, Pau; Tutino, Maria Luisa; Mattanovich, Diethard; Villaverde, Antonio

    2008-01-01

    Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. PMID:18394160

  10. Interspecific protoplast fusion in Streptomyces--selection of thermotolerant antibiotic-producing recombinant.

    PubMed

    Qi, H Y; Zheng, Y X

    1990-01-01

    The thermotolerant fusants were obtained after interspecific protoplast fusion between S. qingfengmyceticus M15S (SMr, stop growth at 39 degrees C, producing qingfingmycin with wide antimicrobial spectrum) and S. hygroscopicus var. jinggangensis *75 (SMs, grow well at 42 degrees C, producing jingganmycin of antifungus) by directly selecting from the regeneration plates containing SM 100 micrograms/ml and incubated at 42 degrees C. The fusion frequency was about 10(-5) -10(-4). The stable thermotolerant recombinants with antimicrobial activity were obtained. The properties of their products were quite different from that of the parents (Qm, Jm). The antimicrobial substance produced by recombinant F6-6 consists of two components: one has acid-alkaline indicator property; the other is fluorescent under UV light. The antimicrobial products of F1-16, F1-38 and FM3-32 have absorption peaks at 274nm, which suggests that a cytosine moiety may be present in their molecules.

  11. Recent development of two chitinase inhibitors, Argifin and Argadin, produced by soil microorganisms

    PubMed Central

    Hirose, Tomoyasu; Sunazuka, Toshiaki; Ōmura, Satoshi

    2010-01-01

    Chitin, the second most abundant polysaccharide in nature, occurs in fungi, some algae and many invertebrates, including insects. Thus, chitin synthesis and degradation could represent specific targets for fungicides and insecticides. Chitinases hydrolyze chitin into oligomers of N-acetyl-d-glucosamine at key points in the life cycles of organisms, consequently, chitinase inhibitors have become subject of increasing interest. This review covers the development of two chitinase inhibitors of natural origin, Argifin and Argadin, isolated from the cultured broth of microorganisms in our laboratory. In particular, the practical total synthesis of these natural products, the synthesis of lead compounds via computer-aided rational molecular design, and discovery methods that generate only highly-active compounds using a kinetic target(chitinase)-guided synthesis approach (termed in situ click chemistry) are described. PMID:20154467

  12. Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity

    DOEpatents

    Premuzic, E.T.; Lin, M.

    1996-02-20

    This invention relates to the preparation of new, modified organisms, through challenge growth processes, that are viable in the extreme temperature, pressure and pH conditions and salt concentrations of an oil reservoir and that are suitable for use in microbial enhanced oil recovery. The modified microorganisms of the present invention are used to enhance oil recovery and remove sulfur compounds and metals from the crude oil. The processes are comprised of steps which successively limit the carbon sources and increase the temperature, pressure and salinity of the media. This is done until microbial strains are obtained that are capable of growing in essentially crude oil as a carbon source and at a temperature range from about 70 C to 90 C, at a pressure range from about 2,000 to 2,500 psi and at a salinity range from about 1.3 to 35%. 68 figs.

  13. Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity

    DOEpatents

    Premuzic, Eugene T.; Lin, Mow

    1996-02-20

    This invention relates to the preparation of new, modified organisms, through challenge growth processes, that are viable in the extreme temperature, pressure and pH conditions and salt concentrations of an oil reservoir and that are suitable for use in microbial enhanced oil recovery. The modified microorganisms of the present invention are used to enhance oil recovery and remove sulfur compounds and metals from the crude oil. The processes are comprised of steps which successively limit the carbon sources and increase the temperature, pressure and salinity of the media. This is done until microbial strains are obtained that are capable of growing in essentially crude oil as a carbon source and at a temperature range from about 70.degree. C. to 90.degree. C., at a pressure range from about 2,000 to 2,500 psi and at a salinity range from about 1.3 to 35%.

  14. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  15. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris.

    PubMed

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J; Pimentel, Luisa; Barrera, Luis A

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  16. In vitro carboxylation of a blood coagulation factor IX precursor produced by recombinant-DNA technology.

    PubMed

    Soute, B A; Balland, A; Faure, T; de la Salle, H; Vermeer, C

    1989-04-25

    Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.

  17. An emerging public health problem: acquired carbapenemase-producing microorganisms are present in food-producing animals, their environment, companion animals and wild birds.

    PubMed

    Guerra, Beatriz; Fischer, Jennie; Helmuth, Reiner

    2014-07-16

    Worldwide, the emergence and global spread of microorganisms with acquired carbapenemases is of great concern. The reservoirs for such organisms are increasing, not only in hospitals, but also in the community and environment. A new and important development is the presence of such organisms in livestock, companion animals and wildlife. During the last three years, carbapenemase-producing Escherichia coli, Salmonella spp. (VIM-1 producers) and Acinetobacter spp. (producing OXA-23 and NDM-1) in livestock animals (poultry, cattle and swine) and their environment have been reported. In addition, the isolation of NDM-1-producing E. coli, OXA-48 in E. coli and Klebsiella pneumoniae or OXA-23 in Acinetobacter spp. from companion animals (cats, dogs or horses) has also been observed. Other reports have described the presence of NDM-1-producing Salmonella isolated from wild birds, as well as OXA-23-like-producing Acinetobacter baumannii in ectoparasites. However, until now carbapenemase producers from foods have not been detected. For humans in contrast carbapenem-producing Salmonella isolates are increasingly reported. The real prevalence of carbapenemase-encoding genes in zoonotic bacteria or commensals from animals is unknown. Consequently, there is a need for intensified surveillance on the occurrence of carbapenemase-producing bacteria in the food chain and other animal sources in order to assist in the formulation of measures to prevent their potential spread.

  18. Activity and characterization of secondary metabolites produced by a new microorganism for control of plant diseases.

    PubMed

    Ko, Wen-Hsiung; Tsou, Yi-Jung; Lin, Mei-Ju; Chern, Lih-Ling

    2010-09-30

    Microorganisms capable of utilizing vegetable tissues for growth in soils were isolated and their vegetable broth cultures were individually sprayed directly on leaves to test their ability to control Phytophthora blight of bell pepper caused by Phytophthora capsici. Liquid culture of Streptomyces strain TKA-5, a previously undescribed species obtained in this study, displayed several desirable disease control characteristics in nature, including high potency, long lasting and ability to control also black leaf spot of spoon cabbage caused by Alternaria brassicicolca. The extract was fungicidal to P. capsici but fungistatic to A. brassicicola. It was stable at high temperature and high pH. However, after exposure to pH 2 for 24h, the extract was no longer inhibitory to P. capsici although it was still strongly inhibitory to A. brassicicola. After treatment with cation or anion exchange resins, the extract lost its inhibitory effect against P. capsici but not A. brassicicola. The results suggest that the extract contained two different kinds of inhibitory metabolites, one against P. capsici with both positive and negative charges on its molecule and another against A. brassicicola with no charges on its molecule. The inhibitory metabolites were soluble in ethanol or methanol but not in water, ether or chloroform. They were dialyzable in the membrane tubing with molecular weight cut-off of 10,000, 1000 or 500 but not 100, indicating that the inhibitors have a molecular weight between 500 and 100. Results also showed that both inhibitors are not proteins. PMID:20580869

  19. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin.

    PubMed

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L

    2013-01-01

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

  20. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    SciTech Connect

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C.; Remington, Mary P.; Pepinsky, R. Blake; Fishman, Paul S.; Brown, Robert H.; Francis, Jonathan W.

    2009-07-31

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  1. Using the second law of thermodynamics for enrichment and isolation of microorganisms to produce fuel alcohols or hydrocarbons.

    PubMed

    Kohn, Richard A; Kim, Seon-Woo

    2015-10-01

    Fermentation of crops, waste biomass, or gases has been proposed as a means to produce desired chemicals and renewable fuels. The second law of thermodynamics has been shown to determine the net direction of metabolite flow in fermentation processes. In this article, we describe a process to isolate and direct the evolution of microorganisms that convert cellulosic biomass or gaseous CO2 and H2 to biofuels such as ethanol, 1-butanol, butane, or hexane (among others). Mathematical models of fermentation elucidated sets of conditions that thermodynamically favor synthesis of desired products. When these conditions were applied to mixed cultures from the rumen of a cow, bacteria that produced alcohols or alkanes were isolated. The examples demonstrate the first use of thermodynamic analysis to isolate bacteria and control fermentation processes for biofuel production among other uses.

  2. Using the second law of thermodynamics for enrichment and isolation of microorganisms to produce fuel alcohols or hydrocarbons.

    PubMed

    Kohn, Richard A; Kim, Seon-Woo

    2015-10-01

    Fermentation of crops, waste biomass, or gases has been proposed as a means to produce desired chemicals and renewable fuels. The second law of thermodynamics has been shown to determine the net direction of metabolite flow in fermentation processes. In this article, we describe a process to isolate and direct the evolution of microorganisms that convert cellulosic biomass or gaseous CO2 and H2 to biofuels such as ethanol, 1-butanol, butane, or hexane (among others). Mathematical models of fermentation elucidated sets of conditions that thermodynamically favor synthesis of desired products. When these conditions were applied to mixed cultures from the rumen of a cow, bacteria that produced alcohols or alkanes were isolated. The examples demonstrate the first use of thermodynamic analysis to isolate bacteria and control fermentation processes for biofuel production among other uses. PMID:26231417

  3. Influence of Space-Flight Factors on the Properties of Microorganisms, Producers of Biologically Active Substances

    NASA Astrophysics Data System (ADS)

    Krasheninnikova, T. K.; Kanaeva, E. N.; Ukraintsev, A. D.; Smolyanaya, G. L.; Kuznetsov, N. V.; Panasyuk, M. I.; Shcherbakov, G. Ya.

    2001-07-01

    The following substances were isolated under the influence of space-flight factors in cosmic experiments aboard the Mirorbital station: an MIB-90 monoisolant, which is distinguished by its morphological and biochemical properties and enhanced productivity, was isolated from the Bacillus thuringiensis ssp. Kurstaki var. Z-52culture, which is a producer of the plant protection agent Lepidocide; and MIA-74 and MIP-89 monoisolants, which are highly active toward heavy petroleum fractions (C23 C33), were isolated from the Arthrobacter OC-1culture, which is a producer of biodegradants for petroleum.

  4. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  5. Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain.

    PubMed

    Zaghloul, Taha I; Embaby, Amira M; Elmahdy, Ahmed R

    2011-02-01

    OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

  6. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms

    PubMed Central

    Gorby, Yuri A.; Yanina, Svetlana; McLean, Jeffrey S.; Rosso, Kevin M.; Moyles, Dianne; Dohnalkova, Alice; Beveridge, Terry J.; Chang, In Seop; Kim, Byung Hong; Kim, Kyung Shik; Culley, David E.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Hill, Eric A.; Shi, Liang; Elias, Dwayne A.; Kennedy, David W.; Pinchuk, Grigoriy; Watanabe, Kazuya; Ishii, Shun’ichi; Logan, Bruce; Nealson, Kenneth H.; Fredrickson, Jim K.

    2006-01-01

    Shewanella oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA, and those that lacked a functional Type II secretion pathway displayed nanowires that were poorly conductive. These mutants were also deficient in their ability to reduce hydrous ferric oxide and in their ability to generate current in a microbial fuel cell. Nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to dissimilatory metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution. PMID:16849424

  7. Cyt toxins produced by Bacillus thuringiensis: a protein fold conserved in several pathogenic microorganisms.

    PubMed

    Soberón, Mario; López-Díaz, Jazmin A; Bravo, Alejandra

    2013-03-01

    Bacillus thuringiensis bacteria produce different insecticidal proteins known as Cry and Cyt toxins. Among them the Cyt toxins represent a special and interesting group of proteins. Cyt toxins are able to affect insect midgut cells but also are able to increase the insecticidal damage of certain Cry toxins. Furthermore, the Cyt toxins are able to overcome resistance to Cry toxins in mosquitoes. There is an increasing potential for the use of Cyt toxins in insect control. However, we still need to learn more about its mechanism of action in order to define it at the molecular level. In this review we summarize important aspects of Cyt toxins produced by Bacillus thuringiensis, including current knowledge of their mechanism of action against mosquitoes and also we will present a primary sequence and structural comparison with related proteins found in other pathogenic bacteria and fungus that may indicate that Cyt toxins have been selected by several pathogenic organisms to exert their virulence phenotypes.

  8. Innovative approaches for effective selection of lipase-producing microorganisms as whole cell catalysts for biodiesel production.

    PubMed

    Ciudad, Gustavo; Reyes, Isaac; Azócar, Laura; Briones, Reinaldo; Jorquera, Milko; Wick, Lukas Y; Navia, Rodrigo

    2011-07-01

    The high cost of commercial lipases limits their industrial application in the production of biodiesel or fatty acid methyl esters (FAME). This disadvantage has encouraged the search for lipase-producing microorganisms (LPMs) as potential whole cell catalysts for FAME production. The aim of this study, therefore, was to evaluate innovative procedures for easy selection and testing of LPMs as a low-cost whole cell catalyst, based on catalytic performance, methanol tolerance and physico-chemical cell surface properties. The latter (in particular the cell surface hydrophobicity and charge) were analyzed because of their crucial role in microbial adhesion to surfaces and the concomitant increase in cell immobilization and bioavailability of hydrophobic substrates. Biocatalysis experiments performed in the presence of nutrient, rapeseed oil and methanol were an effective tool for studying and identifying, in just two experiments, the capacity of different LPMs as biocatalysts in organic media, as well as the methanol tolerance of the cell and the lipase. This indicates the potential for using live microorganisms for FAME production. Another finding was that the inhibitory effect of methanol is more significant for lipase activity than LPM growth, indicating that the way in which alcohol is supplied to the reaction is a crucial step in FAME production by biocatalysts. According to these results, the application of these innovative assessments should simplify the search for new strains which are able to effectively catalyze the FAME production process.

  9. Fluorescence technique for on-line monitoring of state of hydrogen-producing microorganisms

    DOEpatents

    Seibert, Michael; Makarova, Valeriya; Tsygankov, Anatoly A.; Rubin, Andrew B.

    2007-06-12

    In situ fluorescence method to monitor state of sulfur-deprived algal culture's ability to produce H.sub.2 under sulfur depletion, comprising: a) providing sulfur-deprived algal culture; b) illuminating culture; c) measuring onset of H.sub.2 percentage in produced gas phase at multiple times to ascertain point immediately after anerobiosis to obtain H.sub.2 data as function of time; and d) determining any abrupt change in three in situ fluorescence parameters; i) increase in F.sub.t (steady-state level of chlorophyll fluorescence in light adapted cells); ii) decrease in F.sub.m', (maximal saturating light induced fluorescence level in light adapted cells); and iii) decrease in .DELTA.F/F.sub.m'=(F.sub.m'-F.sub.t)/F.sub.m' (calculated photochemical activity of photosystem II (PSII) signaling full reduction of plastoquinone pool between PSII and PSI, which indicates start of anaerobic conditions that induces synthesis of hydrogenase enzyme for subsequent H.sub.2 production that signal oxidation of plastoquinone pool asmain factor to regulate H.sub.2 under sulfur depletion.

  10. Preliminary characterization of biosurfactants produced by microorganisms isolated from refinery wastewaters.

    PubMed

    Yalçin, Emine; Ergene, Aysun

    2010-02-01

    Some bacterial strains isolated from refinery wastewaters were identified as Pseudomonas aeruginosa RWI, Pseudomonas putida RWII, Pseudomonas fluorescens RWIII and Burkholderia cepacia RWIV, and the biosurfactants produced by these strains were coded as BS-I, BS-II, BS-III and BS-IV, respectively. The bacterial strains were characterized by the following biochemical methods: Gram stain, oxidase activity, indol, lactose and growth at 42 degrees C. Biosurfactant production was evaluated by: emulsification activity, surface tension measurement and critical micelle concentration. Chemical characterization of the biosurfactants was done by: FTIR and analysis of carbohydrate, protein and lipid content. The biosurfactants showed good emulsification activity against different hydrocarbon sources. The initial surface tension of culture broth was determined as 67.3 mN/m, and production of BS-I, BS-II, BS-III and BS-IV lowered this value to 35.9, 49.2, 51.6 and 45.7 mN/m, respectively. The critical micelle concentration of the biosurfactants was found to be in the range 10-50 mg/L. From the results of this study it was observed that the refinery wastewaters are a suitable source for isolation of biosurfactant-producing bacteria, but are not a substrate for biosurfactant production.

  11. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

    PubMed

    Einsfeldt, Karen; Baptista, Isis Cavalcante; Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; Costa, Elaine Sobral da; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

  12. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

    PubMed Central

    Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; da Costa, Elaine Sobral; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells. PMID:27253887

  13. The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

    PubMed

    Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

  14. The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

    PubMed

    Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein. PMID:25115849

  15. Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33.

    PubMed

    Akond, Muhammad Ali; Matsuda, Yusuke; Ishimaru, Takayuki; Iwai, Ken; Saito, Akira; Kato, Akio; Tanaka, Shuhei; Kobayashi, Jun; Koga, Daizo

    2014-01-01

    A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of culture medium. The chitinase was purified by column chromatography after Endoglycosidase H treatment and then characterized. It showed properties similar to the original chitinase E purified from the yam tuber reported by Arakane et al. (2000). This Pichia-produced chitinase also showed strong lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability, optimum activity at higher temperature such as 70 °C, and high substrate affinity, indicating that one can use this Pichia-produced yam chitinase as a bio-control agent.

  16. Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

    PubMed Central

    Serra-Moreno, Ruth; Acosta, Sandra; Hernalsteens, Jean Pierre; Jofre, Juan; Muniesa, Maite

    2006-01-01

    Background The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. Results Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. Conclusion This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer. PMID:16984631

  17. Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

    PubMed

    Shene, C; Andrews, B A; Asenjo, J A

    2003-07-01

    Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these

  18. High throughput automated colorimetric method for the screening of l-lactic acid producing microorganisms.

    PubMed

    Liaud, Nadège; Navarro, David; Vidal, Nicolas; Sigoillot, Jean-Claude; Raouche, Sana

    2014-01-01

    Lactic acid is a valuable and fully degradable organic acid with promising applications in poly-lactic acid production (Taskila S and Ojamo, 2013 [1]). Despite their efficiency, the cost of the current lactic acid bio-processes is still an obstacle to this application (Miller et al., 2011 [2]). To ameliorate lactic acid producing strains, researchers are using mutations and metabolic engineering techniques, as well as medium optimization. All these studies necessitate a good and high throughput screening method. Currently, researchers mostly use HPLC methods which often necessitate sample preparation, are not stereospecific and do not allow high throughput. To help optimizing l-lactic acid production, we developed a high throughput colorimetric method inspired by the blood l-lactic acid detection method used for diagnosis (Lin et al., 1999 [3]).•Two sequential enzymatic reactions using l-lactate oxidase, peroxidase and ABTS (2,2'-azino-di-[3-ethylbenzthiazoine-sulfonate]), a chromogenic peroxidase substrate, are used to quantify l-lactate between 13.8 and 90 mg/l.•The accuracy of the method was ascertained before automation.•The method was successfully applied for the direct determination of l-lactate content in fungal culture supernatants.

  19. Genome Sequence of the Streptomycin-Producing Microorganism Streptomyces griseus IFO 13350▿ †

    PubMed Central

    Ohnishi, Yasuo; Ishikawa, Jun; Hara, Hirofumi; Suzuki, Hirokazu; Ikenoya, Miwa; Ikeda, Haruo; Yamashita, Atsushi; Hattori, Masahira; Horinouchi, Sueharu

    2008-01-01

    We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5′-GGA-3′, are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3(2) and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a γ-butyrolactone signaling molecule) regulatory cascade in S. griseus. PMID:18375553

  20. Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4.

    PubMed

    Hong, Eunsoo; Yoon, Sangyoung; Kim, Jinyoung; Kim, Eumin; Kim, Doosub; Rhie, Seunggyo; Ryu, Yeon-woo

    2013-06-01

    Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH(4))(2)SO(4) on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH(4))(2)SO(4), 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.

  1. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications. PMID:25447786

  2. The human potential of a recombinant pandemic influenza vaccine produced in tobacco plants.

    PubMed

    Jul-Larsen, Åsne; Madhun, Abdullah S; Brokstad, Karl A; Montomoli, Emanuele; Yusibov, Vidadi; Cox, Rebecca J

    2012-05-01

    Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNγ and low frequencies of CD4(+) T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B- and T cells. PMID:22634440

  3. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

    PubMed

    Feeney, Lauren; Carvalhal, Veronica; Yu, X Christopher; Chan, Betty; Michels, David A; Wang, Yajun Jennifer; Shen, Amy; Ressl, Jan; Dusel, Brendon; Laird, Michael W

    2013-04-01

    Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

  4. Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity.

    PubMed

    Bruce, C; Fitches, E C; Chougule, N; Bell, H A; Gatehouse, J A

    2011-07-01

    Conotoxins are a diverse collection of more than 50,000 peptides produced by predatory marine snails of the genus Conus in order to immobilize their prey. Many conotoxins modulate the activity of ion channels, and show high specificity to their targets; as a result, some have valuable pharmaceutical applications. However, obtaining active peptide is difficult and to date has only been achieved though natural collection, chemical synthesis, or the use of prokaryotic expression systems, which often have the disadvantage of requiring subsequent steps to correctly fold the peptide. This paper reports the production of a conotoxin, TxVIA from Conus textile, as a biologically active recombinant protein, using the yeast Pichia pastoris as expression host. The presence of the pro-peptide was found to be necessary for the expression of biologically active conotoxin. We also show that TxVIA is not, as previously reported, mollusc-specific, but also shows insecticidal activity when injected into lepidopteran (cabbage moth) and dipteran (house fly) larvae. In contrast, recombinant TxVIA was not found to be molluscicidal to the grey field slug Deroceras reticulatum. PMID:21640131

  5. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

    PubMed

    Wang, Haikuan; Wang, Xiaojie; Li, Xiaolu; Zhang, Yehong; Dai, Yujie; Guo, Changlu; Zheng, Heng

    2012-09-28

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters.

  6. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

    PubMed

    Wang, Haikuan; Wang, Xiaojie; Li, Xiaolu; Zhang, Yehong; Dai, Yujie; Guo, Changlu; Zheng, Heng

    2012-01-01

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters. PMID:23016923

  7. Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

    PubMed Central

    TAKAHASHI, Hitomi; TSUNAZAKI, Makoto; HAMANO, Takashi; TAKAHASHI, Masashi; OKUDA, Kiyoshi; INUMARU, Shigeki; OKANO, Akira; GESHI, Masaya; HIRAKO, Makoto

    2013-01-01

    ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ. PMID:24212505

  8. Proteomic differences in recombinant CHO cells producing two similar antibody fragments

    PubMed Central

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin

    2016-01-01

    ABSTRACT Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  9. Fermentation of a yeast producing A. niger glucose oxidase: scale-up, purification and characterization of the recombinant enzyme.

    PubMed

    De Baetselier, A; Vasavada, A; Dohet, P; Ha-Thi, V; De Beukelaer, M; Erpicum, T; De Clerck, L; Hanotier, J; Rosenberg, S

    1991-06-01

    We have developed a fermentation process to produce up to 3 grams per liter of active, secreted glucose oxidase from a recombinant Saccharomyces cerevisiae. Real-time size-exclusion HPLC analysis is used to monitor enzyme production during fermentation, and purification to more than 95 percent is obtained using only filtration methods. The recombinant enzyme is stable to higher temperatures and a wider pH range than the native Aspergillus niger enzyme, and is free of contaminating amylase, cellulase and catalase.

  10. Inhibition of microorganisms involved in deterioration of an archaeological site by silver nanoparticles produced by a green synthesis method.

    PubMed

    Carrillo-González, Rogelio; Martínez-Gómez, Miriam Araceli; González-Chávez, Ma Del Carmen A; Mendoza Hernández, José Carlos

    2016-09-15

    The Citadel, part of the pre-Hispanic city of Teotihuacan and listed as a World Heritage Site, harbors irreplaceable archaeological walls and murals. This city was abandoned by the 7th century and its potential deterioration represents a noteworthy loss of the world's cultural heritage. This research consisted of isolation and identification of bacteria and fungi contributing to this deterioration from walls of a pre-Hispanic city. In addition, silver nanoparticles (AgNP) produced, using a green synthesis method, were tested as potential inhibitors of microbes. AgNP of different sizes and concentrations were tested using in situ assays. Leaf aqueous extracts from two plants species (Foeniculum vulgare and Tecoma stans) and two extraction procedures were used in the NP synthesis. The potential of AgNP as preventive/corrective treatments to protect stucco materials from biodeterioration, as well as the microbial inhibition on three stone materials (stucco, basalt and calcite) was analyzed. Twenty-three bacterial species belonging to eight genera and fourteen fungal species belonging to seven genera were isolated from colored stains, patinas and biofilms produced on the surfaces of archaeological walls from the pre-Hispanic city, Teotihuacan. AgNP from F. vulgare were more effective for in vitro microbial growth inhibition than those from T. stans. Bacteria were less sensitive to AgNP than fungi; however, sensitivity mainly depended on the microbial strain and the plant extract used to prepare AgNP. The use of AgNP as a preventive or corrective treatment to decrease microbial colonization in three kinds of stone used in historical walls was successful. Calcite was more colonized by Alternaria alternata, but less by Pectobacterium carotovorum. This is the first study at different scales (in vitro and tests on different stone types) of inhibition of biodeterioration-causing microorganisms isolated from an archaeological site by green synthesized AgNP.

  11. Inhibition of microorganisms involved in deterioration of an archaeological site by silver nanoparticles produced by a green synthesis method.

    PubMed

    Carrillo-González, Rogelio; Martínez-Gómez, Miriam Araceli; González-Chávez, Ma Del Carmen A; Mendoza Hernández, José Carlos

    2016-09-15

    The Citadel, part of the pre-Hispanic city of Teotihuacan and listed as a World Heritage Site, harbors irreplaceable archaeological walls and murals. This city was abandoned by the 7th century and its potential deterioration represents a noteworthy loss of the world's cultural heritage. This research consisted of isolation and identification of bacteria and fungi contributing to this deterioration from walls of a pre-Hispanic city. In addition, silver nanoparticles (AgNP) produced, using a green synthesis method, were tested as potential inhibitors of microbes. AgNP of different sizes and concentrations were tested using in situ assays. Leaf aqueous extracts from two plants species (Foeniculum vulgare and Tecoma stans) and two extraction procedures were used in the NP synthesis. The potential of AgNP as preventive/corrective treatments to protect stucco materials from biodeterioration, as well as the microbial inhibition on three stone materials (stucco, basalt and calcite) was analyzed. Twenty-three bacterial species belonging to eight genera and fourteen fungal species belonging to seven genera were isolated from colored stains, patinas and biofilms produced on the surfaces of archaeological walls from the pre-Hispanic city, Teotihuacan. AgNP from F. vulgare were more effective for in vitro microbial growth inhibition than those from T. stans. Bacteria were less sensitive to AgNP than fungi; however, sensitivity mainly depended on the microbial strain and the plant extract used to prepare AgNP. The use of AgNP as a preventive or corrective treatment to decrease microbial colonization in three kinds of stone used in historical walls was successful. Calcite was more colonized by Alternaria alternata, but less by Pectobacterium carotovorum. This is the first study at different scales (in vitro and tests on different stone types) of inhibition of biodeterioration-causing microorganisms isolated from an archaeological site by green synthesized AgNP. PMID:27015961

  12. Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

    PubMed

    McCue, Justin; Kshirsagar, Rashmi; Selvitelli, Keith; Lu, Qi; Zhang, Mingxuan; Mei, Baisong; Peters, Robert; Pierce, Glenn F; Dumont, Jennifer; Raso, Stephen; Reichert, Heidi

    2015-07-01

    Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

  13. Design of a recombinant Escherichia coli for producing L-phenylalanine from glycerol.

    PubMed

    Thongchuang, Mayura; Pongsawasdi, Piamsook; Chisti, Yusuf; Packdibamrung, Kanoktip

    2012-10-01

    A recombinant Escherichia coli was engineered to produce the commercially important amino acid L-phenylalanine (L-Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L-Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L-Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L-Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L-Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5 l stirred-tank fermenter (37 °C, an aeration rate of 1 vvm, an agitation speed of 400 rpm). In the fermenter, the maximum concentration of L-Phe (366 mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L-Phe was only 76 % of the maximum value attained in the fermenter. PMID:22806734

  14. A New Defective Helper RNA to Produce Recombinant Sindbis Virus that Infects Neurons but does not Propagate.

    PubMed

    Kebschull, Justus M; Garcia da Silva, Pedro; Zador, Anthony M

    2016-01-01

    Recombinant Sindbis viruses are important tools in neuroscience because they combine rapid and high transgene expression with a capacity to carry large transgenes. Currently, two packaging systems based on the defective helper (DH) RNAs DH(26S)5'SIN and DH-BB(tRNA;TE12) are available for generating recombinant Sindbis virus that is neurotropic (able to infect neurons and potentially other cells). Both systems produce a fraction of viral particles that can propagate beyond the primary infected neuron. When injected into mouse brain, viruses produced using these DH RNAs produce transgene expression at the injection site, but also elsewhere in the brain. Such ectopic labeling caused recombinant Sindbis viruses to be classified as anterograde viruses with limited retrograde spread, and can complicate the interpretation of neuroanatomical and other experiments. Here we describe a new DH RNA, DH-BB(5'SIN;TE12ORF), that can be used to produce virus that is both neurotropic and propagation-incompetent. We show in mice that DH-BB(5'SIN;TE12ORF)-packaged virus eliminates infection of cells outside the injection site. We also provide evidence that ectopically labeled cells observed in previous experiments with recombinant Sindbis virus resulted from secondary infection by propagation-competent virus, rather than from inefficient retrograde spread. Virus produced with our new packaging system retains all the advantages of previous recombinant Sindbis viruses, but minimizes the risks of confounding results with unwanted ectopic labeling. It should therefore be considered in future studies in which a neurotropic, recombinant Sindbis virus is needed. PMID:27252627

  15. A New Defective Helper RNA to Produce Recombinant Sindbis Virus that Infects Neurons but does not Propagate

    PubMed Central

    Kebschull, Justus M.; Garcia da Silva, Pedro; Zador, Anthony M.

    2016-01-01

    Recombinant Sindbis viruses are important tools in neuroscience because they combine rapid and high transgene expression with a capacity to carry large transgenes. Currently, two packaging systems based on the defective helper (DH) RNAs DH(26S)5’SIN and DH-BB(tRNA;TE12) are available for generating recombinant Sindbis virus that is neurotropic (able to infect neurons and potentially other cells). Both systems produce a fraction of viral particles that can propagate beyond the primary infected neuron. When injected into mouse brain, viruses produced using these DH RNAs produce transgene expression at the injection site, but also elsewhere in the brain. Such ectopic labeling caused recombinant Sindbis viruses to be classified as anterograde viruses with limited retrograde spread, and can complicate the interpretation of neuroanatomical and other experiments. Here we describe a new DH RNA, DH-BB(5’SIN;TE12ORF), that can be used to produce virus that is both neurotropic and propagation-incompetent. We show in mice that DH-BB(5’SIN;TE12ORF)-packaged virus eliminates infection of cells outside the injection site. We also provide evidence that ectopically labeled cells observed in previous experiments with recombinant Sindbis virus resulted from secondary infection by propagation-competent virus, rather than from inefficient retrograde spread. Virus produced with our new packaging system retains all the advantages of previous recombinant Sindbis viruses, but minimizes the risks of confounding results with unwanted ectopic labeling. It should therefore be considered in future studies in which a neurotropic, recombinant Sindbis virus is needed. PMID:27252627

  16. Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, François; Chretien, Nathalie; Welsh, Elizabeth A.; Soares, Jason W.; Karatzas, Costas N.

    2002-01-01

    Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

  17. Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.

    PubMed

    Gludovacz, Elisabeth; Maresch, Daniel; Bonta, Maximilian; Szöllösi, Helen; Furtmüller, Paul G; Weik, Robert; Altmann, Friedrich; Limbeck, Andreas; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-06-10

    Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions.

  18. Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Funk, J.; Biber, N.; Schneider, M.; Hauser, E.; Enzenmüller, S.; Förtsch, C.

    2015-01-01

    In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death. PMID:25824835

  19. A Gateway recombination herpesvirus cloning system with negative selection that produces vectorless progeny.

    PubMed

    Kunec, Dusan; van Haren, Sandra; Burgess, Shane C; Hanson, Larry A

    2009-01-01

    Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines. PMID:18948138

  20. Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli.

    PubMed Central

    Burnette, W N; Arciniega, J L; Mar, V L; Burns, D L

    1992-01-01

    The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation. The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer. Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin. These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component. Images PMID:1587592

  1. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  2. Effects of organic acid producing microorganisms on the biodeterioration of an embedding matrix such as cement and on the transport and/or retardation of radionuclides

    SciTech Connect

    Libert, M.F.; Spor, H.; Sellier, R.; Trescinski, M.

    1993-12-31

    The authors` study concerns the effect of organic acids produced by microorganisms in a deep repository. In a first step, they study the conditions leading to cellulose degradation and organic acids production by cellulotytic microorganism: fermentative conditions lead to an increase of the organic acids production. Secondly, the complexation properties of uranium by these acids are shown. Finally, the effect of the organic acids on the degradation of cement, used as an engineered barrier or as an embedding matrix for nuclear waste is shown. The leaching of calcium is correlated to the pH of the solution and to organic acid production. Biodegradation of cement is expressed in terms of equivalent leach layer thickness in calcium.

  3. Microorganism immobilization

    DOEpatents

    Compere, Alicia L.; Griffith, William L.

    1981-01-01

    Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

  4. Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line.

    PubMed

    Voronina, E V; Seregin, Y A; Litvinova, N A; Shvets, V I; Shukurov, R R

    2016-01-01

    Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions. PMID:27652157

  5. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

    PubMed Central

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L.; Lee, Sang Yup

    2010-01-01

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  6. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.

    PubMed

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L; Lee, Sang Yup

    2010-08-10

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  7. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed

    Kilian, Oliver; Benemann, Christina S E; Niyogi, Krishna K; Vick, Bertrand

    2011-12-27

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology. PMID:22123974

  8. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed Central

    Kilian, Oliver; Benemann, Christina S. E.; Niyogi, Krishna K.; Vick, Bertrand

    2011-01-01

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology. PMID:22123974

  9. [Recombinant strain producing thermostable lipase from Thermomyces lanuginosus immobilized into nanocarbon silica matrices and properties of the prepared biocatalyzers].

    PubMed

    Kovalenko, G A; Beklemishev, A B; Perminova, L V; Chuenko, T V; Ivanov, I D; Moiseenkov, S I; Kuznetsov, V L

    2013-01-01

    Multicomponent composite biocatalyzers with lipolytic activity have been studied. These biocatalyzers were prepared through the immobilization of a recombinant producer strain of thermostable lipase from Thermomyces lanuginosus into SiO2 xerogel, which contains a nanocarbon component, i.e., multilayered carbon nanotubes with varying diameters, and also bulblike structured carbon nanospheres ("nanobulb"). The properties of lipase were studied both in cell suspensions of a recombinant producer strain constructed based on E. coli BL21(DE3) and in the immobilized state with regard to the structure and dispersibility of the nanocarbon component used in the composition of the biocatalyzers. It was shown that the recombinant intracellular lipase exerted its activity in a reaction of tributirin hydrolysis on average comprising 50 U/mg of dried cells and had a high level of thermostability. Upon heating in olive oil at 100 degrees C, the inactivation constant and the period of semi-inactivation comprised 6 x 10(-3) min(-1) and 2 h, respectively, exceeding by one order the thermostability of lipase in a buffer solution. Biocatalyzers that contained aggregated "thick" nanotubes with a diameter of 20-22 nm had the maximum initial activity-250 U/g. PMID:23882949

  10. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

    PubMed Central

    2013-01-01

    Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures. PMID:23557146

  11. Decreased fluidity of cell membranes causes a metal ion deficiency in recombinant Saccharomyces cerevisiae producing carotenoids.

    PubMed

    Liu, Peitong; Sun, Liang; Sun, Yuxia; Shang, Fei; Yan, Guoliang

    2016-04-01

    The genome-wide transcriptional responses of S. cerevisiae to heterologous carotenoid biosynthesis were investigated using DNA microarray analysis. The results show that the genes involved in metal ion transport were specifically up-regulated in the recombinant strain, and metal ions, including Cu(2+), Fe(2+), Mn(2+), and Mg(2+), were deficient in the recombinant strain compared to the ion content of the parent strain. The decrease in metal ions was ascribed to a decrease in cell membrane (CM) fluidity caused by lower levels of unsaturated fatty acids and ergosterol. This was confirmed by the observation that metal ion levels were restored when CM fluidity was increased by supplying linoleic acid. In addition, a 24.3 % increase in the β-carotene concentration was observed. Collectively, our results suggest that heterologous production of carotenoids in S. cerevisiae can induce cellular stress by rigidifying the CM, which can lead to a deficiency in metal ions. Due to the importance of CM fluidity in cellular physiology, maintaining normal CM fluidity might be a potential approach to improving carotenoid production in genetically engineered S. cerevisiae. PMID:26749524

  12. European community and US-FDA approval of recombinant human antithrombin produced in genetically altered goats.

    PubMed

    Adiguzel, Cafer; Iqbal, Omer; Demir, Muzaffer; Fareed, Jawed

    2009-12-01

    Thrombin and factor Xa play a central role in thrombogenesis in both medical and surgical patients. Antithrombin (AT) is the key inhibitor, which controls the action of these enzymes in hypercoagulable states. The AT concentrates prepared from human blood have been used to treat patients with thrombotic disorders and heparin resistance. The AT concentrates are prepared from pooled human plasma and beside limited supply, suffer from viral and other biological contaminants. The availability of recombinant human AT (rhAT) obtained from genetically engineered goats provide a biologically equivalent product that can be used in practically all indications where human AT is indicated including heparin resistance. Moreover, because of its high affinity to heparin and related drugs, recombinant AT can also be developed in further indications. On review of the preclinical and clinical data on the safety and efficacy, the European Union and U.S. Food and Drug Administration (US-FDA) have recently approved the use of rhAT in specified clinical indications.

  13. Nano-structured calcite produced by micro-organisms in ancient and modern loess in Chinese Loess Plateau

    NASA Astrophysics Data System (ADS)

    Xu, H.; Chen, T.; Lu, H.; Wang, X.

    2005-12-01

    The results from transmission electron microscopy (TEM) and field emission gun scanning microscopy (FEG-SEM) investigation show that there are calcite nano-fibers (CNFs) formed during pedogenic process. The CNFs are widely distributed in the loess and red clay samples of Caoxian, Luochuan, Lingtai, Lantian, and Xifeng profiles as well as the samples of modern surface loess soils in Chinese Loess Plateau. Diameters of all the NFCs are about 40 nm, the length of the CNFs ranges from tens nanometer to several micrometers. Elongation direction of NFCs is unusual near parallel (105)* or (115)*. Crystals of NFCs arrange as bird net like and lattice-like frameworks. X-ray EDS spectra show the weak peaks of magnesium, phosphorous, and sulfur. Our investigation indicates that CNFs are in pore space of loess and paleosol and made up most of carbonate except for caliche nodular layers. Concentration of NFCs in the loess layers are significantly higher than those of paleosol layers because of leaching of carbonate in the paleosol forming environment (warn and wet paleoclimate). The "nanobacteria-like CNFs are well crystalline calcite single crystals with smoothes surfaces. The morphologies of CNFs are very unusual and different from the calcite single crystals observed in most geological environments. The CNFs are directly related to microbial activities in both ancient and modern loess. It is proposed that the intervention of organic compounds derived from microbial activities control the formation of the calcite nano-fibers. Both morphology and bulk composition of CNFs indicate that the formation of the CNFs involves bio-organics derived from microorganisms in loess deposit environment. Formation conditions of the calcite nano-fibers may information about paleoclimate, paleo-environment and paleoecology. So, the discovery of CNFs in loess-paloesol sequences can provide a new route for reconstruct paleoclimate by oxygen and carbon isotope from the CNFs.

  14. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose. PMID:23300051

  15. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.

  16. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

    PubMed Central

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  17. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

    PubMed

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-Yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-Liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  18. Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants.

    PubMed

    Dicker, Martina; Tschofen, Marc; Maresch, Daniel; König, Julia; Juarez, Paloma; Orzaez, Diego; Altmann, Friedrich; Steinkellner, Herta; Strasser, Richard

    2016-01-01

    The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

  19. Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

    PubMed Central

    Dicker, Martina; Tschofen, Marc; Maresch, Daniel; König, Julia; Juarez, Paloma; Orzaez, Diego; Altmann, Friedrich; Steinkellner, Herta; Strasser, Richard

    2016-01-01

    The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans. PMID:26858738

  20. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  1. High Prevalence of Infectious Diseases and Drug-Resistant Microorganisms in Asylum Seekers Admitted to Hospital; No Carbapenemase Producing Enterobacteriaceae until September 2015

    PubMed Central

    Ravensbergen, Sofanne J.; Lokate, Mariëtte; Cornish, Darren; Kloeze, Eveline; Ott, Alewijn; Friedrich, Alex W.; van Hest, Rob; Akkerman, Onno W.; de Lange, Wiel C.; van der Werf, Tjip S.; Bathoorn, Erik

    2016-01-01

    Introduction The current refugee crisis emphasizes the need for information on infectious diseases and resistant microorganisms in asylum seekers with possible consequences for public health and infection control. Methods We collected data from asylum seekers admitted to our university hospital or who presented at the Emergency Department (n = 273). We collected general and demographic characteristics including country of origin, the reason of presentation, and the screening results of multi-drug resistant organisms. Results 67% of the patients were male with a median age of the study group of 24 years (IQR 15–33); 48% of the patients had an infectious disease—predominantly malaria with P. vivax or tuberculosis. Patients also reported with diseases which are less common—e.g. leishmaniasis, or even conditions rarely diagnosed in Europe—e.g. louse borne relapsing fever. A carriage rate of 31% for multi-drug resistant microorganisms (MDRO) was observed, with ESBL-expressing E.coli (n = 20) being the most common MDRO. No carriage of Carbapenemase Producing Enterobacteriaceae was found. Conclusion The current refugee crisis in Europe challenges hospitals to quickly identify and respond to communicable diseases and the carriage of MDRO. A rapid response is necessary to optimize the treatment of infectious diseases amongst asylum seekers to maximize infection control. PMID:27144599

  2. Use of Escherichia coli Nissle 1917 producing recombinant colicins for treatment of IBD patients.

    PubMed

    Kotłowski, Roman

    2016-08-01

    Patients with Crohn's Disease and Ulcerative Colitis infected with Adherent-Invasive Escherichia coli strains constitute the largest group among Inflammatory Bowel Disease subjects, when taking into account all known etiological agents of the disease. A possible link between these pathogenic bacteria and inflammation process has gained the confidence in recently published papers. Observed enteric neuroglial cells apoptosis and epithelial gaps of ileum are probably the key manifestations of inflammation, which has been shown in IBD patients in contrary to the samples taken from healthy individuals. The cascade of consecutive events from bacterial infection via inflammation to excessive apoptosis in IBD patients leads up to the aim of our hypothesis about designing of new therapeutic strategy directed to Adherent-Invasive E. coli strains. The main advantage of biological method, which will rely on application of E. coli Nissle 1917 strain as a carrier for specific recombinant colicins against AIEC strains, could probably cause a long-lasting remission of inflammation in CD and UC patients. PMID:27372848

  3. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

    PubMed

    Kim, Min-Ji; Park, Hyun Soo; Seo, Kyung Hye; Yang, Hyo-Jin; Kim, Sook-Kyung; Choi, Jun-Hyuk

    2013-01-01

    High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

  4. Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system.

    PubMed

    Lan, John Chi-Wei; Yeh, Chun-Yi; Wang, Chih-Chi; Yang, Yu-Hsuan; Wu, Ho-Shing

    2013-10-01

    Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg⁻¹, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.

  5. Sequence analysis of the gene for a novel superantigen produced by Yersinia pseudotuberculosis and expression of the recombinant protein

    SciTech Connect

    Yasuhiko, Ito; Abe, Jun; Kohsaka, Takao

    1995-06-01

    We previously reported that the Gram-negative bacterium Yersinia pseudotuberculosis produces a superantigen (YPM, Y. pseudotuberculosis-derived mitogen) that expands T cells bearing V{beta}s 3, 9, 13.1, and 13.2 in an MHC class II-dependent manner. Based on the previously determined N-terminal 23 amino acids of YPM (T-D-Y-D-N-T-L-N-S-I-P-S-L-R-I-P-N-I-A-T-Y-T-G- (one-letter code)), we cloned the ypm gene and analyzed the nucleotide sequence. The gene encodes a 151-amino acid protein with a 20-amino acid signal peptide at its N terminus. The recombinant YPM expressed by the cloned gene exerted a mitogenic activity on human PBMC at a concentration of approximately 1 pg/ml. T cells bearing V{beta} 13.3 were preferentially expanded as well as T cells bearing the same V{beta} repertoires stimulated by native YPM. T cells were stimulated by the recombinant YPM in the presence of either fixed or unfixed HLA class II-transfected mouse fibroblasts. Furthermore, sequence diversity in the junctional region of the TCR {beta}-chain containing the V{beta}3 element could be observed after stimulation by the recombinant YPM. These results indicate that YPM belongs to the category of superantigens and should be included as a novel member. The amino acid sequence of the mature protein showed no significant homology to other superantigens derived from Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes. This observation, together with the substantially smaller m.w. suggest that ypm must have evolved from a different ancestral gene. 67 refs., 7 figs., 5 tabs.

  6. Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology.

    PubMed

    Thomsson, Elisabeth; Persson, Linn; Grahn, Anna; Snäll, Johanna; Ekblad, Maria; Brunhage, Eva; Svensson, Frida; Jern, Christina; Hansson, Gunnar C; Bäckström, Malin; Bergström, Tomas

    2011-07-01

    A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV.

  7. Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem.

    PubMed

    Kagkli, Dafni-Maria; Tâche, Roselyne; Cogan, Timothy M; Hill, Colin; Casaregola, Serge; Bonnarme, Pascal

    2006-11-01

    Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from L-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher L-methionine demethiolating activity, resulting in a direct conversion of L-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.

  8. Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.

    PubMed

    Emmons, Thomas L; Mathis, Karl J; Shuck, Mary E; Reitz, Beverly A; Curran, Daniel F; Walker, Mark C; Leone, Joseph W; Day, Jacqueline E; Bienkowski, Michael J; Fischer, H David; Tomasselli, Alfredo G

    2009-06-01

    Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships

  9. SIV/HIV Nef recombinant virus (SHIVnef) produces simian AIDS in rhesus macaques.

    PubMed

    Mandell, C P; Reyes, R A; Cho, K; Sawai, E T; Fang, A L; Schmidt, K A; Luciw, P A

    1999-12-20

    The simian immunodeficiency virus (SIV) nef gene is an important determinant of viral load and acquired immunodeficiency syndrome (AIDS) in macaques. A role(s) for the HIV-1 nef gene in infection and pathogenesis was investigated by constructing recombinant viruses in which the nef gene of the pathogenic molecular clone SIVmac239 nef was replaced with either HIV-1sf2nef or HIV-1sf33nef. These chimeras, designated SHIV-2nef and SHIV-33nef, expressed HIV-1 Nef protein and replicated efficiently in cultures of rhesus macaque lymphoid cells. In two SHIV-2nef-infected juvenile rhesus macaques and in one of two SHIV-33nef-infected juvenile macaques, virus loads remained at low levels in both peripheral blood and lymph nodes in acute and chronic phases of infection (for >83 weeks). In striking contrast, the second SHIV-33nef-infected macaque showed high virus loads during the chronic stage of infection (after 24 weeks). CD4+ T-cell numbers declined dramatically in this latter animal, which developed simian AIDS (SAIDS) at 47-53 weeks after inoculation; virus was recovered at necropsy at 53 weeks and designated SHIV-33Anef. Sequence analysis of the HIV-1sf33 nef gene in SHIV-33Anef revealed four consistent amino acid changes acquired during passage in vivo. Interestingly, one of these consensus mutations generated a tyr-x-x-leu (Y-X-X-L) motif in the HIV-1sf33 Nef protein. This motif is characteristic of certain endocytic targeting sequences and also resembles a src-homology region-2 (SH-2) motif found in many cellular signaling proteins. Four additional macaques infected with SHIV-33Anef contained high virus loads, and three of these animals progressed to fatal SAIDS. Several of the consensus amino acid changes in Nef, including Y-X-X-L motif, were retained in these recipient animals exhibiting high virus load and disease. In summary, these findings indicate that the SHIV-33Anef chimera is pathogenic in rhesus macaques and that this approach, i.e., construction of

  10. Soft sensor control of metabolic fluxes in a recombinant Escherichia coli fed-batch cultivation producing green fluorescence protein.

    PubMed

    Gustavsson, Robert; Mandenius, Carl-Fredrik

    2013-10-01

    A soft sensor approach is described for controlling metabolic overflow from mixed-acid fermentation and glucose overflow metabolism in a fed-batch cultivation for production of recombinant green fluorescence protein (GFP) in Escherichia coli. The hardware part of the sensor consisted of a near-infrared in situ probe that monitored the E. coli biomass and an HPLC analyzer equipped with a filtration unit that measured the overflow metabolites. The computational part of the soft sensor used basic kinetic equations and summations for estimation of specific rates and total metabolite concentrations. Two control strategies for media feeding of the fed-batch cultivation were evaluated: (1) controlling the specific rates of overflow metabolism and mixed-acid fermentation metabolites at a fixed pre-set target values, and (2) controlling the concentration of the sum of these metabolites at a set level. The results indicate that the latter strategy was more efficient for maintaining a high titer and low variability of the produced recombinant GFP protein.

  11. Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

    PubMed

    Zhou, Yu; Shen, Chaoyun; Zhang, Chao; Zhang, Wei; Wang, Lili; Lan, Ke; Liu, Qingwei; Huang, Zhong

    2016-08-01

    Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development. PMID:27315772

  12. A Cell Line Producing Recombinant Nerve Growth Factor Evokes Growth Responses in Intrinsic and Grafted Central Cholinergic Neurons

    NASA Astrophysics Data System (ADS)

    Ernfors, Patrik; Ebendal, Ted; Olson, Lars; Mouton, Peter; Stromberg, Ingrid; Persson, Hakan

    1989-06-01

    The rat β nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

  13. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli

    PubMed Central

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V.; Patel, Bhumit A.; Yeh, Syun-Ru; Rousseau, Dennis L.; Crane, Brian R.

    2010-01-01

    Over-expression of heme binding proteins in E. coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His- ligated hemes. PMID:20303407

  14. High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

    PubMed Central

    Saez, Natalie J.; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud

    2014-01-01

    Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive

  15. High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.

    PubMed

    Saez, Natalie J; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud

    2014-07-30

    Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive

  16. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    PubMed

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

  17. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    PubMed

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity. PMID:25142154

  18. The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases.

    PubMed

    Pedraza-Reyes, M; Gutiérrez-Corona, F

    1997-10-01

    The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside. These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units. Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.

  19. Quantitative Determination of Bandpasses for Producing Vegetation Indices from Recombined NEON Hyperspectral Imagery

    NASA Astrophysics Data System (ADS)

    Hulslander, D.

    2015-12-01

    Hyperspectral imaging systems can be used to produce spectral reflectance curves giving rich information about composition, relative abundances of materials, mixes and combinations. However, as each spectral return from these systems is a vector with several hundred elements, they can be very difficult to process and analyze, and problemeatic to compare within, across, and between datasets over time and space. Vegetation indices (e.g. NDVI, ARVI, EVI, et al) attempt to combine spectral features in to single-value scores. When derived from calibrated and atmospherically compensated reflectance data, these indices can be quantitatively compared. Historically, these indices have been calculated from multispectral sensor data. These sensors have a handful (4 to 16 or so) of bandbasses ranging from 20 nm to 200 nm FWHM covering specific spectral regions for a variety of reasons, including both intended applications and system limitations. Hyperspectral sensors, however, cover the spectrum with many, many narrow (5 to 10 nm) bandpasses. This allows for analyses using the full, detailed spectral curve, or combination of the bands in to regions by averaging or in to composites using transforms or other techniques. This raises the question of exactly which bands should be used and combined in what manner for ideally deriving well-known vegetation indices typically made from multispectral data. In this study we use derivatives and other curve and signal analysis techniques to analyze vegetation reflectance spectra to quantitatively define optimal bandpasses for several vegetation indices and combine the 5 nm hypserspectral bandpasses of the NEON Imaging Spectrometer to synthesize them.

  20. [Requirements to a medical and biologic assessment and the hygienic control of the food production received from recombinant-DNA microorganisms].

    PubMed

    Sheveleva, S A; Efimmochkina, N R; Nesterenko, L N; Zigangirova, N A; Khovaev, A A; Naroditskiĭ, B S; Ivanov, G E; Tutel'ian, V A; Gintsburg, A L

    2008-01-01

    In work the characteristic of the created in the Russian Federation system of an estimation of safety of the foodstuff received from/or with use of genetically modified microorganisms (GMM) is given, at their admission to realization and the hygienic control of given production over a revolution. It is shown, that strategy of a safety at a stage of registration GMM, the established order and accepted control measures of the foodstuff received from/or with use GMM, in Russia their large-scale commercial use, and the normative-legal and methodical base based on the federal legislation on state regulation in the field of genetically engineering activity, about quality and effectively outstrip safety of foodstuff about protection of the rights of consumers, is harmonized with approaches of the international organizations.

  1. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

    PubMed

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-07-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

  2. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  3. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    PubMed

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

  4. Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

    PubMed

    Fernandez-del-Carmen, Asun; Juárez, Paloma; Presa, Silvia; Granell, Antonio; Orzáez, Diego

    2013-02-20

    The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.

  5. Pilot scale ex-situ bioremediation of heavily PAHs-contaminated soil by indigenous microorganisms and bioaugmentation by a PAHs-degrading and bioemulsifier-producing strain.

    PubMed

    Sun, Guang-Dong; Xu, Yang; Jin, Jing-Hua; Zhong, Zhi-Ping; Liu, Ying; Luo, Mu; Liu, Zhi-Pei

    2012-09-30

    This study aims at the remediation of heavily PAH-contaminated soil containing 375 mg of total PAHs per kilogram dry soil. Pilot scale bioremediation experiments were carried out by three approaches with contaminated soil from abandoned sites of Beijing Coking Plant using outdoor pot trials. The first approach was bioaugmentation with a bacterial strain which degrades PAH and produces bioemulsifier, the second approach comprised of biostimulation of indigenous microorganisms with supplementing nutrients and the last approach involved the combination of both biostimulation and bioaugmentation. An on-site land farming group was set as a control in which the total PAHs and 4-6 ring-PAHs were reduced by 23.4% and 10.1%, respectively after 175 days. Meanwhile, in the first approach group, the total PAHs and 4-6 ring-PAHs were reduced by 26.82% and 35.36%, respectively; in the second approach group both percentages were 33.9% and 11.0%, respectively; while in the third approach group, these pollutants were reduced by 43.9% and 55.0%, respectively. The results obtained suggested that biostimulation and bioaugmentation combined could significantly enhance the removal of PAHs in the contaminated soil.

  6. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    SciTech Connect

    Nguyen, Minh Vu Chuong; Zhang, Leilei; Lhomme, Stanislas; Mouz, Nicolas

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  7. Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.

    PubMed

    Bos, Ineke G A; de Bruin, Eric C; Karuntu, Yani A; Modderman, Piet W; Eldering, Eric; Hack, C Erik

    2003-05-30

    Therapeutic application of the serpin C1-inhibitor (C1-Inh) in inflammatory diseases like sepsis, acute myocardial infarction and vascular leakage syndrome seems promising, but large doses may be required. Therefore, a high-yield recombinant expression system for C1-Inh is very interesting. Earlier attempts to produce high levels of C1-Inh resulted in predominantly inactive C1-Inh. We describe the high yield expression of rhC1-Inh in Pichia pastoris, with 180 mg/l active C1-Inh at maximum. On average, 30 mg/l of 80-100% active C1-Inh was obtained. Progress curves were used to study the interaction with C1s, kallikrein, coagulation factor XIIa and XIa, and demonstrated that rhC1-Inh had the same inhibitory capacity as plasma C1-Inh. Structural integrity, as monitored via heat stability, was comparable despite differences in extent and nature of glycosylation. We conclude that the P. pastoris system is capable of high-level production of functionally and structurally intact human C1 inhibitor.

  8. Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium

    PubMed Central

    Galesi, Adriana L. L.; Aguiar, Marcelo A.; Astray, Renato M.; Augusto, Elisabeth F. P.

    2008-01-01

    Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 × 106 cells mL−1) was only obtained when Pluronic F68® percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10−7 cells. PMID:19003175

  9. Characterization of rabbit polyclonal sera against recombinant Shiga toxin and its subunits for detection of Stx-producing E. coli.

    PubMed

    Oloomi, Mana; Bouzari, Saeid

    2011-03-01

    Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera could also neutralize the cytotoxicity of the shiga toxin on vero cells. The neutralizing ability of the prepared sera was compared for different subunits. The neutralization of toxicity was observed by incubation of raised sera with the rStx or Shiga toxin from wild type strains. The inhibition of cell toxicity was shown by anti-A, anit-B and anti-AB antisera, separately. It was shown that anti-A antibody, more significantly recognized Stx producing strains, comparing to anti-B antibody. These sera from immunized rabbits were also used as specific antibodies in Enzyme-Linked Immunosorbant Assay (ELISA) for detection of Shiga toxin. It was demonstrated that the raised antibodies especially antibody against A subunit could be a useful tool for immunological diagnosis of STEC induced infection.

  10. Classifying Microorganisms.

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn J.; Lang, Michael; Goodmanis, Ben

    2002-01-01

    Focuses on an activity in which students sample air at school and generate ideas about how to classify the microorganisms they observe. The results are used to compare air quality among schools via the Internet. Supports the development of scientific inquiry and technology skills. (DDR)

  11. Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines.

    PubMed

    Yoo, Esther M; Yu, Li J; Wims, Letitia A; Goldberg, David; Morrison, Sherie L

    2010-01-01

    The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. These glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHO cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galalpha(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHO cells contained glycans that were more similar to those found on human IgA. Expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHO cells was significantly lower than that previously reported for serum IgA1. These data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.

  12. Anti-loxoscelic horse serum produced against a recombinant dermonecrotic protein of Brazilian Loxosceles intermedia spider neutralize lethal effects of Loxosceles laeta venom from Peru.

    PubMed

    Duarte, C G; Bonilla, C; Guimarães, G; Machado de Avila, R A; Mendes, T M; Silva, W; Tintaya, B; Yarleque, A; Chávez-Olórtegui, C

    2015-01-01

    In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.

  13. Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

    PubMed

    Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

    2015-02-01

    Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

  14. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    PubMed

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms. PMID:27180265

  15. Industrial Microorganisms.

    ERIC Educational Resources Information Center

    Phaff, Herman J.

    1981-01-01

    Describes industrially important yeasts, molds, bacteria, and actinomycetes. Discussed in detail are microbial products, such as primary metabolites, secondary metabolites, enzymes, and capsular polysaccharides. Traces the historical background of human cell culture, mentioning recombinant DNA research and hybridization of normal mammalian cells…

  16. Development of microorganisms for cellulose-biofuel consolidated bioprocessings: metabolic engineers’ tricks

    PubMed Central

    Mazzoli, Roberto

    2012-01-01

    Cellulose waste biomass is the most abundant and attractive substrate for “biorefinery strategies” that are aimed to produce high-value products (e.g. solvents, fuels, building blocks) by economically and environmentally sustainable fermentation processes. However, cellulose is highly recalcitrant to biodegradation and its conversion by biotechnological strategies currently requires economically inefficient multistep industrial processes. The need for dedicated cellulase production continues to be a major constraint to cost-effective processing of cellulosic biomass. Research efforts have been aimed at developing recombinant microorganisms with suitable characteristics for single step biomass fermentation (consolidated bioprocessing, CBP). Two paradigms have been applied for such, so far unsuccessful, attempts: a) “native cellulolytic strategies”, aimed at conferring high-value product properties to natural cellulolytic microorganisms; b) “recombinant cellulolytic strategies”, aimed to confer cellulolytic ability to microorganisms exhibiting high product yields and titers. By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product (i.e. butanol, ethanol, and hydrogen) biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes) benefit from stronger proximity effects and show enhanced synergy on insoluble substrates (i.e. crystalline cellulose) with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms. PMID:24688667

  17. Mesenchymal Stromal Cells Engineered to Produce IGF-I by Recombinant Adenovirus Ameliorate Liver Fibrosis in Mice

    PubMed Central

    Fiore, Esteban J.; Bayo, Juan M.; Garcia, Mariana G.; Malvicini, Mariana; Lloyd, Rodrigo; Piccioni, Flavia; Rizzo, Manglio; Peixoto, Estanislao; Sola, M. Beatriz; Atorrasagasti, Catalina; Alaniz, Laura; Camilletti, María A.; Enguita, Mónica; Prieto, Jesús; Aquino, Jorge B.

    2015-01-01

    Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis. PMID:25315017

  18. Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.

    PubMed

    Fiore, Esteban J; Bayo, Juan M; Garcia, Mariana G; Malvicini, Mariana; Lloyd, Rodrigo; Piccioni, Flavia; Rizzo, Manglio; Peixoto, Estanislao; Sola, M Beatriz; Atorrasagasti, Catalina; Alaniz, Laura; Camilletti, María A; Enguita, Mónica; Prieto, Jesús; Aquino, Jorge B; Mazzolini, Guillermo

    2015-03-15

    Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.

  19. Dissolved carbon dioxide determines the productivity of a recombinant hemagglutinin component of an influenza vaccine produced by insect cells.

    PubMed

    Meghrous, Jamal; Khramtsov, Nikolai; Buckland, Barry C; Cox, Manon M J; Palomares, Laura A; Srivastava, Indresh K

    2015-11-01

    Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 (<50, 50-100, 100-200, and >200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels > 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.

  20. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    PubMed

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  1. Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins.

    PubMed

    da Cunha, Nicolau B; Vianna, Giovanni R; da Almeida Lima, Thaina; Rech, Elíbio

    2014-01-01

    Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms. PMID:24376137

  2. [Biotechnology using modified microorganisms].

    PubMed

    Deshayes, A F

    1992-11-01

    Few microorganisms, as compare to their high diversity, are used for human needs. They can produce molecules of interest, process fermentation, protect crops, treat wastes or clean environment. Molecular technics and genetic engineering are new tools offer to geneticists which breed microorganisms for years. Using them, it is now possible, theoretically, to introduce any gene in any organism. Some examples are given concerning genetic modifications in yeasts and lactic acid bacteria to optimize agrofood processes and to improve nutritive and flavour characteristics of fermented products like bread, beer, wine, cheese, meat, vegetable juices... In spite of scientific and industrial interest of the new technologies, limiting factors can explain that genetically modified microorganisms are not routinely used in agrofood yet. First, risks assessment on human health and environment are still in debate, but their is a consensus, within the scientific community, to consider that new characteristics of improved microorganisms are more important than the technics used for their construction. Second, regulations turn out to impose constraints susceptible to discourage technological innovations. At least, the public perception about the new technologies appears, actually, as the major factor to limit their development.

  3. Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed.

    PubMed

    Palomares, Oscar; Monsalve, Rafael I; Rodríguez, Rosalía; Villalba, Mayte

    2002-05-01

    2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.

  4. A fluorescent assay amenable to measuring production of beta-D-glucuronides produced from recombinant UDP-glycosyl transferase enzymes.

    PubMed

    Trubetskoy, O V; Shaw, P M

    1999-05-01

    Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

  5. Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale.

    PubMed

    Fibi, M R; Bröker, M; Schulz, R; Johannsen, R; Zettlmeissl, G

    1991-08-01

    Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taq-polymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121 degrees C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10(3) per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120 degrees C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes.

  6. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    PubMed

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  7. Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

    PubMed Central

    Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone. PMID:23300841

  8. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment.

    PubMed

    Lima, Matheus S; Damasio, André R de L; Crnkovic, Paula M; Pinto, Marcelo R; da Silva, Ana M; da Silva, Jean C R; Segato, Fernando; de Lucas, Rosymar C; Jorge, João A; Polizeli, Maria de L T de M

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production.

  9. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment.

    PubMed

    Lima, Matheus S; Damasio, André R de L; Crnkovic, Paula M; Pinto, Marcelo R; da Silva, Ana M; da Silva, Jean C R; Segato, Fernando; de Lucas, Rosymar C; Jorge, João A; Polizeli, Maria de L T de M

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production. PMID:27199917

  10. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment

    PubMed Central

    Lima, Matheus S.; Damasio, André R. de L.; Crnkovic, Paula M.; Pinto, Marcelo R.; da Silva, Ana M.; da Silva, Jean C. R.; Segato, Fernando; de Lucas, Rosymar C.; Jorge, João A.; Polizeli, Maria de L. T. de M.

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60–80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production. PMID:27199917

  11. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  12. Recombinant scorpine produced using SUMO fusion partner in Escherichia coli has the activities against clinically isolated bacteria and inhibits the Plasmodium falciparum parasitemia in vitro.

    PubMed

    Zhang, Chao; He, Xinlong; Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel-nitrilotriacetic acid (Ni2+-NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+-NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  13. Expression and purification of biologically active rat bone morphogenetic protein-4 produced as inclusion bodies in recombinant Escherichia coli.

    PubMed

    Klösch, Burkhard; Fürst, Walter; Kneidinger, Rudolf; Schuller, Monika; Rupp, Barbara; Banerjee, Asmita; Redl, Heinz

    2005-10-01

    Rat bone morphogenetic protein-4 (rBMP-4) cDNA was cloned from rat osteoblasts by RT-PCR and expressed in E. coli. Monomeric, dimeric and polymeric forms of recombinant rat BMP-4 (rrBMP-4) were obtained from inclusion bodies after solubilization with urea. The dimer was separated from the remaining polymer and host cell contaminants using size exclusion chromatography. Furthermore, purified rrBMP-4 was stabilized at low urea concentration (40 mM) and at pH 8.5 through the addition of bovine serum albumin. Both, rrBMP-4 dimer and polymer were biologically active as tested by the induction of alkaline phosphatase activity in MC3T3-E1 cells.

  14. Transgenic Cows That Produce Recombinant Human Lactoferrin in Milk Are Not Protected from Experimental Escherichia coli Intramammary Infection▿

    PubMed Central

    Hyvönen, P.; Suojala, L.; Orro, T.; Haaranen, J.; Simola, O.; Røntved, C.; Pyörälä, S.

    2006-01-01

    This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows. PMID:16954396

  15. Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

    PubMed Central

    Bäckström, Malin; Link, Thomas; Olson, Fredrik J; Karlsson, Hasse; Graham, Rosalind; Picco, Gianfranco; Burchell, Joy; Taylor-Papadimitriou, Joyce; Noll, Thomas; Hansson, Gunnar C

    2003-01-01

    We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer. PMID:12950230

  16. Harmonic 'signatures' of microorganisms.

    PubMed

    Blake-Coleman, B C; Hutchings, M J; Silley, P

    1994-01-01

    The frequency/amplitude effect of various microorganisms exposed to periodic (time varying) electric fields, when proximate to immersed electrodes, has been studied using a novel analytical instrument. The harmonic distribution, in complex signals caused by cells exposed to harmonic free waveforms and occupying part of the electrode/suspension interface volume, was shown to be almost entirely due to the change in the standing interfacial transfer function by the (dielectrically nonlinear) presence of cells. Thus, the characteristic interfacial non-linearity is viewed as variable, being uniquely modulated by the presence of particular cells in the interfacial region. Little can be attributed to bulk (far field) effects. The tendency for subtle (characteristic) signal distortion to occur as a function of particulate (cell or molecular) occupancy of the near electrode interfacial region under controlled current conditions leads to the method of sample characterisation by harmonic (Fourier) analysis. We report here, as a sequel to our original studies (Hutchings et al., 1993; Hutchings and Blake-Coleman, 1993), preliminary results of the harmonic analysis of microbial suspensions under controlled signal conditions using a three-electrode configuration. These data provide three-dimensional graphical representations producing harmonic 'surfaces' for various microorganisms. Thus, cell type differences are characterised by their 'harmonic signature'. The visual distinction provided by these 'surface' forming three-dimensional plots is striking and gives a convincing impression of the ability to identify and enumerate specific microorganisms by acquisition of cell-modulated electrode interfacial Fourier spectra. PMID:8060593

  17. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins

    PubMed Central

    2012-01-01

    After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line. PMID:22531032

  18. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

    PubMed Central

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  19. Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).

    PubMed

    Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

    2013-10-01

    Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality.

  20. Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

    PubMed Central

    Paul, Matthew; Reljic, Rajko; Klein, Katja; Drake, Pascal MW; van Dolleweerd, Craig; Pabst, Martin; Windwarder, Markus; Arcalis, Elsa; Stoger, Eva; Altmann, Friedrich; Cosgrove, Catherine; Bartolf, Angela; Baden, Susan; Ma, Julian K-C

    2014-01-01

    Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb. PMID:25484063

  1. Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Luz, Daniela; Chen, Gang; Maranhão, Andrea Q.; Rocha, Leticia B.; Sidhu, Sachdev; Piazza, Roxane M. F.

    2015-01-01

    Background Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. PMID:25790467

  2. Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein.

    PubMed

    Mukai, Tetsu; Maeda, Yumi; Tamura, Toshiki; Matsuoka, Masanori; Tsukamoto, Yumiko; Makino, Masahiko

    2010-11-15

    To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.

  3. Spontaneous hybrids between native and exotic Rubus in the Western United States produce offspring both by apomixis and by sexual recombination.

    PubMed

    Clark, L V; Jasieniuk, M

    2012-11-01

    Facultative asexual reproduction is a trait commonly found in invasive species. With a combination of sexual and asexual reproductive modes, such species may adapt to new environments via sexual recombination during range expansion, while at the same time having the benefits of asexuality such as the maintenance of fitness effects that depend upon heterozygosity. In the Western United States, native species of Rubus (Rosaceae) reproduce sexually whereas exotic naturalized Rubus species reproduce by pseudogamous apomixis. We hypothesized that new asexual lineages of Rubus could arise from hybridization in this range. To detect hybridization between native and exotic Rubus, we genotyped 579 individuals collected across California, Oregon and Washington with eight nuclear microsatellites and two chloroplast markers. Principal Coordinate Analysis and Bayesian clustering revealed a limited amount of hybridization of the native R. ursinus with the exotic R. armeniacus and R. pensilvanicus, as well as cultivated varieties. Genetic distances between these hybrids and their offspring indicated that both R. ursinus × R. armeniacus and R. ursinus × R. pensilvanicus produced a mix of apomictic and sexual seeds, with sexual seeds being more viable. Although neither of these hybrid types is currently considered invasive, they model the early stages of evolution of new invasive lineages, given the potential for fixed heterosis and the generation of novel genotypes. The hybrids also retain the ability to increase their fitness via sexual recombination and natural selection. Mixed reproductive systems such as those described here may be an important step in the evolution of asexual invasive species.

  4. Spontaneous hybrids between native and exotic Rubus in the Western United States produce offspring both by apomixis and by sexual recombination

    PubMed Central

    Clark, L V; Jasieniuk, M

    2012-01-01

    Facultative asexual reproduction is a trait commonly found in invasive species. With a combination of sexual and asexual reproductive modes, such species may adapt to new environments via sexual recombination during range expansion, while at the same time having the benefits of asexuality such as the maintenance of fitness effects that depend upon heterozygosity. In the Western United States, native species of Rubus (Rosaceae) reproduce sexually whereas exotic naturalized Rubus species reproduce by pseudogamous apomixis. We hypothesized that new asexual lineages of Rubus could arise from hybridization in this range. To detect hybridization between native and exotic Rubus, we genotyped 579 individuals collected across California, Oregon and Washington with eight nuclear microsatellites and two chloroplast markers. Principal Coordinate Analysis and Bayesian clustering revealed a limited amount of hybridization of the native R. ursinus with the exotic R. armeniacus and R. pensilvanicus, as well as cultivated varieties. Genetic distances between these hybrids and their offspring indicated that both R. ursinus × R. armeniacus and R. ursinus × R. pensilvanicus produced a mix of apomictic and sexual seeds, with sexual seeds being more viable. Although neither of these hybrid types is currently considered invasive, they model the early stages of evolution of new invasive lineages, given the potential for fixed heterosis and the generation of novel genotypes. The hybrids also retain the ability to increase their fitness via sexual recombination and natural selection. Mixed reproductive systems such as those described here may be an important step in the evolution of asexual invasive species. PMID:22850699

  5. The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein.

    PubMed

    Danshiitsoodol, Narandalai; de Pinho, Catherine Azzariti; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

    2006-07-01

    Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain. PMID:16756991

  6. Purification and crystallization of the entire recombinant subunit E of the energy producer A(1)A(o) ATP synthase.

    PubMed

    Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

    2010-03-01

    A(1)A(o) ATP synthases are the major energy producers in archaea. Subunit E of the stator domain of the ATP synthase from Pyrococcus horikoshii OT3 was cloned, expressed and purified to homogeneity. The monodispersed protein was crystallized by vapour diffusion. A complete diffraction data set was collected to 3.3 A resolution with 99.4% completeness using a synchrotron-radiation source. The crystals belonged to space group I4, with unit-cell parameters a = 112.51, b = 112.51, c = 96.25 A, and contained three molecules in the asymmetric unit.

  7. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines.

    PubMed

    Khurana, Surender; Larkin, Christopher; Verma, Swati; Joshi, Manju B; Fontana, Juan; Steven, Alasdair C; King, Lisa R; Manischewitz, Jody; McCormick, William; Gupta, Rajesh K; Golding, Hana

    2011-08-01

    Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

  8. [Effect of electromagnetic fields on movement of microorganisms].

    PubMed

    Zel'nichenko, A T; Koval'chuk, V S; Posudin, Iu I

    1988-01-01

    Relationships between the motor activity and orientation of microorganisms and parameters of the electromagnetic field and of the microorganisms themselves were investigated. It has been shown that the type of microorganism and field amplitude produces the strongest influence on the behaviour of microorganisms in the fields. Theoretical relationships of the value of rotating moment and the field parameters, microorganism and environment were obtained. The results of the experiments well agree with the theory. PMID:3224110

  9. Engineering new metabolic capabilities in bacteria: lessons from recombinant cellulolytic strategies.

    PubMed

    Mazzoli, Roberto; Lamberti, Cristina; Pessione, Enrica

    2012-02-01

    Cellulose waste biomass is the most attractive substrate for 'biorefinery strategies' producing high-value products (e.g. fuels or plastics) by fermentation. However, traditional biomass bioconversions are economically inefficient multistep processes. Thus far, no microorganisms able to perform single-step fermentation into products (consolidated bioprocessing; CBP) have been isolated. Metabolic engineering is currently employed to develop recombinant microorganisms suitable for CBP. The heterologous expression of extracellular proteins (e.g. cellulases or hemicellulases) is the key feature of recombinant cellulolytic strategies, conferring cellulolytic ability to microorganisms exhibiting high product yields and titers. Although more molecular tools are becoming available, efficient heterologous expression of secreted proteins is still a challenge. The present review summarizes both bottlenecks and solutions of organism engineering for biomass biorefinery strategies. PMID:21930321

  10. [Modern Approaches to the Creation of Industrial Microorganism Strains].

    PubMed

    Debabov, V G

    2015-04-01

    Microorganism producer strains are the basis of industrial biotechnology. Their properties determine the economical parameters of the production. Methods of rational design (metabolic engineering) and combinatorial methods of mutagenesis and selection (laboratory evolution, adaptive evolution, protein and genomic shuffling) are used for the construction of microorganism strains. Combination of these methods is frequently used. Modern strains usually do not contain plasmids and markers of drug resistance. All changes are introduced into the chromosome by the methods of homologous and site-specific recombination. The sum of such approaches is called recombineering. Gene expression is carried out at the optimal level under the control of promoters of a certain power (frequently regulated). Knowledge of a complete genomic sequence is almost a mandatory condition for the use of methods of metabolic engineering. Bioinformatics significantly assists in the selection of enzymes and the search for necessary genes and metabolic reactions. Measurement of metabolic fluxes largely assists in the construction of strains. The current level of science makes it possible to construct metabolic pathways de novo in strains for the production of chemicals and biofuel. Carbon dioxide has potential as a raw material for microbiological industry; therefore, the study of CO2 fixation by acetogens and electrogens is a promising direction of studies.

  11. Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

    PubMed Central

    Cecchini, Sylvain; Virag, Tamas

    2011-01-01

    Abstract The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 106 cells/ml, ≥1016 purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned. PMID:21381980

  12. Microorganisms detected by enzyme-catalyzed reaction

    NASA Technical Reports Server (NTRS)

    Vango, S. P.; Weetall, H. H.; Weliky, N.

    1966-01-01

    Enzymes detect the presence of microorganisms in soils. The enzyme lysozymi is used to release the enzyme catalase from the microorganisms in a soil sample. The catalase catalyzes the decomposition of added hydrogen peroxide to produce oxygen which is detected manometrically. The partial pressure of the oxygen serves as an index of the samples bacteria content.

  13. Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

    PubMed

    Birlea, Marius; Owens, Gregory P; Eshleman, Emily M; Ritchie, Alanna; Traktinskiy, Igor; Bos, Nathan; Seitz, Scott; Azarkh, Yevgeniy; Mahalingam, Ravi; Gilden, Don; Cohrs, Randall J

    2013-01-01

    Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

  14. Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast. Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway.

    PubMed

    Urban, P; Werck-Reichhart, D; Teutsch, H G; Durst, F; Regnier, S; Kazmaier, M; Pompon, D

    1994-06-15

    Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisiae strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced iron/carbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data. PMID:8026495

  15. Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.

    PubMed

    Jansson, Ronnie; Lau, Cheuk H; Ishida, Takuya; Ramström, Margareta; Sandgren, Mats; Hedhammar, My

    2016-05-01

    Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation. PMID:26814048

  16. Characterization of N-linked glycosylation on recombinant glycoproteins produced in Pichia pastoris using ESI-MS and MALDI-TOF.

    PubMed

    Gong, Bing; Cukan, Michael; Fisher, Richard; Li, Huijuan; Stadheim, Terrance A; Gerngross, Tillman

    2009-01-01

    The production of recombinant therapeutic glycoproteins is an active area of research and drug development. Typically, improvements in therapeutic glycoprotein efficacy have focused on engineering additional N-glycosylation sites into the primary amino acid sequence or attempting to control a particular glycoform profile on a protein through process improvements. Recently, a number of alternative expression systems have appeared that are challenging the dominance of mammalian cell culture. Our laboratory has focused on the re-engineering of the secretory pathway in the yeast Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans. We have demonstrated that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. In this chapter we provide detailed protocols for the analysis of glycosylation on intact glycoproteins by MALDI-TOF and site specific N-glycan occupancy on digested glycoprotein using ESI-MS.

  17. Microorganism Utilization for Synthetic Milk

    NASA Technical Reports Server (NTRS)

    Morford, Megan A.; Khodadad, Christina L.; Caro, Janicce I.; Spencer, LaShelle E.; Richards, Jeffery T.; Strayer, Richard F.; Birmele, Michele N.; Wheeler, Raymond M.

    2014-01-01

    A desired architecture for long duration spaceflight, like aboard the International Space Station or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of the Synthetic Biology project, Cow in a Column, was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel-through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) in order to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms were optimized in the laboratory and the desired end-products, sugars and lipids, were analyzed. Trichoderma reesei, a known cellulolytic fungus, was utilized to drive the production of glucose, with the intent that the produced glucose would serve as the carbon source for milk fat production and be a substitute for the milk sugar lactose. Lipid production would be carried out by Rhodosporidium toruloides, yeast known to accumulate those lipids that are typically found in milk fat. Results showed that glucose and total lipid content were below what was expected during this phase of experimentation. In addition, individual analysis of six fatty acids revealed that the percentage of each fatty acid was lower than naturally produced bovine milk. Overall, this research indicates that microorganisms could be utilized to breakdown inedible solid waste to produce useable products. For future work, the production of the casein protein for milk would require the development of a genetically modified organism, which was beyond the scope of the original project. Additional trials would be needed to further refine the required

  18. Recombinant organisms capable of fermenting cellobiose

    DOEpatents

    Ingram, Lonnie O.; Lai, Xiaokuang; Moniruzzaman, Mohammed; York, Sean W.

    2000-01-01

    This invention relates to a recombinant microorganism which expresses pyruvate decarboxylase, alcohol dehydrogenase, Klebsiella phospho-.beta.-glucosidase and Klebsiella (phosphoenolpyruvate-dependent phosphotransferase system) cellobiose-utilizing Enzyme II, wherein said phospho-.beta.-glucosidase and said (phosphoenolpyruvate-dependent phosphotransferase) cellobiose-utilizing Enzyme II are heterologous to said microorganism and wherein said microorganism is capable of utilizing both hemicellulose and cellulose, including cellobiose, in the production of ethanol.

  19. Microorganisms and Chemical Pollution

    ERIC Educational Resources Information Center

    Alexander, M.

    1973-01-01

    Discusses the importance of microorganisms in chemical pollution and pollution abatement. Selected chemical pollutants are chosen to illustrate that microorganisms synthesize hazardous substances from reasonably innocuous precursors, while others act as excellent environmental decontaminating agents by removing undesirable natural and synthetic…

  20. Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2014-01-01

    Introduction: In order to employ Nicotiana tabacum cells as a profitable natural bioreactor for production of bio-functional "Soluble human TRAIL" (ShTRAIL), endoplasmic reticulum (ER) targeted expression and innovative extraction procedures were exploited. Methods: At first, the ShTRAIL encoding gene was sub-cloned into designed H2 helper vector to equip it with potent TMV omega leader sequences, ER sorting signal peptide, poly-histidine tag and ER retention signal peptide (KDEL). Then, the ER targeted ShTRAIL cassette was sequentially sub-cloned into "CaMV-35S" helper and "pGreen-0179" final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum . Next, the ShTRAIL protein was extracted through both phosphate and innovative ascorbate extraction buffers. Subsequently, oligomerization state of the ShTRAIL was evaluated through cross-linking assay and western blot analysis. Then, semi-quantitative western blot analysis was performed to estimate the ShTRAIL production. Finally, biological activity of the ShTRAIL was evaluated through MTT assay. Results: The phosphate buffer extracted ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy increased the ShTRAIL’s production level up to about 20 μg/g of fresh weight of N. tabacum . MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line. Conclusion: Endoplasmic reticulum expression and reductive ascorbate buffer extraction procedure can be employed to enhance the stability and overall production level of bio-functional recombinant ShTRAIL from transgenic N. tabacum cells. PMID:25337465

  1. Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

    PubMed Central

    Bäckström, B Thomas; Brockelbank, Jane A; Rehm, Bernd HA

    2007-01-01

    Background Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. Results Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were

  2. Immunosorbent Assay Based on Recombinant Hemagglutinin Protein Produced in a High-Efficiency Mammalian Expression System for Surveillance of Measles Immunity

    PubMed Central

    Bouche, Fabienne; Ammerlaan, Wim; Berthet, Francoise; Houard, Sophie; Schneider, Francois; Muller, Claude P.

    1998-01-01

    Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as “gold standards.” In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for

  3. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    PubMed

    Liu, Jun; Luo, Yan; Ge, Hengtao; Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  4. Reconstitution of R-spondin:LGR4:ZNRF3 adult stem cell growth factor signaling complexes with recombinant proteins produced in Escherichia coli.

    PubMed

    Moad, Heather E; Pioszak, Augen A

    2013-10-15

    R-Spondins are secreted glycoproteins (RSPO1-RSPO4) that have proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine-rich repeat (LRR)-containing seven-transmembrane receptors LGR4-LGR6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present a methodology for the bacterial expression and purification of the signaling competent, cysteine-rich Fu1-Fu2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1-14, and the human ZNRF3 ECD. In a cell-based signaling assay, the nonglycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to those of mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nanomolar affinities that decreased in the following order in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: RSPO4 > RSPO2 > RSPO3 > RSPO1. RSPO-receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinities that decreased in the following order: RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and suggest that the stronger signaling potencies of RSPO2 and -3 result from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.

  5. Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

    PubMed Central

    Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals. PMID:23799010

  6. Recombinant zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark

    1996-01-01

    The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  7. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.

    1996-05-07

    The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 2 figs.

  8. Micro-Organ Device

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

    2013-01-01

    A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

  9. Micro-organ device

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); von Gustedt-Gonda, legal representative, Iris (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

    2013-01-01

    A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

  10. Recombinant host cells and media for ethanol production

    DOEpatents

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  11. Measuring micro-organism gas production

    NASA Technical Reports Server (NTRS)

    Wilkins, J. R.; Pearson, A. O.; Mills, S. M.

    1973-01-01

    Transducer, which senses pressure buildup, is easy to assemble and use, and rate of gas produced can be measured automatically and accurately. Method can be used in research, in clinical laboratories, and for environmental pollution studies because of its ability to detect and quantify rapidly the number of gas-producing microorganisms in water, beverages, and clinical samples.

  12. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  13. Fermentations with new recombinant organisms

    SciTech Connect

    Bothast, R.J.; Nichols, N.N.; Dien, B.S.

    1999-10-01

    US fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharomyces cerevisiae and Zymomonas mobilis for pentose utilization. The authors have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21--34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different industrially hardened strains may find separate applications in the fermentation of specific feedstocks.

  14. Activated recombinant adenovirus proteinases

    SciTech Connect

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  15. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  16. Fossil Microorganisms in Archaean

    NASA Technical Reports Server (NTRS)

    Astafleva, Marina; Hoover, Richard; Rozanov, Alexei; Vrevskiy, A.

    2006-01-01

    Ancient Archean and Proterozoic rocks are the model objects for investigation of rocks comprising astromaterials. The first of Archean fossil microorganisms from Baltic shield have been reported at the last SPIE Conference in 2005. Since this confeence biomorphic structures have been revealed in Archean rocks of Karelia. It was determined that there are 3 types of such bion structures: 1. structures found in situ, in other words microorganisms even-aged with rock matrix, that is real Archean fossils biomorphic structures, that is to say forms inhabited early formed rocks, and 3. younger than Archean-Protherozoic minerali microorganisms, that is later contamination. We made attempt to differentiate these 3 types of findings and tried to understand of burial of microorganisms. The structures belongs (from our point of view) to the first type, or real Archean, forms were under examination. Practical investigation of ancient microorganisms from Green-Stone-Belt of Northern Karelia turns to be very perspective. It shows that even in such ancient time as Archean ancient diverse world existed. Moreover probably such relatively highly organized cyanobacteria and perhaps eukaryotic formes existed in Archean world.

  17. Micro-Organ Devices

    NASA Technical Reports Server (NTRS)

    Gonda, Steven R.; Leslie, Julia; Chang, Robert C.; Starly, Binil; Sun, Wei; Culbertson, Christopher; Holtorf, Heidi

    2009-01-01

    Micro-organ devices (MODs) are being developed to satisfy an emerging need for small, lightweight, reproducible, biological-experimentati on apparatuses that are amenable to automated operation and that imp ose minimal demands for resources (principally, power and fluids). I n simplest terms, a MOD is a microfluidic device containing a variety of microstructures and assemblies of cells, all designed to mimic a complex in vivo microenvironment by replicating one or more in vivo micro-organ structures, the architectures and composition of the extr acellular matrices in the organs of interest, and the in vivo fluid flows. In addition to microscopic flow channels, a MOD contains one or more micro-organ wells containing cells residing in microscopic e xtracellular matrices and/or scaffolds, the shapes and compositions o f which enable replication of the corresponding in vivo cell assembl ies and flows.

  18. Microorganisms and Man.

    ERIC Educational Resources Information Center

    Noble, W. C.

    1983-01-01

    Provides information to update Institute of Biology's Studies in Biology No. 111, "Microorganisms and Man," by W. C. Noble and Jay Naidoo (Edward Arnold, 1979). Topics include: (1) food poisoning; (2) airborn infections in man; (3) infection in animals and plants; and (4) biodegradation and biosynthesis. (JN)

  19. Microorganism Utilization for Synthetic Milk Production

    NASA Technical Reports Server (NTRS)

    Birmele, Michele; Morford, Megan; Khodadad, Christina; Spencer, Lashelle; Richards, Jeffrey; Strayer, Richard; Caro, Janicce; Hummerick, Mary; Wheeler, Ray

    2014-01-01

    A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms.

  20. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  1. Application of Recombinant Human Leukemia Inhibitory Factor (LIF) Produced in Rice (Oryza sativa L.) for Maintenance of Mouse Embryonic Stem Cells

    PubMed Central

    Youngblood, Bradford A.; Alfano, Randall; Pettit, Steve C.; Zhang, Deshui; Dallmann, H. Garry; Huang, Ning; MacDonald, Clinton C.

    2014-01-01

    Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial E. coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market. PMID:24380819

  2. Evaluation of an enzyme-linked immunosorbent assay for detection of West Nile virus infection based on a recombinant envelope protein produced in Trichoplusia ni larvae.

    PubMed

    Alonso-Padilla, Julio; Jiménez de Oya, Nereida; Blázquez, Ana-Belén; Loza-Rubio, Elizabeth; Escribano, José M; Saiz, Juan-Carlos; Escribano-Romero, Estela

    2010-06-01

    West Nile virus (WNV), a Flavivirus distributed most widely, is presenting lately variable epidemiological and ecological patterns, including an increasing virulence that has already caused over 1000 human deaths in USA. Currently, diagnosis of WNV is achieved mainly by enzyme-linked immunoassays (ELISAs) based on the use of inactivated whole WNV (iWNV) as antigen, although results have to be confirmed by plaque reduction neutralization tests (PRNTs). Expression of WNV envelope recombinant E (rE) protein and its usefulness as ELISA antigen are described. Production of rE was achieved upon infection of Trichoplusia ni insect larvae with a recombinant baculovirus. Once optimized, the rE-based ELISA was validated with a battery of mouse and equine sera characterized previously. Concordance with the iWNV-based ELISA used routinely was good (95%), as it was with the reference PRNT (90%), with specificity of 94.4% and sensitivity of 88.1%. Production of rE protein in insect larvae allows for an easy, low cost and quite large-scale yield of partially purified antigen which is suitable for serological diagnosis of WNV, without the need for manipulation of large quantities of infective virus.

  3. Inactivation of Microorganisms

    NASA Astrophysics Data System (ADS)

    Alzamora, Stella Maris; Guerrero, Sandra N.; Schenk, Marcela; Raffellini, Silvia; López-Malo, Aurelio

    Minimal processing techniques for food preservation allow better retention of product flavor, texture, color, and nutrient content than comparable conventional treatments. A wide range of novel alternative physical factors have been intensely investigated in the last two decades. These physical factors can cause inactivation of microorganisms at ambient or sublethal temperatures (e.g., high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, and ultraviolet light). These technologies have been reported to reduce microorganism population in foods while avoiding the deleterious effects of severe heating on quality. Among technologies, high-energy ultrasound (i.e., intensities higher than 1 W/cm2, frequencies between 18 and 100 kHz) has attracted considerable interest for food preservation applications (Mason et al., 1996; Povey and Mason, 1998).

  4. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  5. Butanol tolerance in microorganisms

    DOEpatents

    Bramucci, Michael G.; Nagarajan, Vasantha

    2016-03-01

    Provided herein are recombinant yeast host cells and methods for their use for production of fermentation products from a pyruvate utilizing pathway. Yeast host cells provided herein comprise reduced pyruvate decarboxylase activity and modified adenylate cyclase activity. In embodiments, yeast host cells provided herein comprise resistance to butanol and increased biomass production.

  6. Detecting the presence of microorganisms

    NASA Technical Reports Server (NTRS)

    Wilkins, Judd R. (Inventor); Stoner, Glenn E. (Inventor)

    1977-01-01

    The presence of microorganisms in a sample is determined by culturing microorganisms in a growth medium which is in contact with a measuring electrode and a reference electrode and detecting a change in potential between the electrodes caused by the presence of the microorganisms in the medium with a high impedance potentiometer.

  7. Consolidated bioprocessing method using thermophilic microorganisms

    DOEpatents

    Mielenz, Jonathan Richard

    2016-02-02

    The present invention is directed to a method of converting biomass to biofuel, and particularly to a consolidated bioprocessing method using a co-culture of thermophilic and extremely thermophilic microorganisms which collectively can ferment the hexose and pentose sugars produced by degradation of cellulose and hemicelluloses at high substrate conversion rates. A culture medium therefor is also provided as well as use of the methods to produce and recover cellulosic ethanol.

  8. Mass Spectrometer for Airborne Micro-Organisms

    NASA Technical Reports Server (NTRS)

    Sinha, M. P.; Friedlander, S. K.

    1986-01-01

    Bacteria and other micro-organisms identified continously with aid of new technique for producing samples for mass spectrometer. Technique generates aerosol of organisms and feeds to spectrometer. Given species of organism produces characteristic set of peaks in mass spectrum and thereby identified. Technique useful for monitoring bacterial makeup in environmental studies and in places where cleanliness is essential, such as hospital operating rooms, breweries, and pharmaceutical plants.

  9. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  10. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  11. Antifungal and antibacterial activity of marine microorganisms.

    PubMed

    El Amraoui, B; El Amraoui, M; Cohen, N; Fassouane, A

    2014-03-01

    In order to explore marine microorganisms with pharmaceutical potential, marine bacteria, collected from different coastal areas of the Moroccan Atlantic Ocean, were previously isolated from seawater, sediment, marine invertebrates and seaweeds. The antimicrobial activities of these microorganisms were investigated against the pathogens involved in human pathologies. Whole cultures of 34 marine microorganisms were screened for antimicrobial activities using the method of agar diffusion against three Gram-positive bacteria, two Gram-negative bacteria, and against yeast. The results showed that among the 34 isolates studied, 28 (82%) strains have antimicrobial activity against at least one pathogen studied, 11 (32%) strains have antifungal activity and 24 (76%) strains are active against Gram-positive bacteria, while 21 (62%) strains are active against Gram-negative bacteria. Among isolates having antimicrobial activity, 14 were identified and were assigned to the genera Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Chromobacterium, Enterococcus, Pantoea and Pseudomonas. Due to a competitive role for space and nutrient, the marine microorganisms can produce antibiotic substance; therefore, these marine microorganisms were expected to be potential resources of natural antibiotic products.

  12. Recombinant Botulinum Toxoids: A Practical Guide for Production.

    PubMed

    Moreira, Gustavo Marçal S G; Moreira, Clóvis; da Cunha, Carlos Eduardo P; Mendonça, Marcelo; Conceição, Fabricio R

    2016-01-01

    Clostridium botulinum is a Gram-positive, spore-forming, anaerobic bacillus that produces a potent neurotoxin. Botulinum neurotoxins (BoNTs) are classified from serotypes A to H, and even though they have similar mechanisms of action, they show preferential hosts. In veterinary medicine, BoNT serotypes C and D are the most important, once several animal species are susceptible to them. Since BoNTs are the most potent toxins known in nature, the best way to control botulism in animals is through vaccination. However, current commercial vaccines are based on inactivated toxins (toxoids) and cells (bacterins) and present many drawbacks, such as a time-consuming production with variable antigen yield and biosafety risks. Recombinant vaccines, especially those produced by Escherichia coli expression system, have proved to be an interesting alternative to overcome these problems. E. coli is a very well-known microorganism that allows the production of large amounts of nontoxic recombinant antigens in a short period using simple culture medium reducing the production complexity and decreasing most of the biosafety risks involved in the process. We describe herein a method for the production of recombinant vaccines for veterinary medicine application, involving initial steps of gene design up to vaccine formulation and evaluation itself. PMID:27076326

  13. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  14. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals.

    PubMed

    Zeldes, Benjamin M; Keller, Matthew W; Loder, Andrew J; Straub, Christopher T; Adams, Michael W W; Kelly, Robert M

    2015-01-01

    Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

  15. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals

    PubMed Central

    Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.; Straub, Christopher T.; Adams, Michael W. W.; Kelly, Robert M.

    2015-01-01

    Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

  16. Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli

    PubMed Central

    2014-01-01

    Background Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production. Result In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate. Conclusions The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt. PMID:24885047

  17. Conceptualizing "suicidal genetically engineered microorganisms" for bioremediation applications.

    PubMed

    Pandey, Gunjan; Paul, Debarati; Jain, Rakesh K

    2005-02-18

    Use of genetically modified microorganisms (GEMs) for pollution abatement has been limited because of risks associated with their release in the environment. Recent developments in the area of recombinant DNA technologies have paved the way for conceptualizing "suicidal genetically engineered microorganisms" (S-GEMS) to minimize such anticipated hazards and to achieve efficient and safer bioremediation of contaminated sites. Our strategy of designing a novel S-GEM is based on the knowledge of killer-anti-killer gene(s) that would be susceptible to programmed cell death after detoxification of any given contaminated site(s). PMID:15649393

  18. Pentose fermentation by recombinant zymomonas

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  19. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

    1998-03-10

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  20. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  1. Pentose fermentation by recombinant Zymomonas

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

    1998-01-27

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  2. The remote sensing of microorganisms

    NASA Astrophysics Data System (ADS)

    Bramall, Nathan Earl

    An approach for detecting microbial life in extreme environments, including glacial ice and deep water, using autofluorescence is described. The emission spectra for a variety of microorganisms and common minerals were measured for excitation at 224 nm and it is shown that exciting autofluorescence at 224 nm provides adequate separation between mineral and microbial emission spectra to detect a single microorganism in the glacial ice of Antarctica or on mineral surfaces. The different generations of borehole-logging instruments, dubbed 'biospectral loggers' (BSLs), were designed, built and deployed. The first BSL is a prototype that uses ˜375 nm excitation from UV LEDS and was deployed in the ˜1,000 m deep Siple Dome borehole (January, 2002) and in oligotrophic Lake Tahoe (October, 2002), where microbial NADH, chlorophyll, and dissolved organic matter (DOM) fluorescence was measured. The fluorescence red-shifted with depth, suggesting an increase in of terrestrially-derived DOM with depth, perhaps indicating a benthic source and little recent vertical mixing. The second BSL uses a similar optical geometry as the first, but employs a 224 nm HeAg laser to excite protein fluorescence and was deployed at the SPRESO borehole at the South Pole (January; 2004). It recorded a strong microbial signal corresponding to the time period spanning 1,200 B.P. and 900 B.P., which roughly correlates with major desiccation events in both Patagonia and the Antarctic Dry Valleys, suggesting they may have been aeolian sources of aquatic microorganisms for the South Pole. The third BSL, dubbed the mini-BSL, also uses a 224 nm laser for excitation of protein fluorescence, but its novel optical layout enabled its diameter to be reduced to less than 5.1 cm, allowing it to operate in a borehole produced by a modified JPL 'Gopher' drill[16]. High sensitivity was not sacrificed in the miniaturization process as the mBSL is capable of detecting a single B. megatcrium on a background of iron

  3. Marine Microorganisms: perspectives for getting involved in cellulosic ethanol

    PubMed Central

    2012-01-01

    The production of ethanol has been considered as an alternative to replace part of the petroleum derivate. Brazil and the US are the leading producers, but more environmentally friendly alternatives are needed. Lignocellulose has an enormous potential but technology has to be still improve in order to economically produce ethanol. The present paper reviews the potential and problems of this technology and proposes the study of a group of microorganisms with the largest genetic pool, marine microorganism. PMID:22931793

  4. Microorganisms having enhanced resistance to acetate and methods of use

    SciTech Connect

    Brown, Steven D; Yang, Shihui

    2014-10-21

    The present invention provides isolated or genetically modified strains of microorganisms that display enhanced resistance to acetate as a result of increased expression of a sodium proton antiporter. The present invention also provides methods for producing such microbial strains, as well as related promoter sequences and expression vectors. Further, the present invention provides methods of producing alcohol from biomass materials by using microorganisms with enhanced resistance to acetate.

  5. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

    PubMed

    Cavka, Adnan; Alriksson, Björn; Rose, Shaunita H; van Zyl, Willem H; Jönsson, Leif J

    2014-08-01

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention. PMID:24862324

  6. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

    PubMed

    Cavka, Adnan; Alriksson, Björn; Rose, Shaunita H; van Zyl, Willem H; Jönsson, Leif J

    2014-08-01

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

  7. Microorganism Utilization for Synthetic Milk Production

    NASA Technical Reports Server (NTRS)

    Morford, Megan A.; Khodadad, Christina L.; Mccoy, LaShelle E.; Richards, Jeffrey T.; Strayer, Richard F.; Caro, Janicce L.; Hummerick, Mary E.; Birmele, Michele N.; Wheeler, Raymond M.

    2014-01-01

    A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms were optimized in the laboratory and the desired end-products, sugars and lipids, were analyzed. Trichoderma reesei, a known cellulolytic fungus, was utilized to drive the production of glucose, with the intent that the produced glucose would serve as the carbon source for milk fat production and be a substitute for the milk sugar lactose. Lipid production would be carried out by Rhodosporidium toruloides, yeast known to accumulate those lipids that are typically found in milk fat. Results showed that glucose and total lipid content were below what was expected during this phase of experimentation. In addition, individual analysis of six fatty acids revealed that the percentage of each fatty acid was lower than naturally produced bovine milk. Overall, this research indicates that microorganisms could be utilized to breakdown inedible solid waste to produce useable products.

  8. Toolbox for Antibiotics Discovery from Microorganisms.

    PubMed

    Fisch, Katja M; Schäberle, Till F

    2016-09-01

    Microorganisms produce a vast array of biologically active metabolites. Such compounds are applied by humans to positively influence their health and, therefore, natural products serve as drug leads for pharmaceutical and medicinal chemistry. In this minireview, tools for the discovery and the production of potential drug leads are explained. A snapshot is provided, starting from the isolation of new producer strains, across genomic mining of (meta)genomes to identify biosynthetic gene clusters corresponding to natural products, toward heterologous expression to produce potential drug leads. PMID:27311607

  9. Gravitaxis in unicellular microorganisms

    NASA Astrophysics Data System (ADS)

    Häder, D.-P.

    1999-01-01

    Orientation of organisms with respect to the gravitational field of the Earth has been studied for more than 100 years in a number of unicellular microorganisms including flagellates and ciliates. Several hypotheses have been developed how the weak stimulus is perceived. Intracellular statoliths have been found to be involved in gravitaxis of Loxodes, while no specialized organelles have been detected in other ciliates, e.g. Paramecium. Also in the slime mold Physarum no specialized gravireceptors have been identified yet. In the flagellate Euglena gracilis the whole cell body, which is denser than the surrounding medium, seems to act as a statolith pressing onto the lower membrane where it activates mechanosensitive ion channels. Similar results were obtained for the ciliate Paramecium. In contrast to the flagellate Euglena, several ciliates have been found to show gravikinesis, which is defined as a dependence of the swimming velocity on the direction of movement in the gravity field.

  10. Proteolysis in hyperthermophilic microorganisms

    DOE PAGES

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; Levy, Ryan D.; Michel, Joshua K.; Conners, Shannon B.; Kelly, Robert M.

    2002-01-01

    Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus , the crenarchaeote Sulfolobus solfataricus , and the bacterium Thermotoga maritima . An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putativemore » proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.« less

  11. Thermal inactivation of microorganisms.

    PubMed

    Smelt, J P P M; Brul, S

    2014-01-01

    This paper serves as an overview of various aspects of thermal processing. Heat processing of foods has a long history and is still one of the most important preservation methods. To guarantee microbiological safety and stability, large safety margins are often applied in traditional heat processes. Because of the need for more fresh like foods, there is a need for milder preservation methods without compromising on safety and stability. The review deals with heat resistance data and mathematical models that describe heat inactivation. The effects of food composition are not yet fully clear and more knowledge of the cell physiology of the target microorganism could be of help in predicting the effects of food constituents. Finally, special attention has been paid to biological time temperature indicators to enable proper process calculations.

  12. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  13. Thermophilic microorganisms in biomining.

    PubMed

    Donati, Edgardo Rubén; Castro, Camila; Urbieta, María Sofía

    2016-11-01

    Biomining is an applied biotechnology for mineral processing and metal extraction from ores and concentrates. This alternative technology for recovering metals involves the hydrometallurgical processes known as bioleaching and biooxidation where the metal is directly solubilized or released from the matrix for further solubilization, respectively. Several commercial applications of biomining can be found around the world to recover mainly copper and gold but also other metals; most of them are operating at temperatures below 40-50 °C using mesophilic and moderate thermophilic microorganisms. Although biomining offers an economically viable and cleaner option, its share of the world´s production of metals has not grown as much as it was expected, mainly considering that due to environmental restrictions in many countries smelting and roasting technologies are being eliminated. The slow rate of biomining processes is for sure the main reason of their poor implementation. In this scenario the use of thermophiles could be advantageous because higher operational temperature would increase the rate of the process and in addition it would eliminate the energy input for cooling the system (bioleaching reactions are exothermic causing a serious temperature increase in bioreactors and inside heaps that adversely affects most of the mesophilic microorganisms) and it would decrease the passivation of mineral surfaces. In the last few years many thermophilic bacteria and archaea have been isolated, characterized, and even used for extracting metals. This paper reviews the current status of biomining using thermophiles, describes the main characteristics of thermophilic biominers and discusses the future for this biotechnology.

  14. Thermophilic microorganisms in biomining.

    PubMed

    Donati, Edgardo Rubén; Castro, Camila; Urbieta, María Sofía

    2016-11-01

    Biomining is an applied biotechnology for mineral processing and metal extraction from ores and concentrates. This alternative technology for recovering metals involves the hydrometallurgical processes known as bioleaching and biooxidation where the metal is directly solubilized or released from the matrix for further solubilization, respectively. Several commercial applications of biomining can be found around the world to recover mainly copper and gold but also other metals; most of them are operating at temperatures below 40-50 °C using mesophilic and moderate thermophilic microorganisms. Although biomining offers an economically viable and cleaner option, its share of the world´s production of metals has not grown as much as it was expected, mainly considering that due to environmental restrictions in many countries smelting and roasting technologies are being eliminated. The slow rate of biomining processes is for sure the main reason of their poor implementation. In this scenario the use of thermophiles could be advantageous because higher operational temperature would increase the rate of the process and in addition it would eliminate the energy input for cooling the system (bioleaching reactions are exothermic causing a serious temperature increase in bioreactors and inside heaps that adversely affects most of the mesophilic microorganisms) and it would decrease the passivation of mineral surfaces. In the last few years many thermophilic bacteria and archaea have been isolated, characterized, and even used for extracting metals. This paper reviews the current status of biomining using thermophiles, describes the main characteristics of thermophilic biominers and discusses the future for this biotechnology. PMID:27628339

  15. [Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein].

    PubMed

    Makino, Masahiko; Mukai, Tetsu

    2012-09-01

    To activate naïve T cells convincingly using Mycobacterium bovis BCG (BCG), rBCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4+ and CD8+ subsets of naïve T cells than rBCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DC) to induce cytokine production and phenotypic changes, and activated CD4+ T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pre-treatment of DC with chloroquine inhibited both surface expression of MMP-II on DC and the activation of T cells by BCG-D70M-infected APCs. The naïve CD8+ T cell activation was inhibited by treatment of DC with brefeldin A and lactacystin so that the T cells was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naïve CD8+ T cells, effector T cells producing perforin and memory T cells having migration markers, were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70, and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II and urease depletion may provide useful tool for inducing better activation of naïve T cells.

  16. Hydrogen production by photosynthetic microorganisms

    SciTech Connect

    Akano, T.; Fukatsu, K.; Miyasaka, H. |

    1996-12-31

    Hydrogen is a clean energy alternative to the fossil fuels, the main source of greenhouse gas emissions. We developed a stable system for the conversion of solar energy into hydrogen using photosynthetic microorganisms. Our system consists of the following three stages: (1) Photosynthetic starch accumulation in green microalgae (400 L x2); (2) Dark anaerobic fermentation of the algal starch biomass to produce hydrogen and organic compounds (155 L x2); and (3) Further conversion of the organic compounds to produce hydrogen using photosynthetic bacteria (three types of reactors, parallel plate, raceway, and tubular). We constructed a test plant of this process at Nankoh power plant of Kansai Electric Power Company in Osaka, Japan, and carried out a series of tests using CO{sub 2} obtained from a chemical absorption pilot-plant. The photobiological hydrogen production process used a combination of a marine alga, Chlamydomonas sp. MGA 161 and marine photosynthetic bacterium, Rhodopseudomonas sp. W-1S. The dark anaerobic fermentation of algal starch biomass was also investigated. Sustained and stable starch accumulation, starch degradation in the algal cell, and hydrogen production from algal fermentation and photosynthetic bacteria in the light were demonstrated during several experiments. 3 refs., 12 figs., 1 tab.

  17. Identification, Characterization, and Recombinant Expression of Epidermicin NI01, a Novel Unmodified Bacteriocin Produced by Staphylococcus epidermidis That Displays Potent Activity against Staphylococci

    PubMed Central

    Sandiford, Stephanie

    2012-01-01

    We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide. PMID:22155816

  18. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci.

    PubMed

    Sandiford, Stephanie; Upton, Mathew

    2012-03-01

    We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide.

  19. Microorganism lipid droplets and biofuel development.

    PubMed

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-12-01

    Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech 'factories' to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development.

  20. Microorganism lipid droplets and biofuel development

    PubMed Central

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-01-01

    Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech ‘factories’ to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development. [BMB Reports 2013; 46(12): 575-581] PMID:24355300

  1. Radiation sensitivity of hyperthermal composting microorganisms

    NASA Astrophysics Data System (ADS)

    Choi, Jong-Il; Yoon, Min-Chul; Kim, Jae-Hun; Yamashita, Masamichi; Kim, Geun Joong; Lee, Ju-Woon

    In the space station and vehicles designed for long human mission, high-temperature compost is a promising technology for decomposing organic waste and producing the fertilizers. In space, the microorganisms could have the changed biological activities or even be mutated by ionizing irradiation. Therefore, in this study, the effect of gamma irradiation on the sensitivity of bacteria in hyperthermal composting was investigated. The sequence analysis of the amplified 16s rDNA genes and amoA gene were used for the identification of composting microorganisms. Viability of microorganisms in compost soil after gamma irradiation was directly visualized with LIVE/DEAD Baclight viability kit. The dominant bacterial genera are Weissella cibaria and Leuconostoc sp. and fungus genera are Metschnikowia bicuspidate and Pichia guilliermondii, respectively. By the gamma irradiation up to the dose of 1 kGy, the microbial population was not changed. Also, the enzyme activities of amylase and cellulose were sustained by the gamma irradiation. These results show that these hyperthermia microorganisms might have the high resistance to gamma radiation and could be used for agriculture in the Space Station.

  2. Population dynamics of transgenic microorganisms in the different microecosystem conditions

    NASA Astrophysics Data System (ADS)

    Popova, L. Yu.; Lobova, T. I.; Krylova, T. Yu.; Kargatova, T. V.; Maksimova, E. E.; Boyandin, A. N.; Pechurkin, N. S.

    The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap rLux +) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human α 2-interferon Km rInf +), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity.

  3. Population dynamics of transgenic microorganisms in the different microecosystem conditions.

    PubMed

    Popova, L Y; Lobova, T I; Krylova, T Y; Kargatova, T V; Maksimova, E E; Boyandin, A N; Pechurkin, N S

    2001-01-01

    The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity. Grant numbers: 99-04-96017, 25, 00-07-9011.

  4. 'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

    PubMed

    Sharma, Prerna; Guptasarma, Purnananda

    2015-05-01

    Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes. PMID:25824038

  5. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  6. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for...

  7. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for...

  8. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for...

  9. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for...

  10. How could haloalkaliphilic microorganisms contribute to biotechnology?

    PubMed

    Zhao, Baisuo; Yan, Yanchun; Chen, Shulin

    2014-11-01

    Haloalkaliphiles are microorganisms requiring Na(+) concentrations of at least 0.5 mol·L(-1) and an alkaline pH of 9 for optimal growth. Their unique features enable them to make significant contributions to a wide array of biotechnological applications. Organic compatible solutes produced by haloalkaliphiles, such as ectoine and glycine betaine, are correlated with osmoadaptation and may serve as stabilizers of intracellular proteins, salt antagonists, osmoprotectants, and dermatological moisturizers. Haloalkaliphiles are an important source of secondary metabolites like rhodopsin, polyhydroxyalkanoates, and exopolysaccharides that play essential roles in biogeocycling organic compounds. These microorganisms also can secrete unique exoenzymes, including proteases, amylases, and cellulases, that are highly active and stable in extreme haloalkaline conditions and can be used for the production of laundry detergent. Furthermore, the unique metabolic pathways of haloalkaliphiles can be applied in the biodegradation and (or) biotransformation of a broad range of toxic industrial pollutants and heavy metals, in wastewater treatment, and in the biofuel industry.

  11. Microorganisms having enhanced tolerance to inhibitors and stress

    SciTech Connect

    Brown, Steven D.; Yang, Shihui

    2014-07-29

    The present invention provides genetically modified strains of microorganisms that display enhanced tolerance to stress and/or inhibitors such as sodium acetate and vanillin. The enhanced tolerance can be achieved by increasing the expression of a protein of the Sm-like superfamily such as a bacterial Hfq protein and a fungal Sm or Lsm protein. Further, the present invention provides methods of producing alcohol from biomass materials by using the genetically modified microorganisms of the present invention.

  12. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  13. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  14. Recombinant Lactococcus lactis fails to secrete bovine chymosine

    PubMed Central

    Luerce, Tessália Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela Santos

    2014-01-01

    Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein. PMID:25482140

  15. Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen.

    PubMed Central

    Lagranderie, M; Lo-Man, R; Dériaud, E; Gicquel, B; Gheorghiu, M; Leclerc, C

    1997-01-01

    Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various inbred strains of mice to recombinant BCG expressing beta-galactosidase. These experiments demonstrated that BALB/c mice developed strong antibody responses against BCG expressing beta-galactosidase under the control of two different promoters. In contrast, C57BL/6, C3H, and CBA mice produced high anti-beta-galactosidase antibody titers only when immunized with recombinant BCG expressing beta-galactosidase under the control of the pblaF* promoter, which induced the production of high levels of this antigen. This difference in mouse responsiveness to recombinant BCG was not due to innate resistance to BCG infection, since similar immune responses were induced in Ity(r) and Ity(s) congenic strains of mice. In contrast, the analysis of anti-beta-galactosidase antibody responses of H-2 congenic mice in two different genetic backgrounds demonstrated that H-2 genes are involved in the immune responsiveness to beta-galactosidase delivered by recombinant BCG. Together, these results demonstrate that immune responses to an antigen delivered by recombinant BCG are under complex genetic influences which could play a crucial role in the efficiency of future recombinant BCG vaccines. PMID:9234754

  16. Antibiotic resistant in microorganisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antimicrobial agents are necessary for use in veterinary medicine including the production of food producing animals. Antibiotic use is indicated for the treatment of bacterial target organisms and/or disease for which the antibiotic was developed. However, an unintended consequence of antibiotic ...

  17. [Plant-Producers Of Recombinant Cytokines (Review)].

    PubMed

    Burlakovskii, M S; Yemel'yanov, V V; Lutova, L A

    2016-01-01

    Cytokines are a family of signaling polypeptides involved in cell-cell interactions in the process of the immune response, as well as in the regulation of a number of normal physiological functions. Cytokines are used in medicine for the treatment of cancer, immune disorders, viral infections, and other socially significant diseases, but the extent of their use is limited by the high production cost of the active agent. The development of this area of pharmacology is associated with the success of genetic engineering, which allows the production of significant amounts of protein by transgenic organisms. The review discusses the latest advances in the production of various cytokines with the use of genetically modified plants. PMID:27266244

  18. Sensor arrays for detecting microorganisms

    NASA Technical Reports Server (NTRS)

    Lewis, Nathan S. (Inventor); Freund, Michael S. (Inventor)

    2000-01-01

    A sensor array for detecting a microorganism comprising first and second sensors electrically connected to an electrical measuring apparatus, wherein the sensors comprise a region of nonconducting organic material and a region of conducting material compositionally that is different than the nonconducting organic material and an electrical path through the regions of nonconducting organic material and the conducting material. A system for identifying microorganisms using the sensor array, a computer and a pattern recognition algorithm, such as a neural net are also disclosed.

  19. Textiles for protection against microorganism

    NASA Astrophysics Data System (ADS)

    Sauperl, O.

    2016-04-01

    Concerning micro-organisms such as bacteria, viruses and fungi, there is a huge progress in the development of textile materials and procedures which should effectively protect against these various pathogens. In this sense there is especially problematic hospital environment, where it is necessary to take into account properly designed textile material which, when good selected and composed, act as a good barrier against transfer of micro-organisms through material mainly in its wet state. Respect to this it is necessary to be familiar with the rules regarding selection of the input material, the choice of proper yarn construction, the choice of the proper weaving mode, the rules regarding selection of antimicrobial-active compound suitable for (eco-friendly) treatment, and the choice of the most appropriate test method by which it is possible objectively to conclude on the reduction of selected microorganism. As is well known, fabrics are three-dimensional structures with void and non-void areas. Therefore, the physical-chemical properties of the textile material/fabric, the surface characteristics together with the shape of microorganism, and the carriers' characteristics contribute to control the transfer of microorganism through textile material. Therefore, careful planning of textile materials and treatment procedure with the compound which is able to reduce micro-organism satisfactory is particularly important, especially due to the fact that in hospital environment population with impaired immune system is mainly presented.

  20. Genetic and biochemical evidence that recombinant Enterococcus spp. strains expressing gelatinase (GelE) produce bovine milk-derived hydrolysates with high angiotensin converting enzyme-inhibitory activity (ACE-IA).

    PubMed

    Gútiez, Loreto; Borrero, Juan; Jiménez, Juan J; Gómez-Sala, Beatriz; Recio, Isidra; Cintas, Luis M; Herranz, Carmen; Hernández, Pablo E

    2014-06-18

    In this work, genes encoding gelatinase (gelE) and serine proteinase (sprE), two extracellular proteases produced by Enterococcus faecalis DBH18, were cloned in the protein expression vector pMG36c, containing the constitutive P32 promoter, generating the recombinant plasmids pCG, pCSP, and pCGSP encoding gelE, sprE, and gelE-sprE, respectively. Transformation of noncaseinolytic E. faecalis P36, E. faecalis JH2-2, E. faecium AR24, and E. hirae AR14 strains with these plasmids permitted detection of caseinolytic activity only in the strains transformed with pCG or pCGSP. Complementation of a deletion (knockout) mutant of E. faecalis V583 for production of gelatinase (GelE) with pCG unequivocally supported that gelE is responsible for the caseinolytic activity of the transformed strain grown in bovine skim milk (BSM). RP-HPLC-MS/MS analysis of hydrolysates of transformed Enterococcus spp. strains grown in BSM permitted the identification of 38 major peptide fragments including peptides with previously reported angiotensin converting enzyme-inhibitory activity (ACE-IA), antihypertensive activity, and antioxidant activity.

  1. Biomining: metal recovery from ores with microorganisms.

    PubMed

    Schippers, Axel; Hedrich, Sabrina; Vasters, Jürgen; Drobe, Malte; Sand, Wolfgang; Willscher, Sabine

    2014-01-01

    Biomining is an increasingly applied biotechnological procedure for processing of ores in the mining industry (biohydrometallurgy). Nowadays the production of copper from low-grade ores is the most important industrial application and a significant part of world copper production already originates from heap or dump/stockpile bioleaching. Conceptual differences exist between the industrial processes of bioleaching and biooxidation. Bioleaching is a conversion of an insoluble valuable metal into a soluble form by means of microorganisms. In biooxidation, on the other hand, gold is predominantly unlocked from refractory ores in large-scale stirred-tank biooxidation arrangements for further processing steps. In addition to copper and gold production, biomining is also used to produce cobalt, nickel, zinc, and uranium. Up to now, biomining has merely been used as a procedure in the processing of sulfide ores and uranium ore, but laboratory and pilot procedures already exist for the processing of silicate and oxide ores (e.g., laterites), for leaching of processing residues or mine waste dumps (mine tailings), as well as for the extraction of metals from industrial residues and waste (recycling). This chapter estimates the world production of copper, gold, and other metals by means of biomining and chemical leaching (bio-/hydrometallurgy) compared with metal production by pyrometallurgical procedures, and describes new developments in biomining. In addition, an overview is given about metal sulfide oxidizing microorganisms, fundamentals of biomining including bioleaching mechanisms and interface processes, as well as anaerobic bioleaching and bioleaching with heterotrophic microorganisms.

  2. Biomining: metal recovery from ores with microorganisms.

    PubMed

    Schippers, Axel; Hedrich, Sabrina; Vasters, Jürgen; Drobe, Malte; Sand, Wolfgang; Willscher, Sabine

    2014-01-01

    Biomining is an increasingly applied biotechnological procedure for processing of ores in the mining industry (biohydrometallurgy). Nowadays the production of copper from low-grade ores is the most important industrial application and a significant part of world copper production already originates from heap or dump/stockpile bioleaching. Conceptual differences exist between the industrial processes of bioleaching and biooxidation. Bioleaching is a conversion of an insoluble valuable metal into a soluble form by means of microorganisms. In biooxidation, on the other hand, gold is predominantly unlocked from refractory ores in large-scale stirred-tank biooxidation arrangements for further processing steps. In addition to copper and gold production, biomining is also used to produce cobalt, nickel, zinc, and uranium. Up to now, biomining has merely been used as a procedure in the processing of sulfide ores and uranium ore, but laboratory and pilot procedures already exist for the processing of silicate and oxide ores (e.g., laterites), for leaching of processing residues or mine waste dumps (mine tailings), as well as for the extraction of metals from industrial residues and waste (recycling). This chapter estimates the world production of copper, gold, and other metals by means of biomining and chemical leaching (bio-/hydrometallurgy) compared with metal production by pyrometallurgical procedures, and describes new developments in biomining. In addition, an overview is given about metal sulfide oxidizing microorganisms, fundamentals of biomining including bioleaching mechanisms and interface processes, as well as anaerobic bioleaching and bioleaching with heterotrophic microorganisms. PMID:23793914

  3. Screening for improved isoprenoid biosynthesis in microorganisms.

    PubMed

    Emmerstorfer-Augustin, Anita; Moser, Sandra; Pichler, Harald

    2016-10-10

    The production of isoprenoids in recombinant microbes for flavor & fragrance, pharmaceutical, agricultural or fuel applications is a booming research field. Isoprenoid extraction from natural resources and chemical synthesis is frequently neither ecological nor commercially profitable. However, recombinant microbes also show severe limitations in specific isoprenoid synthesis. Therefore, diverse directed evolution strategies have been developed for recombinant microbes. The focus has been laid either on the overall engineering of recombinant hosts or on the improvement of isoprenoid synthases. Currently, the most prominent and advanced approaches are based on carotenoid-producing strains, which can be screened by simple colorimetric readout. Other screening strategies are based on spectrophotometric analyses of colored by-products, fluorescence applications, growth selection and, to a minor extent, the use of biosensors indicating the pool of isoprenoid precursors. Although the number of approaches is still small, we observe a trend towards rigorous and highly creative assays that, however, often rely on the indirect detection of the evolved enzyme activities or host strains. We conclude that the use of whole-cellular systems is clearly favored over cell extracts and predict that next-generation screening assays need to be developed towards broader applicability and more direct assessment of isoprenoid production levels.

  4. Engineered biosealant strains producing inorganic and organic biopolymers.

    PubMed

    Bergdale, Terran E; Pinkelman, Rebecca J; Hughes, Stephen R; Zambelli, Barbara; Ciurli, Stefano; Bang, Sookie S

    2012-10-31

    Microbiologically induced calcium carbonate precipitation (MICCP) is a naturally occurring biological process that has shown its potential in remediation of a wide range of structural damages including concrete cracks. In this study, genetically engineered microorganisms, capable of producing extracellular polymeric substances (EPSs) as well as inducing MICCP, were developed based on the assumption that the complex of inorganic CaCO(3) and organic EPS would provide a stronger matrix than MICCP alone as biosealant. In order to develop a recombinant biosealant microorganism, the entire Sporosarcina pasteurii urease gene sequences including ureA, ureB, ureC, ureD, ureE, ureF, and ureG from plasmid pBU11 were sub-cloned into the shuttle vector, pUCP18. The newly constructed plasmid, pUBU1, was transformed into two Pseudomonas aeruginosa strains, 8821 and PAO1, to develop recombinants capable of inducing calcite precipitation in addition to their own ability to produce EPS. Nickel-dependent urease activities were expressed from the recombinant P. aeruginosa 8821 (pUBU1) and P. aeruginosa PAO1 (pUBU1), at 99.4% and 60.9% of the S. pasteurii urease activity, respectively, in a medium containing 2mM NiCl(2). No urease activities were detected from the wild type P. aeruginosa 8821 and P. aeruginosa PAO1 under the same growth conditions. Recombinant Pseudomonas strains induced CaCO(3) precipitation at a comparable rate as S. pasteurii and scanning electron microscopy evidenced the complex of CaCO(3) crystals and EPS layers surrounding the cells. The engineered strains produced in this study are expected to serve as a valuable reference to future biosealants that could be applied in the environment. However, the pathogenic potential of P. aeruginosa, used here only as a model system to show the proof of principle, prevents the use of this recombinant organism as a biosealant. In practical applications, other recombinant organisms should be used. PMID:22789480

  5. Screening of biosurfactants from cloud microorganisms

    NASA Astrophysics Data System (ADS)

    Sancelme, Martine; Canet, Isabelle; Traikia, Mounir; Uhliarikova, Yveta; Capek, Peter; Matulova, Maria; Delort, Anne-Marie; Amato, Pierre

    2015-04-01

    The formation of cloud droplets from aerosol particles in the atmosphere is still not well understood and a main source of uncertainties in the climate budget today. One of the principal parameters in these processes is the surface tension of atmospheric particles, which can be strongly affected by trace compounds called surfactants. Within a project devoted to bring information on atmospheric surfactants and their effects on cloud droplet formation, we focused on surfactants produced by microorganisms present in atmospheric waters. From our unique collection of microorganisms, isolated from cloud water collected at the Puy-de-Dôme (France),1 we undertook a screening of this bank for biosurfactant producers. After extraction of the supernatants of the pure cultures, surface tension of crude extracts was determined by the hanging drop technique. Results showed that a wide variety of microorganisms are able to produce biosurfactants, some of them exhibiting strong surfactant properties as the resulting tension surface decreases to values less then 35 mN.m-1. Preliminary analytical characterization of biosurfactants, obtained after isolation from overproducing cultures of Rhodococcus sp. and Pseudomonas sp., allowed us to identify them as belonging to two main classes, namely glycolipids and glycopeptides. 1. Vaïtilingom, M.; Attard, E.; Gaiani, N.; Sancelme, M.; Deguillaume, L.; Flossmann, A. I.; Amato, P.; Delort, A. M. Long-term features of cloud microbiology at the puy de Dôme (France). Atmos. Environ. 2012, 56, 88-100. Acknowledgements: This work is supported by the French-USA ANR SONATA program and the French-Slovakia programs Stefanik and CNRS exchange.

  6. Marine microorganisms as potential biofactories for synthesis of metallic nanoparticles.

    PubMed

    Manivasagan, Panchanathan; Nam, Seung Yun; Oh, Junghwan

    2016-11-01

    The use of marine microorganisms as potential biofactories for green synthesis of metallic nanoparticles is a relatively new field of research with considerable prospects. This method is eco-friendly, time saving, and inexpensive and can be easily scaled up for large-scale synthesis. The increasing need to develop simple, nontoxic, clean, and environmentally safe production methods for nanoparticles and to decrease environmental impact, minimize waste, and increase energy productivity has become important in this field. Marine microorganisms are tiny organisms that live in marine ecosystems and account for >98% of biomass of the world's ocean. Marine microorganisms synthesize metallic nanoparticles either intracellularly or extracellularly. Marine microbially-produced metallic nanoparticles have received considerable attention in recent years because of their expected impact on various applications such as medicine, energy, electronic, and space industries. The present review discusses marine microorganisms as potential biofactories for the green synthesis of metallic nanoparticles and their potential applications. PMID:26920850

  7. Marine microorganisms as potential biofactories for synthesis of metallic nanoparticles.

    PubMed

    Manivasagan, Panchanathan; Nam, Seung Yun; Oh, Junghwan

    2016-11-01

    The use of marine microorganisms as potential biofactories for green synthesis of metallic nanoparticles is a relatively new field of research with considerable prospects. This method is eco-friendly, time saving, and inexpensive and can be easily scaled up for large-scale synthesis. The increasing need to develop simple, nontoxic, clean, and environmentally safe production methods for nanoparticles and to decrease environmental impact, minimize waste, and increase energy productivity has become important in this field. Marine microorganisms are tiny organisms that live in marine ecosystems and account for >98% of biomass of the world's ocean. Marine microorganisms synthesize metallic nanoparticles either intracellularly or extracellularly. Marine microbially-produced metallic nanoparticles have received considerable attention in recent years because of their expected impact on various applications such as medicine, energy, electronic, and space industries. The present review discusses marine microorganisms as potential biofactories for the green synthesis of metallic nanoparticles and their potential applications.

  8. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  9. Screening For Alcohol-Producing Microbes

    NASA Technical Reports Server (NTRS)

    Schubert, Wayne W.

    1988-01-01

    Dye reaction rapidly identifies alcohol-producing microbial colonies. Method visually detects alcohol-producing micro-organisms, and distinguishes them from other microbial colonies that do not produce alcohol. Method useful for screening mixed microbial populations in environmental samples.

  10. Innovation by homologous recombination.

    PubMed

    Trudeau, Devin L; Smith, Matthew A; Arnold, Frances H

    2013-12-01

    Swapping fragments among protein homologs can produce chimeric proteins with a wide range of properties, including properties not exhibited by the parents. Computational methods that use information from structures and sequence alignments have been used to design highly functional chimeras and chimera libraries. Recombination has generated proteins with diverse thermostability and mechanical stability, enzyme substrate specificity, and optogenetic properties. Linear regression, Gaussian processes, and support vector machine learning have been used to model sequence-function relationships and predict useful chimeras. These approaches enable engineering of protein chimeras with desired functions, as well as elucidation of the structural basis for these functions.

  11. PCB breakdown by anaerobic microorganisms

    SciTech Connect

    Not Available

    1989-03-01

    Recently, altered PCB cogener distribution patterns observed in anaerobic sediment samples from the upper Hudson River are being attributed to biologically mediated reductive dechlorination. The authors report their successful demonstration of biologically mediated reductive dechlorination of an Aroclor mixture. In their investigation, they assessed the ability of microorganisms from PCB-contaminated Hudson River sediments (60-562 ppm PCBs) to dechlorinate Aroclor 1242 under anaerobic conditions by eluting microorganisms from the PCB- contaminated sediments and transferring them to a slurry of reduced anaerobic mineral medium and PCB-free sediments in tightly stoppered bottles. They observed dechlorination to be the most rapid at the highest PCB concentration tried by them.

  12. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  13. Production of hydroxycitric acid by microorganisms.

    PubMed

    Hida, Hiroyuki; Yamada, Takashi; Yamada, Yasuhiro

    2005-08-01

    Hydroxycitric acid (HCA) is a major acid component of the tropical plants Garcinia cambogia and Hibiscus subdariffa. (2S,3S)-HCA from G. cambogia was shown to be a potent inhibitor of ATP citrate lyase (EC4.1.3.8), which catalyzes the extramitochondrial cleavage of citrate to oxaloacetate and acetyl-CoA. (2S,3R)-HCA from H. subdariffa inhibits alpha-amylase and alpha-glucosidase, leading to reduction of carbohydrate metabolism. The availability of HCA is limited by the restricted habitat of the plants as well as the difficulty of stereoselective organic synthesis. Hence, we screened microorganisms producing HCA to find an alternative source of optically pure bulk HCA. Two strains, Streptomyces sp. U121 and Bacillus megaterium G45C, were screened by HPLC analysis. Particular metabolites were purified from their culture broths and compared with authentic HCA from plants. NMR studies indicated that the products are identical to Hibiscus-type HCA. This is the first report showing isolation of microorganisms producing HCA. PMID:16116285

  14. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  15. Quantity analysis of micro-organisms in bottled water

    NASA Astrophysics Data System (ADS)

    Li, Juan; Li, Xiangyong

    2008-12-01

    Water is necessary to human being and all kinds of animals and plants. In recently years, Bottled Water become the main drinking water whatever for families or for institutions. But most of them have no conception of the water's safety or quality. To have conceptions of the count and distributing of the microorganisms in bucket pure water, we use fluorescent microscope counting stained with SYBR Green I to research the microorganisms (including virus) quantity in Bottled Water for six samples produced in different place. Analyzing shows that the quantity of the microorganisms in these water are different. Some up to 11.912×106 virus/ m L. The quality of Bottled Water needs to be improved. And the quantity of microorganisms in the water is different with different ways to keep the water. At the same time, it shows that fluorescent microscope counting stained with SYBR Green I method is simple and high sensitive to such low microorganisms quantity water sample. It can be used in the microorganisms dynamic quantity research in drinking water.

  16. Absolute configuration of hydroxycitric acid produced by microorganisms.

    PubMed

    Hida, Hiroyuki; Yamada, Takashi; Yamada, Yasuhiro

    2006-08-01

    Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer. PMID:16926511

  17. Comparative Analyses of Exoproteinases Produced by Three Phytopathogenic Microorganisms

    PubMed Central

    Valueva, Tatiana A.; Kudryavtseva, Natalia N.; Sof'in, Alexis V.; Revina, Tatiana A.; Gvozdeva, Ekaterina L.; Ievleva, Elena V.

    2011-01-01

    Proteinases secreted by the oomycete Phytophthora infestans (Mont.) de Bary, Rhizoctonia solani, and Fusarium culmorum belonging to different families of fungi have been studied to determine if the exoenzyme secretion depends on the environmental conditions and the phylogenetic position of the pathogen. The substrate specificity of the extracellular proteinases of F. culmorum, R. solani, and P. infestans and their sensitivity to the action of synthetic and protein inhibitors suggest that they contain trypsin-like and subtilisin-like enzymes regardless of culture medium composition. The relation of trypsin-like and subtilisin-like enzymes is dependent on the culture medium composition, especially on the form of nitrogen nutrition, particularly in the case of the exoenzymes secreted by R. solani. Phylogenetic analyses have shown that the exoproteinase set of ascomycetes and oomycetes has more similarities than basidiomycetes although they are more distant relatives. Our data suggests that the multiple proteinases secreted by pathogenic fungi could play different roles in pathogenesis, increasing the adaptability and host range, or could have different functions in survival in various ecological habitats outside the host. PMID:22567343

  18. Comparative analyses of exoproteinases produced by three phytopathogenic microorganisms.

    PubMed

    Valueva, Tatiana A; Kudryavtseva, Natalia N; Sof'in, Alexis V; Revina, Tatiana A; Gvozdeva, Ekaterina L; Ievleva, Elena V

    2011-01-01

    Proteinases secreted by the oomycete Phytophthora infestans (Mont.) de Bary, Rhizoctonia solani, and Fusarium culmorum belonging to different families of fungi have been studied to determine if the exoenzyme secretion depends on the environmental conditions and the phylogenetic position of the pathogen. The substrate specificity of the extracellular proteinases of F. culmorum, R. solani, and P. infestans and their sensitivity to the action of synthetic and protein inhibitors suggest that they contain trypsin-like and subtilisin-like enzymes regardless of culture medium composition. The relation of trypsin-like and subtilisin-like enzymes is dependent on the culture medium composition, especially on the form of nitrogen nutrition, particularly in the case of the exoenzymes secreted by R. solani. Phylogenetic analyses have shown that the exoproteinase set of ascomycetes and oomycetes has more similarities than basidiomycetes although they are more distant relatives. Our data suggests that the multiple proteinases secreted by pathogenic fungi could play different roles in pathogenesis, increasing the adaptability and host range, or could have different functions in survival in various ecological habitats outside the host. PMID:22567343

  19. Smaller Fleas: Viruses of Microorganisms

    PubMed Central

    Hyman, Paul; Abedon, Stephen T.

    2012-01-01

    Life forms can be roughly differentiated into those that are microscopic versus those that are not as well as those that are multicellular and those that, instead, are unicellular. Cellular organisms seem generally able to host viruses, and this propensity carries over to those that are both microscopic and less than truly multicellular. These viruses of microorganisms, or VoMs, in fact exist as the world's most abundant somewhat autonomous genetic entities and include the viruses of domain Bacteria (bacteriophages), the viruses of domain Archaea (archaeal viruses), the viruses of protists, the viruses of microscopic fungi such as yeasts (mycoviruses), and even the viruses of other viruses (satellite viruses). In this paper we provide an introduction to the concept of viruses of microorganisms, a.k.a., viruses of microbes. We provide broad discussion particularly of VoM diversity. VoM diversity currently spans, in total, at least three-dozen virus families. This is roughly ten families per category—bacterial, archaeal, fungal, and protist—with some virus families infecting more than one of these microorganism major taxa. Such estimations, however, will vary with further discovery and taxon assignment and also are dependent upon what forms of life one includes among microorganisms. PMID:24278736

  20. Automated microorganism Sample Collection Module

    NASA Technical Reports Server (NTRS)

    Gall, L. S.; Graham, M. D.; Umbreit, W.

    1969-01-01

    Modified Gelman Sampler obtains representative sample of microorganism population. Proposed Sample Collection Module is based on direct inoculation of selected solid growth media encased in a cartridge at all times except during inoculation. Cartridge can be handled with no danger of contamination to sample or operator.

  1. Smaller fleas: viruses of microorganisms.

    PubMed

    Hyman, Paul; Abedon, Stephen T

    2012-01-01

    Life forms can be roughly differentiated into those that are microscopic versus those that are not as well as those that are multicellular and those that, instead, are unicellular. Cellular organisms seem generally able to host viruses, and this propensity carries over to those that are both microscopic and less than truly multicellular. These viruses of microorganisms, or VoMs, in fact exist as the world's most abundant somewhat autonomous genetic entities and include the viruses of domain Bacteria (bacteriophages), the viruses of domain Archaea (archaeal viruses), the viruses of protists, the viruses of microscopic fungi such as yeasts (mycoviruses), and even the viruses of other viruses (satellite viruses). In this paper we provide an introduction to the concept of viruses of microorganisms, a.k.a., viruses of microbes. We provide broad discussion particularly of VoM diversity. VoM diversity currently spans, in total, at least three-dozen virus families. This is roughly ten families per category-bacterial, archaeal, fungal, and protist-with some virus families infecting more than one of these microorganism major taxa. Such estimations, however, will vary with further discovery and taxon assignment and also are dependent upon what forms of life one includes among microorganisms.

  2. Development of natural anti-tumor drugs by microorganisms.

    PubMed

    Chang, Chia-Che; Chen, Wei-Chuan; Ho, Tsing-Fen; Wu, Ho-Shing; Wei, Yu-Hong

    2011-05-01

    Discoveries of tumor-resistant pharmacological drugs have mainly resulted from screening of natural products and their analogs. Some are also discovered incidentally when studying organisms. The great biodiversity of microorganisms raises the possibility of producing secondary metabolites (e.g., mevastatin, lovastatin, epothilone, salinosporamide A) to cope with adverse environments. Recently, natural plant pigments with anti-tumor activities such as β-carotene, lycopene, curcumin and anthocyanins have been proposed. However, many plants have a long life cycle. Therefore, pigments from microorganisms represent another option for the development of novel anti-tumor drugs. Prodigiosin (PG) is a natural red pigment produced by microorganisms, i.e., Serratia marcescens and other gram-negative bacteria. The anti-tumor potential of PG has been widely demonstrated. The families of PG (PGs), which share a common pyrrolylpyrromethene (PPM) skeleton, are produced by various bacteria. PGs are bioactive pigments and are known to exert immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. Currently the most common strain used for producing PGs is S. marcescens. However, few reports have discussed PGs production. This review therefore describes the development of an anti-tumor drug, PG, that can be naturally produced by microorganisms, and evaluates the microbial production system, fermentation strategies, purification and identification processes. The application potential of PGs is also discussed. PMID:21277252

  3. Development of natural anti-tumor drugs by microorganisms.

    PubMed

    Chang, Chia-Che; Chen, Wei-Chuan; Ho, Tsing-Fen; Wu, Ho-Shing; Wei, Yu-Hong

    2011-05-01

    Discoveries of tumor-resistant pharmacological drugs have mainly resulted from screening of natural products and their analogs. Some are also discovered incidentally when studying organisms. The great biodiversity of microorganisms raises the possibility of producing secondary metabolites (e.g., mevastatin, lovastatin, epothilone, salinosporamide A) to cope with adverse environments. Recently, natural plant pigments with anti-tumor activities such as β-carotene, lycopene, curcumin and anthocyanins have been proposed. However, many plants have a long life cycle. Therefore, pigments from microorganisms represent another option for the development of novel anti-tumor drugs. Prodigiosin (PG) is a natural red pigment produced by microorganisms, i.e., Serratia marcescens and other gram-negative bacteria. The anti-tumor potential of PG has been widely demonstrated. The families of PG (PGs), which share a common pyrrolylpyrromethene (PPM) skeleton, are produced by various bacteria. PGs are bioactive pigments and are known to exert immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. Currently the most common strain used for producing PGs is S. marcescens. However, few reports have discussed PGs production. This review therefore describes the development of an anti-tumor drug, PG, that can be naturally produced by microorganisms, and evaluates the microbial production system, fermentation strategies, purification and identification processes. The application potential of PGs is also discussed.

  4. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  5. L-methionine degradation potentialities of cheese-ripening microorganisms.

    PubMed

    Bonnarme, P; Lapadatescu, C; Yvon, M; Spinnler, H E

    2001-11-01

    Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase

  6. Recombinant Salmonella typhimurium outer membrane protein A is recognized by synovial fluid CD8 cells and stimulates synovial fluid mononuclear cells to produce interleukin (IL)-17/IL-23 in patients with reactive arthritis and undifferentiated spondyloarthropathy.

    PubMed

    Chaurasia, S; Shasany, A K; Aggarwal, A; Misra, R

    2016-08-01

    In developing countries, one-third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D-sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8(+) T cells showed significant intracellular interferon (IFN)-γ to Omp crude lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8(+) CD69(+) IFN-γ T cells to rOmpA-sal, whereas only four showed reactivity to rOmpD-sal. The mean stimulation index was significantly greater in rOmpA-sal than rOmpD-sal [3·0 (1·5-6·5) versus 1·5 (1·0-2·75), P < 0·005]. Similarly, using enzyme-linked immunospot (ELISPOT) in these 17 patients, the mean spots of IFN-γ-producing SFMCs were significantly greater in rOmpA-sal than rOmpD-sal [44·9 (3·5-130·7) versus 19·25 (6-41), P < 0·05]. SFMCs stimulated by rOmpA-sal produced significantly more proinflammatory cytokines than rOmpD-sal: IFN-γ [1·44 (0·39-20·42) versus 0·72 (0·048-9·15) ng/ml, P < 0·05], interleukin (IL)-17 [28·60 (6·15-510·86) versus 11·84 (6·83-252·62) pg/ml, P < 0·05], IL-23 [70·19 (15-1161·16) versus 28·25 (> 15-241·52) pg/ml, P < 0·05] and IL-6 [59·78 (2·03-273·36) versus 10·17 (0·004-190·19) ng/ml, P < 0·05]. The rOmpA-sal-specific CD8(+) T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8(+) T cells and drives SFMC to produce increased cytokines of the IL-17/IL-23 axis which contribute to the pathogenesis of Salmonella-triggered ReA. PMID:27060348

  7. Current Drive in Recombining Plasma

    SciTech Connect

    P.F. Schmit and N.J. Fisch

    2012-05-15

    The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

  8. Drug resistance in eukaryotic microorganisms.

    PubMed

    Fairlamb, Alan H; Gow, Neil A R; Matthews, Keith R; Waters, Andrew P

    2016-06-24

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies.

  9. Drug resistance in eukaryotic microorganisms.

    PubMed

    Fairlamb, Alan H; Gow, Neil A R; Matthews, Keith R; Waters, Andrew P

    2016-01-01

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies. PMID:27572976

  10. Phosphate Biomineralization of Cambrian Microorganisms

    NASA Technical Reports Server (NTRS)

    McKay, David S.; Rozanov, Alexei Yu.; Hoover, Richard B.; Westall, Frances

    1998-01-01

    As part of a long term study of biological markers (biomarkers), we are documenting a variety of features which reflect the previous presence of living organisms. As we study meteorites and samples returned from Mars, our main clue to recognizing possible microbial material may be the presence of biomarkers rather than the organisms themselves. One class of biomarkers consists of biominerals which have either been precipitated directly by microorganisms, or whose precipitation has been influenced by the organisms. Such microbe-mediated mineral formation may include important clues to the size, shape, and environment of the microorganisms. The process of fossilization or mineralization can cause major changes in morphologies and textures of the original organisms. The study of fossilized terrestrial organisms can help provide insight into the interpretation of mineral biomarkers. This paper describes the results of investigations of microfossils in Cambrian phosphate-rich rocks (phosphorites) that were found in Khubsugul, Northern Mongolia.

  11. Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk.

    PubMed

    Baldassarre, Hernan; Hockley, Duncan K; Doré, Monique; Brochu, Eric; Hakier, Bernard; Zhao, Xin; Bordignon, Vilceu

    2008-02-01

    The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1-5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.

  12. [Redox-sensors of microorganisms].

    PubMed

    Lushchak, V I

    2008-01-01

    This review summarizes available literature data on the existence and operation of redox sensors of microorganisms. It is partially focused on the activation by hyrdrogen peroxide OxyR protein and by superoxide anion SoxR protein in bacteria Escherichia coli and the activation by hyrdrogen peroxide and superoxide anion of Orp1-Yap1 protein system in yeast Saccharomyces cerevisiae. The similarities and peculiarities of redox signal sensing in pro- and eukaryotes have been discussed. PMID:19140447

  13. Phylogenetic relationships among subsurface microorganisms

    SciTech Connect

    Nierzwicki-Bauer, S.A.

    1991-01-01

    This report summarizes the progress made from 6/90--3/91 toward completion of our project, Phylogenetic Relationships among subsurface microorganisms. 16S rRNA was sequenced, and based on the sequence the SMCC isolates were assigned to preliminary groups. Microorganisms were obtained at various depths at the Savannah River Site, including the Surface (0 m), Congaree (91 m), and Middendorf (244 m, 259 m, 265 m). Sequence data from four isolates from the Congaree formation indicate these microorganisms can be divided into Pseudomonas spp. or Acinetobacter spp. Three 16S rRNA probes were synthesized based on sequence data. The synthesized probes were tested through in situ hybridization. Optimal conditions for in situ hybridization were determined. Because stability of RNA-DNA hybrids is dependent on hybridization stringency, related organisms can be differentiated using a single probe under different strigencies. The results of these hybridizations agree with results obtained by Balkwill and Reeves using restriction fragment length polymorphism analysis. The RNA content of a cell reflects its metabolic state. Cells which are starved for four days are not detectable with the homologous 16S rRNA probe. However, within 15 minutes of refeeding, detectable rRNA appeared. This suggests that organisms which are undetectable in environmental samples due to starvation may be detectable after addition of nutrients. Stepwise addition of specific nutrients could indicate which nutrients are rate limiting for growth. Preliminary experiments with soil samples from the Hanford Site indicate indigenous microorganisms can be detected by oligionucleotide probes. Further, using multiple probes based on universal sequences increases the number of organisms detected. Double label experiments, using a rhodamine-labelled oligionucleotide probe with free coumarin succinimidyl ester will allow simultaneous detection of total bacteria and specific 16S rRNA containing bacteria. 4 tabs. (MHB)

  14. Planetary protection Approaching uncultivable microorganisms

    NASA Astrophysics Data System (ADS)

    Nellen, J.; Rettberg, P.; Horneck, G.; Streit, W. R.

    2006-01-01

    With ESA's MiDiv project [ Rettberg, P., Fritze, D., Verbarg, S., Nellen, J., Horneck, G., Stackebrandt, E., Kminek, G. Final report ESA project MiDiv, ESA contract number 17538/03/NL/VS] the exobiology group at the German Aerospace Centre (DLR) in Cologne started to investigate the microbial diversity found on spacecrafts and in assembly halls used by Europe [ Rettberg, P., Nellen, J., Horneck, G., Fritze, D., Verbarg, S., Stackebrandt, E., Kminek, G. Determination of the microbial diversity of spacecraft assembly facilities: first results of the ESA project MiDiv. Adv. Space Res., 2005]. So far this examination was limited to cultivable microorganisms, probably excluding the majority of organisms present. To approach the microbial diversity of uncultivable microorganisms, new methods, have to be implemented into the analysing process. Therefore, we describe in this paper the adaptation of an existing protocol (surface sampling with swabs) to be used with a method based on the detection of DNA-fragments by polymerase chain reaction (PCR). Our data indicate that it is feasible to adapt the standardized sample taking process to be used with the PCR-method. While using a classical swabbing/cultivation approach for the detection of microorganisms on a surface, two impairing factors have to be accounted for. First, not all bacteria present on a surface will be taken up by the swab and second, not all the bacteria taken up will be released again from the swab. This will lead to a diminished overall estimation of the microorganisms present on a surface. Our studies further suggested that the estimated data had to be corrected by a factor of 2-3 to correspond with the actual numbers of spores spotted.

  15. Microorganisms resistant to free-living amoebae.

    PubMed

    Greub, Gilbert; Raoult, Didier

    2004-04-01

    Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens.

  16. Microorganisms Resistant to Free-Living Amoebae

    PubMed Central

    Greub, Gilbert; Raoult, Didier

    2004-01-01

    Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human “Troy,” and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens. PMID:15084508

  17. DIALYSIS FLASK FOR CONCENTRATED CULTURE OF MICROORGANISMS

    PubMed Central

    Gerhardt, Philipp; Gallup, D. M.

    1963-01-01

    Gerhardt, Philipp (The University of Michigan, Ann Arbor), and D. M. Gallup. Dialysis flask for concentrated culture of microorganisms. J. Bacteriol 86:919–929. 1963.—A twin-chambered dialysis flask was designed with a supported membrane clamped between a reservoir of medium in the bottom and a small volume of culture above, the unit being mounted on a shaking machine to provide aeration and agitation. The performance of different dialysis membranes and membrane filters was compared in glucose-diffusion and bacterial-culture tests. Some of the variables in dialysis culture were assessed and the growth response was characterized, with Serratia marcescens as the test organism. The general usefulness and concentrating effect of dialysis culture were demonstrated in trials with 16 representative types of microorganisms. Dialysis culture was shown to be especially suitable for producing dense populations of cells or their macromolecular products in an environment free from complex medium constituents, for removing toxic products that limit growth or fermentation, and for supplying oxygen by diffusion without the damage from usual aeration procedures. Images PMID:14080802

  18. Minerals and Microorganisms in Evaporite Environments

    NASA Astrophysics Data System (ADS)

    Morris, P. A.; Brigmon, R. L.

    2010-12-01

    Traditional analysis of evaporite environments have either focused on the geology or the halophilic organisms. It is relatively rare that the two have been combined and even rarer that both disciplines have been incorporated in comparing evaporite sites. The variation in evaporite environments does influence microbial ecology and fossilization processes as each site varies in pH, temperature, presence or absence springs, and spring chemistry. Understanding the evaporite environments is important for planetary scientists as they serve as analogs for evaluating extraterrestrial materials, including the potential for water and ultimately life. For example Mars lander, rover and orbital missions have identified the evaporite signatures of gypsum, carbonates and chlorides, all indicating that water existed at sometime in the planets geological history. Terrestrial evaporite sites all possess halophilic tolerant life. In some instances such as the Dead Sea, Israel, it is restricted to microbial life, but in other sites there are higher life forms. The microbes associated with these evaporite sites can produce biofilms as a method to develop their own microenvironments. Microorganisms can be observed colonizing specific ecological niches or gradients can be created by these environments. These gradients occur due the localized drying and weathering patterns that create different soil chemistry. The microorganisms in turn colonize specific areas more suitable to their specific metabolic needs. For example, under anaerobic conditions with sulfur and methane prevalent methanogenic and/or sulfur reducing microbial species may be observed. However, under similar chemistry environments with the exception of aerobic conditions sulfur oxidizer and/or methanotrophic microorganism may occur. Because of their conspicuous colored pigments purple sulfur bacteria are frequently observed in anoxic zones of lakes, sulfur springs, and stratified evaporite crusts. Some of these bacteria

  19. [Questions safety and tendency of using genetically modified microorganisms in food, food additives and food derived].

    PubMed

    Khovaev, A A

    2008-01-01

    In this article analysis questions of using genetically modified microorganisms in manufacture food production, present new GMM used in manufacture -food ferments; results of medical biological appraisal/ microbiological and genetic expert examination/ of food, getting by use microorganisms or there producents with indication modern of control methods.

  20. Predatory Microorganisms Would Help Reclaim Water

    NASA Technical Reports Server (NTRS)

    Benjaminson, Morris A.; Lehrer, Stanley

    1995-01-01

    Wastewater-reclamation systems of proposed type use predatory, nonpathogenic microorganisms to consume pathogenic microorganisms. Unlike some other wastewater-reclamation systems, these systems do not require use of toxic chemicals, intense heat, or ionizing radiation (conductivity rays or ultraviolet) to destroy microorganisms.

  1. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Recipient microorganisms. 725.420 Section 725.420 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient...

  2. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Microorganism identity. 725.85 Section... to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to... specific microorganism identity at the time of submission of the information. This claim will apply only...

  3. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Microorganism identity. 725.85 Section... to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to... specific microorganism identity at the time of submission of the information. This claim will apply only...

  4. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Microorganism identity. 725.85 Section... to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to... specific microorganism identity at the time of submission of the information. This claim will apply only...

  5. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Microorganism identity. 725.85 Section... ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Confidentiality and Public Access to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior...

  6. A new ultrasound based method for rapid microorganism detection

    NASA Astrophysics Data System (ADS)

    Shukla, Shiva Kant; Segura, Luis Elvira; Sánchez, Carlos José Sierra; López, Pablo Resa

    2012-05-01

    A new method for rapid detection of catalase positive microorganisms by using an ultrasonic measuring method is proposed in this work. The developed technique is based on the detection of oxygen bubbles produced by the hydrolysis of hydrogen peroxide induced by the enzyme catalase which is present in many microorganisms. The bubbles are trapped in a media based on agar gel which was especially developed for microbiological evaluation. It is found that microorganism concentrations of the order of 105 c.f.u./ml can be detected by using this method. The results obtained show up that the proposed method is competitive with other modern commercial methods like luminescence by ATP system. The method can also be used for characterization of enzyme activity.

  7. Mini-review: Inhibition of biofouling by marine microorganisms.

    PubMed

    Dobretsov, Sergey; Abed, Raeid M M; Teplitski, Max

    2013-01-01

    Any natural or artificial substratum exposed to seawater is quickly fouled by marine microorganisms and later by macrofouling species. Microfouling organisms on the surface of a substratum form heterogenic biofilms, which are composed of multiple species of heterotrophic bacteria, cyanobacteria, diatoms, protozoa and fungi. Biofilms on artificial structures create serious problems for industries worldwide, with effects including an increase in drag force and metal corrosion as well as a reduction in heat transfer efficiency. Additionally, microorganisms produce chemical compounds that may induce or inhibit settlement and growth of other fouling organisms. Since the last review by the first author on inhibition of biofouling by marine microbes in 2006, significant progress has been made in the field. Several antimicrobial, antialgal and antilarval compounds have been isolated from heterotrophic marine bacteria, cyanobacteria and fungi. Some of these compounds have multiple bioactivities. Microorganisms are able to disrupt biofilms by inhibition of bacterial signalling and production of enzymes that degrade bacterial signals and polymers. Epibiotic microorganisms associated with marine algae and invertebrates have a high antifouling (AF) potential, which can be used to solve biofouling problems in industry. However, more information about the production of AF compounds by marine microorganisms in situ and their mechanisms of action needs to be obtained. This review focuses on the AF activity of marine heterotrophic bacteria, cyanobacteria and fungi and covers publications from 2006 up to the end of 2012.

  8. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    SciTech Connect

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  9. Early photosynthetic microorganisms and environmental evolution

    NASA Technical Reports Server (NTRS)

    Golubic, S.

    1980-01-01

    Microfossils which are preserved as shrivelled kerogenous residues provide little information about cellular organization and almost none about the metabolic properties of the organisms. The distinction between prokaryotic vs eukaryotic, and phototrophic vs chemo- and organotrophic fossil microorganisms rests entirely on morphological comparisons with recent counterparts. The residual nature of the microbial fossil record promotes the conclusion that it must be biased toward (a) most abundant organisms, (b) those most resistant to degradation, and (c) those inhabiting environments with high preservation potential e.g., stromatolites. These criteria support the cyanophyte identity of most Precambrian microbial fossils on the following grounds: (1) as primary producers they dominate prokaryotic communities in modern extreme environments, e.g., intertidal zone; (2) several morphological counterparts of modern cyanophytes and microbial fossils have been established based on structure, cell division patterns and degradation sequences. The impact of anaerobic and oxygenic microbial photosynthesis on the evolution of Precambrian environments is discussed.

  10. Cellulases from psychrophilic microorganisms: a review.

    PubMed

    Kasana, Ramesh C; Gulati, Arvind

    2011-12-01

    Cellulases are hydrolytic enzymes that catalyze total hydrolysis of cellulose into sugars. Cellulases are produced by various groups of microorganisms and animals; however, psychrophiles are the ideal candidates for the production of enzymes active at low temperature and stable under alkaline conditions, in the presence of oxidants and detergents, which are in large demand as laundry additives. The cellulases from psychrophiles also find application in environmental bioremediation, food industry and molecular biology. Research work on cellulase has been done over the last six decades, but there is no exclusive review available on the cellulases from psychrophiles. This review is an attempt to fill this gap by providing all the relevant information exclusively for cellulases from psychrophiles, with a focus on the present status of knowledge on their activity, molecular characteristics, gene cloning, statistical experimental designs, crystal structure, and strategies for the improvement of psychrophilic cellulases.

  11. Laboratory studies of ocean mixing by microorganisms

    NASA Astrophysics Data System (ADS)

    Martinez-Ortiz, Monica; Dabiri, John O.

    2011-11-01

    Ocean mixing plays a major role in nutrient and energy transport and is an important input to climate models. Recent studies suggest that the contribution of fluid transport by swimming microorganisms to ocean mixing may be of the same order of magnitude as winds and tides. An experimental setup has been designed in order to study the mixing efficiency of vertical migration of plankton. To this end, a stratified water column is created to model the ocean's density gradient. The vertical migration of Artemia Salina (brine shrimp) within the water column is controlled via luminescent signals on the top and bottom of the column. By fluorescently labelling portions of the water column, the stirring of the density gradient by the animals is visualized and quantified. Preliminary results show that the vertical movement of these organisms produces enhanced mixing relative to control cases in which only buoyancy forces and diffusion are present.

  12. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  13. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  14. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  15. Plant Drought Tolerance Enhancement by Trehalose Production of Desiccation-Tolerant Microorganisms

    PubMed Central

    Vílchez, Juan I.; García-Fontana, Cristina; Román-Naranjo, Desireé; González-López, Jesús; Manzanera, Maximino

    2016-01-01

    A collection of desiccation-tolerant xeroprotectant-producing microorganisms was screened for their ability to protect plants against drought, and their role as plant growth-promoting rhizobacteria was investigated in two different crops (tomato and pepper). The most commonly described biochemical mechanisms for plant protection against drought by microorganisms including the production of phytohormones, antioxidants and xeroprotectants were analyzed. In particular, the degree of plant protection against drought provided by these microorganisms was characterized. After studying the findings and comparing them with results of the closest taxonomic relatives at the species and strain levels, we propose that trehalose produced by these microorganisms is correlated with their ability to protect plants against drought. This proposal is based on the increased protection of plants against drought by the desiccation-sensitive microorganism Pseudomonas putida KT2440, which expresses the otsAB genes for trehalose biosynthesis in trans. PMID:27746776

  16. Biodiversity of the oleaginous microorganisms in Tibetan Plateau.

    PubMed

    Li, Shi Lin; Lin, Qiang; Li, Xin Ran; Xu, Hui; Yang, Yun Xi; Qiao, Dai Rong; Cao, Yi

    2012-04-01

    Microbial lipids, which are also known as single cell oils (SCO), are produced by oleaginous microorganisms including oleaginous bacteria, yeast, fungus and algae through converting carbohydrates into lipids under certain conditions. Due to its unique environment having extremely low temperature and anoxia, the Tibetan Plateau is amongst the regions with numerous rare ecotypes such as arid desert, salt marsh, alpine permafrost, hot spring, and lawn. By using a rapid, convenient screening method, we identified 31 strains of oleaginous microorganisms from different habitats in the Tibetan Plateau, which include wetlands, lawn, hot spring, alpine permafrost, and saline-alkali soil. Molecular identity analysis showed that they belong to 15 different species, 7 of which are reported for the first time as lipid-producing microorganisms, that is, Cladosporium sp., Gibberella fujikuro, Ochrobactrum sp., Plectosphaerella sp., Tilletiopsis albescens, Backusella ctenidia, and Davidiella tassiana. The distribution of the oleaginous microorganisms varies with habitats. 11 strains were found in hot spring (35.5%), 10 in farmland (32.3%), 6 in lawn (19.4%), 2 in sand (6.4%), 1 in wetland (3.2%), and 1 in permafrost (3.2%). Carbon utilization analysis indicated that most of these filamentous fungi can use xylose and carboxymethyl cellulose (CMC) as carbon source, where Backusella ctenidia, Fusarium sp. and Gibberella fujikuroi have the strongest capability.

  17. Cellulolytic Microorganisms from Thermal Environments

    SciTech Connect

    Vishnivetskaya, Tatiana A; Raman, Babu; Phelps, Tommy Joe; Podar, Mircea; Elkins, James G

    2012-01-01

    Thermal, anaerobic environments rich in decaying plant material are a potential source of novel cellulolytic bacteria. Samples collected from geothermal aquifers in the Yellowstone National Park (YNP) were used to select for cellulolytic thermophiles. Laboratory enrichments on dilute-acid pretreated plant biomass (switchgrass, Populus), and crystalline cellulose (Avicel) resulted in the isolation of 247 environmental clones. The majority of individual clones were affiliated with the cellulolytic bacteria of phylum Firmicutes, followed by xylanolytic and saccharolytic members of the phylum Dictyoglomi. Among the Firmicutes, the clones were affiliated with the genera Caldicellulosiruptor (54.4%), Caloramator (11.5%), Thermoanaerobacter (8.8%), Thermovenabulum (4.1%), and Clostridium (2.0%). From established anaerobic thermophilic enrichments a total of 81 single strains of the genera Caldicellulosiruptor (57%) and Thermoanaerobacter (43%) were isolated. With continuous flow enrichment on Avicel, increases in the relative abundance of Caloramator sp. was observed over clones detected from the Caldicellulosiruptor. Complex communities of interacting microorganisms bring about cellulose decomposition in nature, therefore using up-to-date approaches may yield novel cellulolytic microorganisms with high activity and a rapid rate of biomass conversion to biofuels.

  18. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  19. Initiation of meiotic recombination in Ustilago maydis.

    PubMed

    Kojic, Milorad; Sutherland, Jeanette H; Pérez-Martín, José; Holloman, William K

    2013-12-01

    A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.

  20. Genetic recombination and molecular evolution.

    PubMed

    Charlesworth, B; Betancourt, A J; Kaiser, V B; Gordo, I

    2009-01-01

    Reduced rates of genetic recombination are often associated with reduced genetic variability and levels of adaptation. Several different evolutionary processes, collectively known as Hill-Robertson (HR) effects, have been proposed as causes of these correlates of recombination. Here, we use DNA sequence polymorphism and divergence data from the noncrossing over dot chromosome of Drosophila to discriminate between two of the major forms of HR effects: selective sweeps and background selection. This chromosome shows reduced levels of silent variability and reduced effectiveness of selection. We show that neither model fits the data on variability. We propose that, in large genomic regions with restricted recombination, HR effects among nonsynonymous mutations undermine the effective strength of selection, so that their background selection effects are weakened. This modified model fits the data on variability and also explains why variability in very large nonrecombining genomes is not completely wiped out. We also show that HR effects of this type can produce an individual selection advantage to recombination, as well as greatly reduce the mean fitness of nonrecombining genomes and genomic regions.

  1. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

    PubMed Central

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G.

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

  2. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives.

    PubMed

    Jia, Baolei; Jeon, Che Ok

    2016-08-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design. PMID:27581654

  3. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system.

    PubMed

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.

  4. Recombinant expression and inhibition mechanism analysis of pectin methylesterase from Aspergillus flavus.

    PubMed

    Jiang, Xiuping; Jia, Qiulei; Chen, Lei; Chen, Qi; Yang, Qing

    2014-06-01

    Phytopathogenic microorganisms can produce pectin methylesterase (PME) to degrade plant cell walls during plant invasion. This enzyme is thought to be a virulence factor of phytopathogens. In this work, PME from Aspergillus flavus (AFPME) was expressed in Pichia pastoris and an in vitro inhibitor study was performed. The purified AFPME with a yield of 52.2% was resolved as one band with a molecular mass of c. 40 kDa by SDS-PAGE. Optimal activity of the enzyme occurred at a temperature of 55 °C and a pH of 4.8. Epigallocatechin gallate (EGCG) strongly inhibited the activity of recombinant AFPME. The molecular docking analysis indicated that EGCG could form hydrogen bonds and π-π interactions with some amino acid residues in the active site of AFPME. Our studies provide a novel strategy for the control of the plant invasion of A. flavus. PMID:24766423

  5. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system.

    PubMed

    Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G

    2013-01-01

    The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

  6. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives

    PubMed Central

    2016-01-01

    The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design. PMID:27581654

  7. Co-factor activated recombinant adenovirus proteinases

    SciTech Connect

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  8. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  9. Swimming of a Ciliated Microorganism

    NASA Astrophysics Data System (ADS)

    Guo, Hanliang; Kanso, Eva

    2013-11-01

    We propose a 2D model to consider the locomotion of a ciliated microorganism in a viscous fluid. The model consists of a circular body whose boundary is covered by a finite number of cilia. Stokes paradox does not hold due to the self-propelling nature of the organism. Using a regularized Stokeslet method, we determine numerically the time-dependent swimming motion for prescribed kinematics (undulatory beat) of the individual cilium. Phase differences between neighboring cilia result in metachronal waves characteristic of biological cilia. We compare our results based on the discrete cilia approach with the envelope model proposed by JR Blake. We then study the net locomotion as function of the metachronal wave. We find that, for a given geometry and cilia density, there is an optimal wave number (phase difference) for locomotion in terms of velocity of propulsion and efficiency.

  10. Functional Basis of Microorganism Classification

    PubMed Central

    Zhu, Chengsheng; Delmont, Tom O.; Vogel, Timothy M.; Bromberg, Yana

    2015-01-01

    Correctly identifying nearest “neighbors” of a given microorganism is important in industrial and clinical applications where close relationships imply similar treatment. Microbial classification based on similarity of physiological and genetic organism traits (polyphasic similarity) is experimentally difficult and, arguably, subjective. Evolutionary relatedness, inferred from phylogenetic markers, facilitates classification but does not guarantee functional identity between members of the same taxon or lack of similarity between different taxa. Using over thirteen hundred sequenced bacterial genomes, we built a novel function-based microorganism classification scheme, functional-repertoire similarity-based organism network (FuSiON; flattened to fusion). Our scheme is phenetic, based on a network of quantitatively defined organism relationships across the known prokaryotic space. It correlates significantly with the current taxonomy, but the observed discrepancies reveal both (1) the inconsistency of functional diversity levels among different taxa and (2) an (unsurprising) bias towards prioritizing, for classification purposes, relatively minor traits of particular interest to humans. Our dynamic network-based organism classification is independent of the arbitrary pairwise organism similarity cut-offs traditionally applied to establish taxonomic identity. Instead, it reveals natural, functionally defined organism groupings and is thus robust in handling organism diversity. Additionally, fusion can use organism meta-data to highlight the specific environmental factors that drive microbial diversification. Our approach provides a complementary view to cladistic assignments and holds important clues for further exploration of microbial lifestyles. Fusion is a more practical fit for biomedical, industrial, and ecological applications, as many of these rely on understanding the functional capabilities of the microbes in their environment and are less concerned

  11. Functional Basis of Microorganism Classification.

    PubMed

    Zhu, Chengsheng; Delmont, Tom O; Vogel, Timothy M; Bromberg, Yana

    2015-08-01

    Correctly identifying nearest "neighbors" of a given microorganism is important in industrial and clinical applications where close relationships imply similar treatment. Microbial classification based on similarity of physiological and genetic organism traits (polyphasic similarity) is experimentally difficult and, arguably, subjective. Evolutionary relatedness, inferred from phylogenetic markers, facilitates classification but does not guarantee functional identity between members of the same taxon or lack of similarity between different taxa. Using over thirteen hundred sequenced bacterial genomes, we built a novel function-based microorganism classification scheme, functional-repertoire similarity-based organism network (FuSiON; flattened to fusion). Our scheme is phenetic, based on a network of quantitatively defined organism relationships across the known prokaryotic space. It correlates significantly with the current taxonomy, but the observed discrepancies reveal both (1) the inconsistency of functional diversity levels among different taxa and (2) an (unsurprising) bias towards prioritizing, for classification purposes, relatively minor traits of particular interest to humans. Our dynamic network-based organism classification is independent of the arbitrary pairwise organism similarity cut-offs traditionally applied to establish taxonomic identity. Instead, it reveals natural, functionally defined organism groupings and is thus robust in handling organism diversity. Additionally, fusion can use organism meta-data to highlight the specific environmental factors that drive microbial diversification. Our approach provides a complementary view to cladistic assignments and holds important clues for further exploration of microbial lifestyles. Fusion is a more practical fit for biomedical, industrial, and ecological applications, as many of these rely on understanding the functional capabilities of the microbes in their environment and are less concerned with

  12. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  13. A FIELD STUDY WITH GENETICALLY ENGINEERED ALFALFA INOCULATED WITH RECOMBINANT SINORHIZOBIUM MELILOTI: EFFECTS ON THE SOIL ECOSYSTEM

    EPA Science Inventory

    The agricultural use of genetically engineered plants and microorganisms has become increasingly common. Because genetically engineered plants and microorganisms can produce compounds foreign to their environment, there is concern that they may become established outside of thei...

  14. Microorganisms in metalworking fluids: current issues in research and management.

    PubMed

    Trafny, Elżbieta A

    2013-03-01

    The microbial contamination of water miscible metalworking fluids (MWFs) is a serious problem in metal industry. A good maintenance of MWF re-circulation systems can extend the lifetime of coolants and ensure the quality of the tools produced. In MWFs, as in the other water-based environments, microorganisms usually live in the form of biofilms, the communities of bacteria and fungi attached to the surface of sumps, metal parts and also to each other. Biofilms exhibit very high resistance to biocides. The effect of biocides that are used as additives to MWFs to control the growth of the bacterial and fungal microbiomes (microorganisms characteristic to the individual coolant system) have become the subject of research only in recent years. There are also only sparse reports on the impact of biocides on microorganisms growing in biofilms in MWF installations. Fast growing mycobacteria are important members of these biofilm communities. Their presence has recently been linked with the occurrence of cases of hypersensitivity pneumonitis, a serious respiratory disorder in the metal industry employees. The new, relatively fast and inexpensive techniques to assess the species diversity within MWF microbiomes and their population size should be developed in order to control the microorganisms' proliferation in MWFs and to diminish the occupational exposure to harmful bioaerosols in metal industry. PMID:23526197

  15. Development of Novel Drugs from Marine Surface Associated Microorganisms

    PubMed Central

    Penesyan, Anahit; Kjelleberg, Staffan; Egan, Suhelen

    2010-01-01

    While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future. PMID:20411108

  16. Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland.

    PubMed

    Baldassarre, Hernan; Hockley, Duncan K; Olaniyan, Benjamen; Brochu, Eric; Zhao, Xin; Mustafa, Arif; Bordignon, Vilceu

    2008-10-01

    The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na(+), K(+), assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the

  17. Recombination and assortment in the macronucleus of Tetrahymena thermophila: a theoretical study by computer simulation.

    PubMed

    Doerder, F P; Diblasi, S L

    1984-12-01

    The compound nature of the macronucleus of Tetrahymena thermophila presents multiple opportunities for recombination between genes on the same macronuclear chromosome. Such recombinants should be detectable through their assortment at subsequent amitotic macronuclear divisions. Thus, a macronucleus that is initially AB/ab should produce recombinant assortees of the genotypes Ab/aB. Computer simulation shows that, when the recombination frequency is two or fewer times per cell cycle, recombinant assortees are produced at experimentally measurable frequencies of less than 40%. At higher recombination frequencies, linked genes appear to assort independently. The simulations also show that recombination during macronuclear development can be distinguished from recombination in subsequent cell cycles only if the first appearance of recombinant assortees is 100 or more fissions after conjugation. The use of macronuclear recombination and assortment as a means of mapping macronuclear genes is severely constrained by the large variances in assortment outcomes; with experimentally small sample sizes, such mapping is impossible.

  18. Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein.

    PubMed

    Lundgren, A; Leach, S; Tobias, J; Carlin, N; Gustafsson, B; Jertborn, M; Bourgeois, L; Walker, R; Holmgren, J; Svennerholm, A-M

    2013-02-01

    We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB. PMID:23306362

  19. Electron Recombination in a Dense Hydrogen Plasma

    SciTech Connect

    Jana, M.R.; Johnstone, C.; Kobilarcik, T.; Koizumi, G.M.; Moretti, A.; Popovic, M.; Tollestrup, A.V.; Yonehara, K.; Leonova, M.A.; Schwarz, T.A.; Chung, M.; /Unlisted /IIT, Chicago /Fermilab /MUONS Inc., Batavia /Turin Polytechnic

    2012-05-01

    A high pressure hydrogen gas filled RF cavity was subjected to an intense proton beam to study the evolution of the beam induced plasma inside the cavity. Varying beam intensities, gas pressures and electric fields were tested. Beam induced ionized electrons load the cavity, thereby decreasing the accelerating gradient. The extent and duration of this degradation has been measured. A model of the recombination between ionized electrons and ions is presented, with the intent of producing a baseline for the physics inside such a cavity used in a muon accelerator. Analysis of the data taken during the summer of 2011 shows that self recombination takes place in pure hydrogen gas. The decay of the number of electrons in the cavity once the beam is turned off indicates self recombination rather than attachment to electronegative dopants or impurities. The cross section of electron recombination grows for larger clusters of hydrogen and so at the equilibrium of electron production and recombination in the cavity, processes involving H{sub 5}{sup +} or larger clusters must be taking place. The measured recombination rates during this time match or exceed the analytic predicted values. The accelerating gradient in the cavity recovers fully in time for the next beam pulse of a muon collider. Exactly what the recombination rate is and how much the gradient degrades during the 60 ns muon collider beam pulse will be extrapolated from data taken during the spring of 2012.

  20. Biofouling of marbles by oxygenic photosynthetic microorganisms.

    PubMed

    Karaca, Zeki; Öztürk, Ayten; Çolak, Emel

    2015-08-01

    Phototrophic microorganisms disfigure the surfaces of different types of stone. Stone structure is damaged by the activity of photoautotrophic and other microorganisms. However, to date few, investigations have been undertaken into the relationship between microorganisms and the properties of different types of marble. In this study, biological activity of photoautotrophic microorganisms on three types of marble (Yatagan White, Giallo Anticato and Afyon White) was investigated under laboratory conditions over a short period of time. The three types of marble supported the growth of phototrophic microbial communities on their outer and inner layers, turning their original colour from white to a yellowish green colour. The porosity of the marble types facilitated filamentous microbial growth in the presence of water. Scanning electron microscope analysis revealed the accumulation of aggregates such as small spherical, fibrillar, calcified globular bodies on the inner surfaces of the marbles. This suggests that the microscopic characteristics of particular marble types may stimulate the growth of certain types of microorganisms.

  1. Systems Biology of Industrial Microorganisms

    NASA Astrophysics Data System (ADS)

    Papini, Marta; Salazar, Margarita; Nielsen, Jens

    The field of industrial biotechnology is expanding rapidly as the chemical industry is looking towards more sustainable production of chemicals that can be used as fuels or building blocks for production of solvents and materials. In connection with the development of sustainable bioprocesses, it is a major challenge to design and develop efficient cell factories that can ensure cost efficient conversion of the raw material into the chemical of interest. This is achieved through metabolic engineering, where the metabolism of the cell factory is engineered such that there is an efficient conversion of sugars, the typical raw materials in the fermentation industry, into the desired product. However, engineering of cellular metabolism is often challenging due to the complex regulation that has evolved in connection with adaptation of the different microorganisms to their ecological niches. In order to map these regulatory structures and further de-regulate them, as well as identify ingenious metabolic engineering strategies that full-fill mass balance constraints, tools from systems biology can be applied. This involves both high-throughput analysis tools like transcriptome, proteome and metabolome analysis, as well as the use of mathematical modeling to simulate the phenotypes resulting from the different metabolic engineering strategies. It is in fact expected that systems biology may substantially improve the process of cell factory development, and we therefore propose the term Industrial Systems Biology for how systems biology will enhance the development of industrial biotechnology for sustainable chemical production.

  2. [Genome editing of industrial microorganism].

    PubMed

    Zhu, Linjiang; Li, Qi

    2015-03-01

    Genome editing is defined as highly-effective and precise modification of cellular genome in a large scale. In recent years, such genome-editing methods have been rapidly developed in the field of industrial strain improvement. The quickly-updating methods thoroughly change the old mode of inefficient genetic modification, which is "one modification, one selection marker, and one target site". Highly-effective modification mode in genome editing have been developed including simultaneous modification of multiplex genes, highly-effective insertion, replacement, and deletion of target genes in the genome scale, cut-paste of a large DNA fragment. These new tools for microbial genome editing will certainly be applied widely, and increase the efficiency of industrial strain improvement, and promote the revolution of traditional fermentation industry and rapid development of novel industrial biotechnology like production of biofuel and biomaterial. The technological principle of these genome-editing methods and their applications were summarized in this review, which can benefit engineering and construction of industrial microorganism.

  3. Electrical Retrieval of Living Microorganisms from Cryopreserved Marine Sponges Using a Potential-Controlled Electrode.

    PubMed

    Koyama, Sumihiro; Nishi, Shinro; Tokuda, Maki; Uemura, Moeka; Ishikawa, Yoichi; Seya, Takeshi; Chow, Seinen; Ise, Yuji; Hatada, Yuji; Fujiwara, Yoshihiro; Tsubouchi, Taishi

    2015-10-01

    The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at -80 °C. A -0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms. PMID:26242755

  4. Electrical Retrieval of Living Microorganisms from Cryopreserved Marine Sponges Using a Potential-Controlled Electrode.

    PubMed

    Koyama, Sumihiro; Nishi, Shinro; Tokuda, Maki; Uemura, Moeka; Ishikawa, Yoichi; Seya, Takeshi; Chow, Seinen; Ise, Yuji; Hatada, Yuji; Fujiwara, Yoshihiro; Tsubouchi, Taishi

    2015-10-01

    The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at -80 °C. A -0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.

  5. Recombinant baculovirus isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2007-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac or BaculoDirect, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay. Methods unique to the Bac-to-Bac system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  6. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  7. Aspergillus: sex and recombination.

    PubMed

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli.

  8. Aspergillus: sex and recombination.

    PubMed

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli. PMID:25118872

  9. Recombinant clostridia that fix CO2 and CO and uses thereof

    DOEpatents

    Papoutsakis, Eleftherios T.; Al-Hinai, Mohab Ali; Jones, Shawn Williams; Indurthi, Dinesh Chanukya; Mitchell, Daniel Knox; Fast, Alan

    2014-06-24

    The present invention relates a recombinant Clostridium expressing one or more heterologous Wood-Ljungdahl (WL) genes. In particular, the recombinant Clostridium produces a metabolite at an increased level. The present invention also relates to a method for producing a metabolite by the recombinant Clostridium.

  10. Recombinant allergens: what does the future hold?

    PubMed

    Valenta, Rudolf; Niespodziana, Katarzyna; Focke-Tejkl, Margit; Marth, Katharina; Huber, Hans; Neubauer, Angela; Niederberger, Verena

    2011-04-01

    This year we are celebrating not only the centenary of allergen-specific immunotherapy but also the 10-year anniversary of the first administration of recombinant allergen-based vaccines to allergic patients. By using recombinant DNA technology, defined and safe allergy vaccines can be produced that allow us to overcome many, if not all, of the problems associated with the use of natural allergen extracts, such as insufficient quality, allergenic activity, and poor immunogenicity. Here we provide an update of clinical studies with recombinant allergen-based vaccines, showing that some of these vaccines have undergone successful clinical evaluation up to phase III studies. Furthermore, we introduce a strategy for allergen-specific immunotherapy based on recombinant fusion proteins consisting of viral carrier proteins and allergen-derived peptides without allergenic activity, which holds the promise of being free of side effects and eventually being useful for prophylactic vaccination.

  11. Halophilic microorganisms in deteriorated historic buildings: insights into their characteristics.

    PubMed

    Adamiak, Justyna; Otlewska, Anna; Gutarowska, Beata; Pietrzak, Anna

    2016-01-01

    Historic buildings are constantly being exposed to numerous climatic changes such as damp and rainwater. Water migration into and out of the material's pores can lead to salt precipitation and the so-called efflorescence. The structure of the material may be seriously threatened by salt crystallization. A huge pressure is produced when salt hydrates occupy larger spaces, which leads at the end to cracking, detachment and material loss. Halophilic microorganisms have the ability to adapt to high salinity because of the mechanisms of inorganic salt (KCl or NaCl) accumulation in their cells at concentrations isotonic to the environment, or compatible solutes uptake or synthesis. In this study, we focused our attention on the determination of optimal growth conditions of halophilic microorganisms isolated from historical buildings in terms of salinity, pH and temperature ranges, as well as biochemical properties and antagonistic abilities. Halophilic microorganisms studied in this paper could be categorized as a halotolerant group, as they grow in the absence of NaCl, as well as tolerate higher salt concentrations (Staphylococcus succinus, Virgibacillus halodenitrificans). Halophilic microorganisms have been also observed (Halobacillus styriensis, H. hunanensis, H. naozhouensis, H. litoralis, Marinococcus halophilus and yeast Sterigmatomyces halophilus). With respect to their physiological characteristics, cultivation at a temperature of 25-30°C, pH 6-7, NaCl concentration for halotolerant and halophilic microorganisms, 0-10% and 15-30%, respectively, provides the most convenient conditions. Halophiles described in this study displayed lipolytic, glycolytic and proteolytic activities. Staphylococcus succinus and Marinococcus halophilus showed strong antagonistic potential towards bacteria from the Bacillus genus, while Halobacillus litoralis displayed an inhibiting ability against other halophiles. PMID:26894235

  12. DEVELOPMENT OF MICROORGANISMS WITH IMPROVED TRANSPORT AND BIOSURFACTANT ACTIVITY FOR ENHANCED OIL RECOVERY

    SciTech Connect

    M.J. McInerney; R.M. Knapp; D.P. Nagle, Jr.; Kathleen Duncan; N. Youssef; M.J. Folmsbee; S. Maudgakya

    2003-06-26

    production. As an initial step in the search for a better biosurfactant-producing microorganism, 157 bacterial strains were screened for biosurfactant production under both aerobic and anaerobic conditions. A hundred and forty seven strains produced either equal or higher amounts of biosurfactant compared to B. mojavensis JF-2 and the 10 best strains were chosen for further study. In an attempt to increase biosurfactant production, a genetic recombination experiment was conducted by mixing germinating spores of four of the best strains with B. mojavensis JF-2. Biosurfactant production was higher with the mixed spore culture than in the cocultures containing B. mojavensis JF-2 and each of the other 4 strains or in a mixed culture containing all five strains that had not undergone genetic exchange. Four isolates were obtained from the mixed spores culture that gave higher biosurfactant production than any of the original strains. Repetitive sequence-based polymerase chain reaction analysis showed differences in the band pattern for these strains compared to the parent strains, suggesting the occurrence of genetic recombination. We have a large collection of biosurfactant-producing microorganisms and a natural mechanism to improve biosurfactant production in these organisms.

  13. Possibilities for extremophilic microorganisms in microbial electrochemical systems.

    PubMed

    Dopson, Mark; Ni, Gaofeng; Sleutels, Tom H J A

    2016-03-01

    Microbial electrochemical systems exploit the metabolism of microorganisms to generate electrical energy or a useful product. In the past couple of decades, the application of microbial electrochemical systems has increased from the use of wastewaters to produce electricity to a versatile technology that can use numerous sources for the extraction of electrons on the one hand, while on the other hand these electrons can be used to serve an ever increasing number of functions. Extremophilic microorganisms grow in environments that are hostile to most forms of life and their utilization in microbial electrochemical systems has opened new possibilities to oxidize substrates in the anode and produce novel products in the cathode. For example, extremophiles can be used to oxidize sulfur compounds in acidic pH to remediate wastewaters, generate electrical energy from marine sediment microbial fuel cells at low temperatures, desalinate wastewaters and act as biosensors of low amounts of organic carbon. In this review, we will discuss the recent advances that have been made in using microbial catalysts under extreme conditions and show possible new routes that extremophilic microorganisms open for microbial electrochemical systems. PMID:26474966

  14. Possibilities for extremophilic microorganisms in microbial electrochemical systems

    PubMed Central

    Dopson, Mark; Ni, Gaofeng; Sleutels, Tom HJA

    2015-01-01

    Microbial electrochemical systems exploit the metabolism of microorganisms to generate electrical energy or a useful product. In the past couple of decades, the application of microbial electrochemical systems has increased from the use of wastewaters to produce electricity to a versatile technology that can use numerous sources for the extraction of electrons on the one hand, while on the other hand these electrons can be used to serve an ever increasing number of functions. Extremophilic microorganisms grow in environments that are hostile to most forms of life and their utilization in microbial electrochemical systems has opened new possibilities to oxidize substrates in the anode and produce novel products in the cathode. For example, extremophiles can be used to oxidize sulfur compounds in acidic pH to remediate wastewaters, generate electrical energy from marine sediment microbial fuel cells at low temperatures, desalinate wastewaters and act as biosensors of low amounts of organic carbon. In this review, we will discuss the recent advances that have been made in using microbial catalysts under extreme conditions and show possible new routes that extremophilic microorganisms open for microbial electrochemical systems. PMID:26474966

  15. Use of Stable Isotopes in Forensic Analysis of Microorganisms

    SciTech Connect

    Kreuzer-Martin, Helen W.; Hegg, Eric L.

    2012-01-18

    The use of isotopic signatures for forensic analysis of biological materials is well-established, and the same general principles that apply to interpretation of stable isotope content of C, N, O, and H apply to the analysis of microorganisms. Heterotrophic microorganisms derive their isotopic content from their growth substrates, which are largely plant and animal products, and the water in their culture medium. Thus the isotope signatures of microbes are tied to their growth environment. The C, N, O, and H isotope ratios of spores have been demonstrated to constitute highly discriminating signatures for sample matching. They can rule out specific samples of media and/or water as possible production media, and can predict isotope ratio ranges of the culture media and water used to produce a given sample. These applications have been developed and tested through analyses of approximately 250 samples of Bacillus subtilis spores and over 500 samples of culture media, providing a strong statistical basis for data interpretation. A Bayesian statistical framework for integrating stable isotope data with other types of signatures derived from microorganisms has been able to characterize the culture medium used to produce spores of various Bacillus species, leveraging isotopic differences in different medium types and demonstrating the power of data integration for forensic investigations.

  16. [Molecular karyotyping of eukaryotic microorganisms].

    PubMed

    Nasonova, E S

    2012-01-01

    In many fungi and protists small size and weak morphological differentiation of chromosomes embarrass the study of karyotypes using microscopical tools. Molecular karyotyping based on the fractionation of intact chromosomal DNAs by pulsed field gel electrophoresis (PFGE) provides an alternative approach to the analysis of chromosomal sets in such organisms. To assign the bands observed in PFGE gel to the individual chromosomes the following methods of chromosome identification are applied: densitometric analysis of the bands; Southern hybridization with chromosome- and telomere-specific probes, which often is combined with comparative karyotyping of a series of strains with pronounced size polymorphism of chromosomes; comparison of the patterns of restriction fragments of chromosomal DNAs fractioned by KARD 2-D PFGE; comparison with the strains with well-studied interchromosomal rearrangements. Besides estimation of the number and the size of chromosomes, molecular karyotyping allows assessment of haploid genome size and ploidy level, study of genome dynamics, identification of chromosomal rearrangements and associated chromosomal polymorphism. The analysis of karyotype and dynamics of the genomes is important for the study of intra- and interspecial variability, investigation of the chromosome evolution in closely related species and elaboration of the models of speciation. The comparison of molecular karyotypes among isolates of different origin is of great practical importance for clinical diagnostics and for agricultural microbiology. In this review we discuss: 1) the methods of karyotyping and their application to the analysis of chromosomal sets in eukaryotic microorganisms; 2) the specificity of the methods used for extraction and fractionation of intact chromosomal DNAs; 3) the reasons for difficulties in interpretation of molecular karyotypes and the ways of their overcoming; 4) fields of application of molecular karyotyping; 5) the definition of

  17. Internally controlled PCR system for detection of airborne microorganisms.

    PubMed

    Usachev, Evgeny V; Agranovski, Igor E

    2012-05-01

    Recently, we reported the outcomes of feasibility studies of a technological approach allowing rapid detection of a wide range of bioaerosols by combining a personal bioaerosol sampler with a real-time PCR technology. The protocol was found suitable for detection of targeted microorganisms within relatively short time periods. Considering the crucial importance of the PCR procedure quality control, the current paper reports the results of the development of an internally controlled PCR system for utilization by the above technology. The suggested strategy is based on utilization of only two fluorescent dyes, which are used respectively for target and internal amplification control (IAC) DNA amplification. A bacteriophage T4 and recombinant phage fd (M13) were used in this research as target and IAC, respectively. The constructed IAC was added directly to the collection liquid of the personal bioaerosol sampler enabling quality control to be present throughout the entire sampling-analysis procedures. For performance evaluation, serial ten-fold dilutions of T4 phage were aerosolized and sampled over a 10 minutes time period. The results showed that T4 phage could be reliably detected at the concentration of around 200 PFU per litre of air over the 10 minutes sampling period. The developed PCR assay demonstrated high specificity and no cross reaction. It is concluded that the recombinant phage fd is suitable for utilization as an internal control enabling to significantly minimize false negative results for bioaerosol detection procedures. PMID:22565862

  18. Patents in therapeutic recombinant protein production using mammalian cells.

    PubMed

    Picanco-Castro, Virginia; de Freitas, Marcela Cristina Correa; Bomfim, Aline de Sousa; de Sousa Russo, Elisa Maria

    2014-01-01

    The industrial production of recombinant proteins preferentially requires the generation of stable cell lines expressing proteins in a quick, relatively facile, and a reproducible manner. Different methods are used to insert exogenous DNA into the host cell, and choosing the appropriate producing cell is of paramount importance for the efficient production and quality of the recombinant protein. This review addresses the advances in recombinant protein production in mammalian cell lines, according to key patents from the last 30 years.

  19. Evolution of Resistance in Culex quinquefasciatus (Say) Selected With a Recombinant Bacillus thuringiensis Strain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, From Lysinibacillus sphaericus

    PubMed Central

    Wirth, Margaret C.; Walton, William E.; Federici, Brian A.

    2015-01-01

    Fourth instars of Culex quinquefasciatus (Say) (Diptera: Culicidae) were selected with a recombinant bacterial strain synthesizing the mosquitocidal proteins from Lysinibacillus sphaericus (Bin) and Cry11Ba and Cyt1Aa from Bacillus thuringiensis. Selection was initiated in Generation 1 with a concentration of 0.04 μg/ml, which rose to a maximum selection concentration of 8.0 μg/ml in Generation 14, followed by an unexpected, rapid increase in mortality in Generation 15. Subsequently, a selection concentration of 0.8 μg/ml was determined to be survivable. During this same period, resistance rose to nearly 1,000-fold (by Generation 12) and declined to 18.8-fold in Generation 19. Resistance remained low and fluctuated between 5.3 and 7.3 up to Generation 66. The cross-resistance patterns and interactions among the component proteins were analyzed to identify possible causes of this unusual pattern of evolution. Poor activity in the mid-range concentrations and lower-than-expected synergistic interactions were identified as potential sources of the early resistance. These findings should be considered in the development of genetically engineered strains intended to control nuisance and vector mosquitoes. PMID:26336254

  20. Metabolic Engineering of Microorganisms for the Production of Higher Alcohols

    PubMed Central

    Choi, Yong Jun; Lee, Joungmin; Jang, Yu-Sin

    2014-01-01

    ABSTRACT Due to the increasing concerns about limited fossil resources and environmental problems, there has been much interest in developing biofuels from renewable biomass. Ethanol is currently used as a major biofuel, as it can be easily produced by existing fermentation technology, but it is not the best biofuel due to its low energy density, high vapor pressure, hygroscopy, and incompatibility with current infrastructure. Higher alcohols, including 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, which possess fuel properties more similar to those of petroleum-based fuel, have attracted particular interest as alternatives to ethanol. Since microorganisms isolated from nature do not allow production of these alcohols at high enough efficiencies, metabolic engineering has been employed to enhance their production. Here, we review recent advances in metabolic engineering of microorganisms for the production of higher alcohols. PMID:25182323

  1. Biological Synthesis of Nanoparticles from Plants and Microorganisms.

    PubMed

    Singh, Priyanka; Kim, Yu-Jin; Zhang, Dabing; Yang, Deok-Chun

    2016-07-01

    Nanotechnology has become one of the most promising technologies applied in all areas of science. Metal nanoparticles produced by nanotechnology have received global attention due to their extensive applications in the biomedical and physiochemical fields. Recently, synthesizing metal nanoparticles using microorganisms and plants has been extensively studied and has been recognized as a green and efficient way for further exploiting microorganisms as convenient nanofactories. Here, we explore and detail the potential uses of various biological sources for nanoparticle synthesis and the application of those nanoparticles. Furthermore, we highlight recent milestones achieved for the biogenic synthesis of nanoparticles by controlling critical parameters, including the choice of biological source, incubation period, pH, and temperature.

  2. Microbial antigenic variation mediated by homologous DNA recombination

    PubMed Central

    Vink, Cornelis; Rudenko, Gloria; Seifert, H. Steven

    2012-01-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and which are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum and Mycoplasma spp.), fungi (like Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved. PMID:22212019

  3. Experimental measurement of the flow field around a freely swimming microorganism

    NASA Astrophysics Data System (ADS)

    Polin, Marco; Drescher, Knut; Goldstein, Raymond; Michel, Nicolas; Tuval, Idan

    2010-03-01

    Despite their small size, the fluid flows produced by billions of microscopic swimmers in nature can have dramatic macroscopic effects (e.g. biogenic mixing in the ocean). Understanding the flow structure of a single swimming microorganism is essential to explain and model these macroscopic phenomena. Here we report the first detailed measurement of the flow field around an isolated, freely swimming microorganism, the spherical alga Volvox, and discuss the implications of this measurement for other species.

  4. Generation of PHB from Spent Sulfite Liquor Using Halophilic Microorganisms

    PubMed Central

    Weissgram, Michaela; Gstöttner, Janina; Lorantfy, Bettina; Tenhaken, Raimund; Herwig, Christoph; Weber, Hedda K.

    2015-01-01

    Halophilic microorganisms thrive at elevated concentrations of sodium chloride up to saturation and are capable of growing on a wide variety of carbon sources like various organic acids, hexose and also pentose sugars. Hence, the biotechnological application of these microorganisms can cover many aspects, such as the treatment of hypersaline waste streams of different origin. Due to the fact that the high osmotic pressure of hypersaline environments reduces the risk of contamination, the capacity for cost-effective non-sterile cultivation can make extreme halophilic microorganisms potentially valuable organisms for biotechnological applications. In this contribution, the stepwise use of screening approaches, employing design of experiment (DoE) on model media and subsequently using industrial waste as substrate have been implemented to investigate the applicability of halophiles to generate PHB from the industrial waste stream spent sulfite liquor (SSL). The production of PHB on model media as well as dilutions of industrial substrate in a complex medium has been screened for by fluorescence microscopy using Nile Blue staining. Screening was used to investigate the ability of halophilic microorganisms to withstand the inhibiting substances of the waste stream without negatively affecting PHB production. It could be shown that neither single inhibiting substances nor a mixture thereof inhibited growth in the investigated range, hence, leaving the question on the inhibiting mechanisms open. However, it could be demonstrated that some haloarchaea and halophilic bacteria are able to produce PHB when cultivated on 3.3% w/w dry matter spent sulfite liquor, whereas H. halophila was even able to thrive on 6.6% w/w dry matter spent sulfite liquor and still produce PHB.

  5. Generation of PHB from Spent Sulfite Liquor Using Halophilic Microorganisms

    PubMed Central

    Weissgram, Michaela; Gstöttner, Janina; Lorantfy, Bettina; Tenhaken, Raimund; Herwig, Christoph; Weber, Hedda K.

    2015-01-01

    Halophilic microorganisms thrive at elevated concentrations of sodium chloride up to saturation and are capable of growing on a wide variety of carbon sources like various organic acids, hexose and also pentose sugars. Hence, the biotechnological application of these microorganisms can cover many aspects, such as the treatment of hypersaline waste streams of different origin. Due to the fact that the high osmotic pressure of hypersaline environments reduces the risk of contamination, the capacity for cost-effective non-sterile cultivation can make extreme halophilic microorganisms potentially valuable organisms for biotechnological applications. In this contribution, the stepwise use of screening approaches, employing design of experiment (DoE) on model media and subsequently using industrial waste as substrate have been implemented to investigate the applicability of halophiles to generate PHB from the industrial waste stream spent sulfite liquor (SSL). The production of PHB on model media as well as dilutions of industrial substrate in a complex medium has been screened for by fluorescence microscopy using Nile Blue staining. Screening was used to investigate the ability of halophilic microorganisms to withstand the inhibiting substances of the waste stream without negatively affecting PHB production. It could be shown that neither single inhibiting substances nor a mixture thereof inhibited growth in the investigated range, hence, leaving the question on the inhibiting mechanisms open. However, it could be demonstrated that some haloarchaea and halophilic bacteria are able to produce PHB when cultivated on 3.3% w/w dry matter spent sulfite liquor, whereas H. halophila was even able to thrive on 6.6% w/w dry matter spent sulfite liquor and still produce PHB. PMID:27682089

  6. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  7. Recombination Pattern Reanalysis of Some HIV-1 Circulating Recombination Forms Suggest the Necessity and Difficulty of Revision

    PubMed Central

    Jia, Lei; Li, Lin; Li, Hanping; Liu, Siyang; Wang, Xiaolin; Bao, Zuoyi; Li, Tianyi; Zhuang, Daomin; Liu, Yongjian; Li, Jingyun

    2014-01-01

    Background Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs), like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results. Methods Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08), BG recombinants (CRF23 and CRF24), and BF recombinants (CRF38 and CRF44). They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3. Results The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i) length of inserted fragments; and (ii) number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution. Conclusion Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also

  8. Metabolic engineering of Escherichia coli to produce zeaxanthin.

    PubMed

    Li, Xi-Ran; Tian, Gui-Qiao; Shen, Hong-Jie; Liu, Jian-Zhong

    2015-04-01

    Zeaxanthin is a high-value carotenoid that is used in nutraceuticals, cosmetics, food, and animal feed industries. Zeaxanthin is chemically synthesized or purified from microorganisms as a natural product; however, increasing demand requires development of alternative sources such as heterologous biosynthesis by recombinant bacteria. For this purpose, we molecularly engineered Escherichia coli to optimize the synthesis of zeaxanthin from lycopene using fusion protein-mediated substrate channeling as well as by the introduction of tunable intergenic regions. The tunable intergenic regions approach was more efficient compared with protein fusion for coordinating expression of lycopene β-cyclase gene crtY and β-carotene 3-hydroxylase gene crtZ. The influence of the substrate channeling effect suggests that the reaction catalyzed by CrtZ is the rate-limiting step in zeaxanthin biosynthesis. Then Pantoea ananatis, Pantoea agglomerans and Haematococcus pluvialis crtZ were compared. Because P. ananatis crtZ is superior to that of P. agglomerans or H. pluvialis for zeaxanthin production, we used it to generate a recombinant strain of E. coli BETA-1 containing pZSPBA-2(P37-crtZPAN) that produced higher amounts of zeaxanthin (11.95 ± 0.21 mg/g dry cell weight) than other engineered E. coli strains described in the literature.

  9. Metabolic engineering of Escherichia coli to produce zeaxanthin.

    PubMed

    Li, Xi-Ran; Tian, Gui-Qiao; Shen, Hong-Jie; Liu, Jian-Zhong

    2015-04-01

    Zeaxanthin is a high-value carotenoid that is used in nutraceuticals, cosmetics, food, and animal feed industries. Zeaxanthin is chemically synthesized or purified from microorganisms as a natural product; however, increasing demand requires development of alternative sources such as heterologous biosynthesis by recombinant bacteria. For this purpose, we molecularly engineered Escherichia coli to optimize the synthesis of zeaxanthin from lycopene using fusion protein-mediated substrate channeling as well as by the introduction of tunable intergenic regions. The tunable intergenic regions approach was more efficient compared with protein fusion for coordinating expression of lycopene β-cyclase gene crtY and β-carotene 3-hydroxylase gene crtZ. The influence of the substrate channeling effect suggests that the reaction catalyzed by CrtZ is the rate-limiting step in zeaxanthin biosynthesis. Then Pantoea ananatis, Pantoea agglomerans and Haematococcus pluvialis crtZ were compared. Because P. ananatis crtZ is superior to that of P. agglomerans or H. pluvialis for zeaxanthin production, we used it to generate a recombinant strain of E. coli BETA-1 containing pZSPBA-2(P37-crtZPAN) that produced higher amounts of zeaxanthin (11.95 ± 0.21 mg/g dry cell weight) than other engineered E. coli strains described in the literature. PMID:25533633

  10. PARTICLE-ASSOCIATED MICROORGANISMS IN STORMWATER RUNOFF

    EPA Science Inventory

    This research investigated the effects of blending and chemical addition before analysis of the concentration of microorganisms in stormwater runoff to determine whether clumped or particle-associated organisms play a significant role. All organisms, except for Escherichia coli, ...

  11. Detection of microorganisms using terahertz metamaterials.

    PubMed

    Park, S J; Hong, J T; Choi, S J; Kim, H S; Park, W K; Han, S T; Park, J Y; Lee, S; Kim, D S; Ahn, Y H

    2014-05-16

    Microorganisms such as fungi and bacteria cause many human diseases and therefore rapid and accurate identification of these substances is essential for effective treatment and prevention of further infections. In particular, contemporary microbial detection technique is limited by the low detection speed which usually extends over a couple of days. Here we demonstrate that metamaterials operating in the terahertz frequency range shows promising potential for use in fabricating the highly sensitive and selective microbial sensors that are capable of high-speed on-site detection of microorganisms in both ambient and aqueous environments. We were able to detect extremely small amounts of the microorganisms, because their sizes are on the same scale as the micro-gaps of the terahertz metamaterials. The resonant frequency shift of the metamaterials was investigated in terms of the number density and the dielectric constants of the microorganisms, which was successfully interpreted by the change in the effective dielectric constant of a gap area.

  12. Detection of microorganisms using terahertz metamaterials

    NASA Astrophysics Data System (ADS)

    Park, S. J.; Hong, J. T.; Choi, S. J.; Kim, H. S.; Park, W. K.; Han, S. T.; Park, J. Y.; Lee, S.; Kim, D. S.; Ahn, Y. H.

    2014-05-01

    Microorganisms such as fungi and bacteria cause many human diseases and therefore rapid and accurate identification of these substances is essential for effective treatment and prevention of further infections. In particular, contemporary microbial detection technique is limited by the low detection speed which usually extends over a couple of days. Here we demonstrate that metamaterials operating in the terahertz frequency range shows promising potential for use in fabricating the highly sensitive and selective microbial sensors that are capable of high-speed on-site detection of microorganisms in both ambient and aqueous environments. We were able to detect extremely small amounts of the microorganisms, because their sizes are on the same scale as the micro-gaps of the terahertz metamaterials. The resonant frequency shift of the metamaterials was investigated in terms of the number density and the dielectric constants of the microorganisms, which was successfully interpreted by the change in the effective dielectric constant of a gap area.

  13. Detection of microorganisms using terahertz metamaterials

    PubMed Central

    Park, S. J.; Hong, J. T.; Choi, S. J.; Kim, H. S.; Park, W. K.; Han, S. T.; Park, J. Y.; Lee, S.; Kim, D. S.; Ahn, Y. H.

    2014-01-01

    Microorganisms such as fungi and bacteria cause many human diseases and therefore rapid and accurate identification of these substances is essential for effective treatment and prevention of further infections. In particular, contemporary microbial detection technique is limited by the low detection speed which usually extends over a couple of days. Here we demonstrate that metamaterials operating in the terahertz frequency range shows promising potential for use in fabricating the highly sensitive and selective microbial sensors that are capable of high-speed on-site detection of microorganisms in both ambient and aqueous environments. We were able to detect extremely small amounts of the microorganisms, because their sizes are on the same scale as the micro-gaps of the terahertz metamaterials. The resonant frequency shift of the metamaterials was investigated in terms of the number density and the dielectric constants of the microorganisms, which was successfully interpreted by the change in the effective dielectric constant of a gap area. PMID:24832607

  14. Automated systems for identification of microorganisms.

    PubMed Central

    Stager, C E; Davis, J R

    1992-01-01

    Automated instruments for the identification of microorganisms were introduced into clinical microbiology laboratories in the 1970s. During the past two decades, the capabilities and performance characteristics of automated identification systems have steadily progressed and improved. This article explores the development of the various automated identification systems available in the United States and reviews their performance for identification of microorganisms. Observations regarding deficiencies and suggested improvements for these systems are provided. PMID:1498768

  15. Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

    PubMed

    Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

    2014-01-01

    For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

  16. [Secretion of proteolytic enzymes by three phytopathogenic microorganisms].

    PubMed

    2013-01-01

    Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts. PMID:25508654

  17. [Secretion of proteolytic enzymes by three phytopathogenic microorganisms].

    PubMed

    Kudriavtseva, N N; Sof'in, A V; Revina, T A; Gvozdeva, E L; Ievleva, E V; Valueva, T A

    2013-01-01

    Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts. PMID:25474875

  18. Progress and challenges in the engineering of non-cellulolytic microorganisms for consolidated bioprocessing.

    PubMed

    den Haan, Riaan; van Rensburg, Eugéne; Rose, Shaunita H; Görgens, Johann F; van Zyl, Willem H

    2015-06-01

    Lignocellulosic biomass is an abundant, renewable feedstock for the production of fuels and chemicals, if an efficient and affordable conversion technology can be established to overcome its recalcitrance. Consolidated bioprocessing (CBP) featuring enzyme production, substrate hydrolysis and fermentation in a single step is a biologically mediated conversion approach with outstanding potential if a fit-for-purpose microorganism(s) can be developed. Progress in developing CBP-enabling microorganisms is ongoing by engineering (i) naturally cellulolytic microorganisms for improved product-related properties or (ii) non-cellulolytic organisms exhibiting high product yields to heterologously produce different combinations of cellulase enzymes. We discuss progress on developing yeast and bacteria for the latter strategy and consider further challenges that require attention to bring this technology to market.

  19. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  20. Production of bulk chemicals via novel metabolic pathways in microorganisms.

    PubMed

    Shin, Jae Ho; Kim, Hyun Uk; Kim, Dong In; Lee, Sang Yup

    2013-11-01

    Metabolic engineering has been playing important roles in developing high performance microorganisms capable of producing various chemicals and materials from renewable biomass in a sustainable manner. Synthetic and systems biology are also contributing significantly to the creation of novel pathways and the whole cell-wide optimization of metabolic performance, respectively. In order to expand the spectrum of chemicals that can be produced biotechnologically, it is necessary to broaden the metabolic capacities of microorganisms. Expanding the metabolic pathways for biosynthesizing the target chemicals requires not only the enumeration of a series of known enzymes, but also the identification of biochemical gaps whose corresponding enzymes might not actually exist in nature; this issue is the focus of this paper. First, pathway prediction tools, effectively combining reactions that lead to the production of a target chemical, are analyzed in terms of logics representing chemical information, and designing and ranking the proposed metabolic pathways. Then, several approaches for potentially filling in the gaps of the novel metabolic pathway are suggested along with relevant examples, including the use of promiscuous enzymes that flexibly utilize different substrates, design of novel enzymes for non-natural reactions, and exploration of hypothetical proteins. Finally, strain optimization by systems metabolic engineering in the context of novel metabolic pathways constructed is briefly described. It is hoped that this review paper will provide logical ways of efficiently utilizing 'big' biological data to design and develop novel metabolic pathways for the production of various bulk chemicals that are currently produced from fossil resources. PMID:23280013

  1. Production of bulk chemicals via novel metabolic pathways in microorganisms.

    PubMed

    Shin, Jae Ho; Kim, Hyun Uk; Kim, Dong In; Lee, Sang Yup

    2013-11-01

    Metabolic engineering has been playing important roles in developing high performance microorganisms capable of producing various chemicals and materials from renewable biomass in a sustainable manner. Synthetic and systems biology are also contributing significantly to the creation of novel pathways and the whole cell-wide optimization of metabolic performance, respectively. In order to expand the spectrum of chemicals that can be produced biotechnologically, it is necessary to broaden the metabolic capacities of microorganisms. Expanding the metabolic pathways for biosynthesizing the target chemicals requires not only the enumeration of a series of known enzymes, but also the identification of biochemical gaps whose corresponding enzymes might not actually exist in nature; this issue is the focus of this paper. First, pathway prediction tools, effectively combining reactions that lead to the production of a target chemical, are analyzed in terms of logics representing chemical information, and designing and ranking the proposed metabolic pathways. Then, several approaches for potentially filling in the gaps of the novel metabolic pathway are suggested along with relevant examples, including the use of promiscuous enzymes that flexibly utilize different substrates, design of novel enzymes for non-natural reactions, and exploration of hypothetical proteins. Finally, strain optimization by systems metabolic engineering in the context of novel metabolic pathways constructed is briefly described. It is hoped that this review paper will provide logical ways of efficiently utilizing 'big' biological data to design and develop novel metabolic pathways for the production of various bulk chemicals that are currently produced from fossil resources.

  2. Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris MutS strain

    PubMed Central

    Markošová, Kristína; Weignerová, Lenka; Rosenberg, Michal; Křen, Vladimír; Rebroš, Martin

    2015-01-01

    Pichia pastoris is currently one of the most preferred microorganisms for recombinant enzyme production due to its efficient expression system. The advantages include the production of high amounts of recombinant proteins containing the appropriate posttranslational modifications and easy cultivation conditions. α-L-Rhamnosidase is a biotechnologically important enzyme in food and pharmaceutical industry, used for example in debittering of citrus fruit juices, rhamnose pruning from naringin, or enhancement of wine aromas, creating a demand for the production of an active and stable enzyme. The production of recombinant α-L-rhamnosidase cloned in the MutS strain of P. pastoris KM71H was optimized. The encoding gene is located under the control of the AOX promoter, which is induced by methanol whose concentration is instrumental for these strain types. Fermentation was upscaled in bioreactors employing various media and several methanol-feeding strategies. It was found that fed batch with BSM media was more effective compared to BMMH (Buffered Methanol-complex Medium) media due to lower cost and improved biomass formation. In BSM (Basal Salt Medium) medium, the dry cell weight reached approximately 60 g/L, while in BMMH it was only 8.3 g/L, without additional glycerol, which positively influenced the amount of enzyme produced. New methanol feeding strategy, based on the level of dissolved oxygen was developed in this study. This protocol that is entirely independent on methanol monitoring was up scaled to a 19.5-L fermenter with 10-L working volume with the productivity of 13.34 mgprot/L/h and specific activity of α-L-rhamnosidase of 82 U/mg. The simplified fermentation protocol was developed for easy and effective fermentation of P. pastoris MutS based on dissolved oxygen monitoring in the induction phase of an enzyme production. PMID:26539173

  3. Production of a recombinant type 1 antifreeze protein analogue by L. lactis and its applications on frozen meat and frozen dough.

    PubMed

    Yeh, Chuan-Mei; Kao, Bi-Yu; Peng, Hsuan-Jung

    2009-07-22

    In this study, a novel recombinant type I antifreeze protein analogue (rAFP) was produced and secreted by Lactococcus lactis, a food-grade microorganism of major commercial importance. Antifreeze proteins are potent cryogenic protection agents for the cryopreservation of food and pharmaceutical materials. A food-grade expression and fermentation system (BSE- and antibiotic-free) for the production and secretion of high levels of rAFP was developed. Lyophilized, crude rAFP produced by L. lactis was tested in a frozen meat and frozen dough processing model. The frozen meat treated with the antifreeze protein showed less drip loss, less protein loss, and a high score on juiciness by sensory evaluation. Frozen dough treated with the rAFP showed better fermentation capacity than untreated frozen dough. Breads baked from frozen dough treated with rAFP acquired the same consumer acceptance as fresh bread. PMID:19545118

  4. Utilization of Site-Specific Recombination in Biopharmaceutical Production.

    PubMed

    Ahmadi, Maryam; Damavandi, Narges; Akbari Eidgahi, Mohammad Reza; Davami, Fatemeh

    2016-01-01

    Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons.

  5. Utilization of Site-Specific Recombination in Biopharmaceutical Production

    PubMed Central

    Ahmadi, Maryam; Damavandi, Narges; Akbari, Mohammad Reza; Davami, Fatemeh

    2016-01-01

    Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons. PMID:26602035

  6. Microorganisms in the atmosphere over Antarctica.

    PubMed

    Pearce, David A; Bridge, Paul D; Hughes, Kevin A; Sattler, Birgit; Psenner, Roland; Russell, Nick J

    2009-08-01

    Antarctic microbial biodiversity is the result of a balance between evolution, extinction and colonization, and so it is not possible to gain a full understanding of the microbial biodiversity of a location, its biogeography, stability or evolutionary relationships without some understanding of the input of new biodiversity from the aerial environment. In addition, it is important to know whether the microorganisms already present are transient or resident - this is particularly true for the Antarctic environment, as selective pressures for survival in the air are similar to those that make microorganisms suitable for Antarctic colonization. The source of potential airborne colonists is widespread, as they may originate from plant surfaces, animals, water surfaces or soils and even from bacteria replicating within the clouds. On a global scale, transport of air masses from the well-mixed boundary layer to high-altitude sites has frequently been observed, particularly in the warm season, and these air masses contain microorganisms. Indeed, it has become evident that much of the microbial life within remote environments is transported by air currents. In this review, we examine the behaviour of microorganisms in the Antarctic aerial environment and the extent to which these microorganisms might influence Antarctic microbial biodiversity.

  7. Recombinant protein production and insect cell culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

    1993-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

  8. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  9. Combating Antimicrobial Resistance in Foodborne Microorganisms.

    PubMed

    Lai, Edward P C; Iqbal, Zafar; Avis, Tyler J

    2016-02-01

    This review addresses an important public health hazard affecting food safety. Antimicrobial agents are used in foods to reduce or eliminate microorganisms that cause disease. Many traditional organic compounds, novel synthetic organic agents, natural products, peptides, and proteins have been extensively studied for their effectiveness as antimicrobial agents against foodborne Campylobacter spp., Escherichia coli, Listeria spp. and Salmonella. However, antimicrobial resistance can develop in microorganisms, enhancing their ability to withstand the inhibiting or killing action of antimicrobial agents. Knowledge gaps still exist with regard to the actual chemical and microbiological mechanisms that must be identified to facilitate the search for new antimicrobial agents. Technical implementation of antimicrobial active packing films and coatings against target microorganisms must also be improved for extended product shelf life. Recent advances in antimicrobial susceptibility testing can provide researchers with new momentum to pursue their quest for a resistance panacea.

  10. Selective enumeration of probiotic microorganisms in cheese.

    PubMed

    Karimi, Reza; Mortazavian, Amir M; Amiri-Rigi, Atefeh

    2012-02-01

    Cheese is a dairy product which has a good potential for delivery of probiotic microorganisms into the human intestine. To be considered to offer probiotic health benefits, probiotics must remain viable in food products above a threshold level (e.g., 10(6) cfu g(-1)) until the time of consumption. In order to ensure that a minimal number of probiotic bacteria is present in the cheese, reliable methods for enumeration are required. The choice of culture medium for selective enumeration of probiotic strains in combination with starters depends on the product matrix, the target group and the taxonomic diversity of the bacterial background flora in the product. Enumeration protocol should be designed as a function of the target microorganism(s) to be quantified in the cheese. An overview of some series of culture media for selective enumeration of commercial probiotic cultures is presented in this review.

  11. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  12. The Genetic Programming of Industrial Microorganisms.

    ERIC Educational Resources Information Center

    Hopwood, David A.

    1981-01-01

    Traces the development of the field of industrial microbial genetics, describing a range of techniques for genetic programing. Includes a discussion of site-directed mutagenesis, protoplast fusion, and recombinant DNA manipulations. (CS)

  13. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  14. Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide.

    PubMed

    Keller, Matthew W; Schut, Gerrit J; Lipscomb, Gina L; Menon, Angeli L; Iwuchukwu, Ifeyinwa J; Leuko, Therese T; Thorgersen, Michael P; Nixon, William J; Hawkins, Aaron S; Kelly, Robert M; Adams, Michael W W

    2013-04-01

    Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

  15. Metabolic activity of microorganisms in evaporites

    NASA Technical Reports Server (NTRS)

    Rothschild, L. J.; Giver, L. J.; White, M. R.; Mancinelli, R. L.

    1994-01-01

    Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative. Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months. In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen. Denitrification was not observed. Our results quantified the slow microbial activity that can occur in salt crystals. Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community.

  16. Physiologically anaerobic microorganisms of the deep subsurface

    SciTech Connect

    Stevens, S.E. Jr.; Chung, K.T.

    1991-06-01

    This study seeks to determine numbers, diversity, and morphology of anaerobic microorganisms in 15 samples of subsurface material from the Idaho National Engineering Laboratory, in 18 samples from the Hanford Reservation and in 1 rock sample from the Nevada Test Site; set up long term experiments on the chemical activities of anaerobic microorganisms based on these same samples; work to improve methods for the micro-scale determination of in situ anaerobic microbial activity;and to begin to isolate anaerobes from these samples into axenic culture with identification of the axenic isolates.

  17. Screening and Separation of Microorganisms Degrading PCBs.

    PubMed Central

    Bokvajova, A; Burkhard, J; Demnerova, K; Pazlarova, J

    1994-01-01

    We performed an assay to assess the polychlorinated biphenyl (PCB) degradative capability and congener specificity of aerobic microorganisms. Microbial strains were isolated and separated from different types of soils in the Czech Republic, and their PCB-degrading abilities were compared. An industrial mixture of PCB congeners ranging from dichloro- to hexachlorobiphenyl and representing various chlorination patterns was used throughout. The PCB degradative ability of microorganisms was determined by gas chromatography after 7 days of incubation. The degree of degradation was found to depend on the number of chlorine substituents. PMID:9679114

  18. Functional Properties of Microorganisms in Fermented Foods

    PubMed Central

    Tamang, Jyoti P.; Shin, Dong-Hwa; Jung, Su-Jin; Chae, Soo-Wan

    2016-01-01

    Fermented foods have unique functional properties imparting some health benefits to consumers due to presence of functional microorganisms, which possess probiotics properties, antimicrobial, antioxidant, peptide production, etc. Health benefits of some global fermented foods are synthesis of nutrients, prevention of cardiovascular disease, prevention of cancer, gastrointestinal disorders, allergic reactions, diabetes, among others. The present paper is aimed to review the information on some functional properties of the microorganisms associated with fermented foods and beverages, and their health-promoting benefits to consumers. PMID:27199913

  19. Effect of microwaves on microorganisms in foods

    SciTech Connect

    Fung, D.Y.C.; Cunningham, F.E.

    1980-08-01

    The microbial safety of foods cooked in microwave ovens was investigated. The mechanisms of microwave destruction of microorganisms were examined. Effects of time and temperature on microorganisms in different food systems were described. Studies showed that: microwave heating of food is more ''''food dependent'' than conventional heating; recommended microwave treatment time for some foods may not destroy high levels of bacteria; use of microwaves in combination with conventional heating methods results in more uniform heating of foods and destruction of bacteria; and microwaves exert different killing effects on individual bacterial species. (78 references, 2 tables)

  20. [Metagenomics in studying gastrointestinal tract microorganism].

    PubMed

    Xu, Bo; Yang, Yunjuan; Li, Junjun; Tang, Xianghua; Mu, Yuelin; Huang, Zunxi

    2013-12-01

    Animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. The gut microbial community of humans and animals has received significant attention from researchers because of its association with health and disease. The application of metagenomics technology enables researchers to study not only the microbial composition but also the function of microbes in the gastrointestinal tract. In this paper, combined with our own findings, we summarized advances in studying gastrointestinal tract microorganism with metagenomics and the bioinformatics technology.

  1. Metabolic activity of microorganisms in evaporites.

    PubMed

    Rothschild, L J; Giver, L J; White, M R; Mancinelli, R L

    1994-06-01

    Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative. Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months. In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen. Denitrification was not observed. Our results quantified the slow microbial activity that can occur in salt crystals. Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community. PMID:11539827

  2. Metabolic activity of microorganisms in evaporites.

    PubMed

    Rothschild, L J; Giver, L J; White, M R; Mancinelli, R L

    1994-06-01

    Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative. Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months. In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen. Denitrification was not observed. Our results quantified the slow microbial activity that can occur in salt crystals. Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community.

  3. Recombinant allergen-based provocation testing☆

    PubMed Central

    Niederberger, Verena; Eckl-Dorna, Julia; Pauli, Gabrielle

    2014-01-01

    Over the last 25 years, recombinant allergens from all important allergen sources have been cloned and are now available as recombinant proteins. These molecules can be produced in practically unlimited amounts without biological or batch-to-batch variability. It has been shown in provocation tests that recombinant allergens have similar clinical effects as their natural counterparts. With the help of these tools it is possible to reveal the precise reactivity profiles of patients and to uncover and differentiate cross-reactivity from genuine sensitization to an allergen source. Although it has been shown some time ago that it would be possible to replace crude allergen extracts with recombinant allergens for skin prick testing, and even though the use of allergen components can improve routine diagnosis, these tools are still not available for clinical routine applications. The use of provocation tests is a crucial step in the development of new, hypoallergenic vaccines for therapy of allergic disease. Here we describe important provocation methods (skin prick test, intradermal test, atopy patch test, nasal provocation, colonoscopic provocation test) and give an overview of the clinical provocation studies which have been performed with recombinant allergens so far. PMID:23920475

  4. [Mineral salt-dependent inhibition of light emission from the luminescent microorganism Escherichia coli Z9051].

    PubMed

    Boiandin, A N; Popova, L Iu

    2001-01-01

    The influence of some mineral salts on the recombinant strain Escherichia coli Z9051 was investigated. It was shown that the composition (NaCl, Na2SO4, MgCl2 and MgSO4) and concentration (5 and 10%) of the salts substantially affect the expression of genes for the luminescence system of light-emitting bacteria cloned in the plasmid under the control of the lac-promoter. In some cases, the luminescence level of the microorganism in the presence of salts was similar to the luminescence level under catabolite repression by glucose, the more strong influence of the salts exceeding the effect of catabolite repression. The possibility of adaptation of the genetically modified microorganism to the salinity factor is discussed. PMID:11357338

  5. Advances in engineered microorganisms for improving metabolic conversion via microgravity effects.

    PubMed

    Huangfu, Jie; Zhang, Genlin; Li, Jun; Li, Chun

    2015-01-01

    As an extreme and unique environment, microgravity has significant effects on microbial cellular processes, such as cell growth, gene expression, natural pathways and biotechnological products. Application of microgravity effects to identify the regulatory elements in reengineering microbial hosts will draw much more attention in further research. In this commentary, we discuss the microgravity effects in engineered microorganisms for improving metabolic conversion, including cell growth kinetics, antimicrobial susceptibility, resistance to stresses, secondary metabolites production, recombinant protein production and enzyme activity, as well as gene expression changes. Application of microgravity effects in engineered microorganisms could provide valuable platform for innovative approaches in bioprocessing technology to largely improve the metabolic conversion efficacy of biopharmaceutical products. PMID:26038088

  6. Advances in engineered microorganisms for improving metabolic conversion via microgravity effects

    PubMed Central

    Huangfu, Jie; Zhang, Genlin; Li, Jun; Li, Chun

    2015-01-01

    As an extreme and unique environment, microgravity has significant effects on microbial cellular processes, such as cell growth, gene expression, natural pathways and biotechnological products. Application of microgravity effects to identify the regulatory elements in reengineering microbial hosts will draw much more attention in further research. In this commentary, we discuss the microgravity effects in engineered microorganisms for improving metabolic conversion, including cell growth kinetics, antimicrobial susceptibility, resistance to stresses, secondary metabolites production, recombinant protein production and enzyme activity, as well as gene expression changes. Application of microgravity effects in engineered microorganisms could provide valuable platform for innovative approaches in bioprocessing technology to largely improve the metabolic conversion efficacy of biopharmaceutical products PMID:26038088

  7. Diversity of anaerobic halophilic microorganisms

    NASA Astrophysics Data System (ADS)

    Oren, Aharon; Oremland, Roland S.

    2000-12-01

    Life in the presence of high salt concentrations is compatible with life in the absence of oxygen. Halophilic and halotolerant anaerobic prokaryotes are found both in the archaeal and in the bacterial domain, and they display a great metabolic diversity. Many of the representatives of the Halobacteriales (Archaea), which are generally considered aerobes, have the potential of anaerobic growth. Some can use alternative electron acceptors such as nitrate, fumarate, dimethylsulfoxide or trimethylamine-N-oxide Halobacterium salinarum can also grow fermentatively on L-arginine, and bacteriorhodopsin-containing cells may even grow anaerobically, energized by light. Obligatory anaerobic halophilic methanogenic Archaea also exist. The bacterial domain contains many anaerobic halophiles, including sulfate reducers. There is also a group of specialized obligatory anaerobic Bacteria, phylogenetically clustering in the low G + C branch of the Firmicutes. Most representatives of this group (order Haloanaerobiales, families Haloanaerobiaceae and Halobacteroidaceae) are fermentative, using a variety of carbohydrates and amino acids. One species combines the potential for anaerobic growth at high salt concentrations with a preference for high temperatures. Others are homoacetogens; Acetohalobium arabaticum can grow anaerobically as a chemolithotroph, producing acetate from hydrogen and CO2. The Haloanaerobiales accumulate high concentrations of K+ and Cl- in their cytoplasm, thereby showing a strategy of salt adaptation similar to that used by the Halobacteriales. Recently a new representative of the Haloanaerobiales was isolated from bottom sediments of the Dead Sea (strain DSSe1), which grows anaerobically by oxidation of glycerol to acetate and CO2 while reducing selenate to selenite and elementary selenium. Other electron acceptors supporting anaerobic growth of this strain are nitrate and trimethylamine-N-oxide. The versatility of life at high salt concentrations with respect

  8. Recombinant bacteria for mosquito control.

    PubMed

    Federici, B A; Park, H-W; Bideshi, D K; Wirth, M C; Johnson, J J

    2003-11-01

    Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria. PMID:14506223

  9. Dissociative recombination in planetary ionospheres

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1993-01-01

    Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.

  10. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  11. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  12. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  13. Yeasts in table olive processing: desirable or spoilage microorganisms?

    PubMed

    Arroyo-López, F N; Romero-Gil, V; Bautista-Gallego, J; Rodríguez-Gómez, F; Jiménez-Díaz, R; García-García, P; Querol, A; Garrido-Fernández, A

    2012-11-01

    Yeasts are unicellular eukaryotic microorganisms isolated from many foods, and are commonly found in table olive processing where they can play a double role. On one hand, these microorganisms can produce spoilage of fruits due to the production of bad odours and flavours, the accumulation of CO(2) leading to swollen containers, the clouding of brines, the softening of fruits and the degradation of lactic acid, which is especially harmful during table olive storage and packaging. But on the other hand, fortunately, yeasts also possess desirable biochemical activities (lipase, esterase, β-glucosidase, catalase, production of killer factors, etc.) with important technological applications in this fermented vegetable. Recently, the probiotic potential of olive yeasts has begun to be evaluated because many species are able to resist the passage through the gastrointestinal tract and show beneficial effects on the host. In this way, yeasts may improve consumers' health by decreasing cholesterol levels, inhibiting pathogens, degrading non assimilated compounds, producing antioxidants and vitamins, adhering to intestinal cells or by maintaining epithelial barrier integrity. Many yeast species, usually also found in table olive processing, such as Wicherhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens and Kluyveromyces lactis, have been reported to exhibit some of these properties. Thus, the selection of the most appropriate strains to be used as starters, alone or in combination with lactic acid bacteria, is a promising research line to develop in a near future which might improve the added value of the commercialized product. PMID:23141644

  14. Oil Production by a Consortium of Oleaginous Microorganisms grown on primary effluent wastewater

    SciTech Connect

    Hall, Jacqueline; Hetrick, Mary; French, Todd; Hernandez, Rafael; Donaldson, Janet; Mondala, Andro; Holmes, William

    2011-01-01

    Municipal wastewater could be a potential growth medium that has not been considered for cultivating oleaginous microorganisms. This study is designed to determine if a consortium of oleaginous microorganism can successfully compete for carbon and other nutrients with the indigenous microorganisms contained in primary effluent wastewater. RESULTS: The oleaginous consortium inoculated with indigenous microorganisms reached stationary phase within 24 h, reaching a maximum cell concentration of 0.58 g L -1. Water quality post-oleaginous consortium growth reached a maximum chemical oxygen demand (COD) reduction of approximately 81%, supporting the consumption of the glucose within 8 h. The oleaginous consortium increased the amount of oil produced per gram by 13% compared with indigenous microorganisms in raw wastewater. Quantitative polymerase chain reaction (qPCR) results show a substantial population increase in bacteria within the first 24 h when the consortium is inoculated into raw wastewater. This result, along with the fatty acid methyl esters (FAMEs) results, suggests that conditions tested were not sufficient for the oleaginous consortium to compete with the indigenous microorganisms.

  15. QA-RecombineIt: a server for quality assessment and recombination of protein models

    PubMed Central

    Pawlowski, Marcin; Bogdanowicz, Albert; Bujnicki, Janusz M.

    2013-01-01

    QA-RecombineIt provides a web interface to assess the quality of protein 3D structure models and to improve the accuracy of models by merging fragments of multiple input models. QA-RecombineIt has been developed for protein modelers who are working on difficult problems, have a set of different homology models and/or de novo models (from methods such as I-TASSER or ROSETTA) and would like to obtain one consensus model that incorporates the best parts into one structure that is internally coherent. An advanced mode is also available, in which one can modify the operation of the fragment recombination algorithm by manually identifying individual fragments or entire models to recombine. Our method produces up to 100 models that are expected to be on the average more accurate than the starting models. Therefore, our server may be useful for crystallographic protein structure determination, where protein models are used for Molecular Replacement to solve the phase problem. To address the latter possibility, a special feature was added to the QA-RecombineIt server. The QA-RecombineIt server can be freely accessed at http://iimcb.genesilico.pl/qarecombineit/. PMID:23700309

  16. Photosynthetic microorganisms in cold environments

    NASA Astrophysics Data System (ADS)

    Kviderova, Jana; Hajek, Josef; Elster, Josef; Bartak, Milos; Vaczi, Peter; Nedbalova, Linda

    The polar regions are considered as a model of extraterrestrial ecosystems. Depending on the average temperature, temperature variation and water availability, these conditions could be used as a model of Mars or Europa (e.g. (Elster and Benson, 2004). Two cases are presented: 1) Stable temperature and water availability The environment of cryosestic communities, i.e. organisms living in snow, is characterized by very stable temperature; the diurnal variations do not exceed 1 -2 ° C (Kváderová, 2010) and a are not usually exposed to freeze/thaw. Water is not usually limiting since the water content could reach up to 54 % (Nedbalová et al., 2008). The windblown sediments are important a source of nutrient and could provide protection against the excess of radiation. The nutrient concentrations in the snow are low are depleted rapidly when massive algal blooms forms. Such environment could be found near Mars polar caps or in Europa ice cover. The snow algae are the most important primary producers in snow. Their adaptation strategy is dependent on the developmental stages; the motile stages avoid the harsh conditions (e.g. high light) and sessile stages acclimatize to actual conditions. The main genera Chlamydomonas and Chloromonas (both Chlorophyta) are psychrophilic. Their growth optimum temperature is lower than 15 ° C and their growth is inhibited at temperatures above 20 ° C. 2) Unstable temperature and water availability The deglaciated surfaces, inhabited by lichen communities, are typical by variation in temper-ature and moisture. The temperature could range several tens ° C within a short time and the water availability is usually very limited. Due to temperature variation, the lichens are subjected to many freeze/thaw cycles. Such environments could be found in Martian deserts. The lichens are symbotic organisms composed of a mycobiont (heterotrophic fungi) and photo-bionts (algae and/or cyanobacteria). Majority of lichens are dehydrated in the field

  17. Metagenomics: Application of Genomics to Uncultured Microorganisms

    PubMed Central

    Handelsman, Jo

    2004-01-01

    Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies. PMID:15590779

  18. Engineered microorganisms having resistance to ionic liquids

    DOEpatents

    Ruegg, Thomas Lawrence; Thelen, Michael P.

    2016-03-22

    The present invention provides for a method of genetically modifying microorganisms to enhance resistance to ionic liquids, host cells genetically modified in accordance with the methods, and methods of using the host cells in a reaction comprising biomass that has been pretreated with ionic liquids.

  19. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    .... (iii) Upon receipt of a request described in § 725.15(b), EPA may require the submitter who originally...)(2) of this section, EPA will issue for publication in the Federal Register notice described in § 725... microorganism which is described by a generic name on the public Inventory may submit an inquiry to EPA...

  20. Ecophysiology of psychrophilic and psychrotolerant microorganisms.

    PubMed

    Bölter, M

    2004-07-01

    This review describes psychrophilic and psychrotolerant microorganisms, which are abundant in different kinds of environments. Their ecophysiological properties and strategies for survival are reviewed in relation to their occurrences in marine and terrestrial environments, with special reference to the deep-sea, the sea ice and the permafrost soils. PMID:15559973

  1. Mechanisms of nickel toxicity in microorganisms

    PubMed Central

    Macomber, Lee

    2014-01-01

    Summary Nickel has long been known to be an important human toxicant, including having the ability to form carcinomas, but until recently nickel was believed to be an issue only to microorganisms living in nickel-rich serpentine soils or areas contaminated by industrial pollution. This assumption was overturned by the discovery of a nickel defense system (RcnR/RcnA) found in microorganisms that live in a wide range of environmental niches, suggesting that nickel homeostasis is a general biological concern. To date, the mechanisms of nickel toxicity in microorganisms and higher eukaryotes are poorly understood. In this review, we summarize nickel homeostasis processes used by microorganisms and highlight in vivo and in vitro effects of exposure to elevated concentrations of nickel. On the basis of this evidence we propose four mechanisms of nickel toxicity: 1) nickel replaces the essential metal of metalloproteins, 2) nickel binds to catalytic residues of non-metalloenzymes; 3) nickel binds outside the catalytic site of an enzyme to inhibit allosterically, and 4) nickel indirectly causes oxidative stress. PMID:21799955

  2. Mass spectrometry for rapid characterization of microorganisms.

    PubMed

    Demirev, Plamen A; Fenselau, Catherine

    2008-01-01

    Advances in instrumentation, proteomics, and bioinformatics have contributed to the successful applications of mass spectrometry (MS) for detection, identification, and classification of microorganisms. These MS applications are based on the detection of organism-specific biomarker molecules, which allow differentiation between organisms to be made. Intact proteins, their proteolytic peptides, and nonribosomal peptides have been successfully utilized as biomarkers. Sequence-specific fragments for biomarkers are generated by tandem MS of intact proteins or proteolytic peptides, obtained after, for instance, microwave-assisted acid hydrolysis. In combination with proteome database searching, individual biomarker proteins are unambiguously identified from their tandem mass spectra, and from there the source microorganism is also identified. Such top-down or bottom-up proteomics approaches permit rapid, sensitive, and confident characterization of individual microorganisms in mixtures and are reviewed here. Examples of MS-based functional assays for detection of targeted microorganisms, e.g., Bacillus anthracis, in environmental or clinically relevant backgrounds are also reviewed. PMID:20636075

  3. Caenorhabditis briggsae Recombinant Inbred Line Genotypes Reveal Inter-Strain Incompatibility and the Evolution of Recombination

    PubMed Central

    Ross, Joseph A.; Koboldt, Daniel C.; Staisch, Julia E.; Chamberlin, Helen M.; Gupta, Bhagwati P.; Baird, Scott E.; Haag, Eric S.

    2011-01-01

    The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes. PMID:21779179

  4. Advancing metabolic engineering through systems biology of industrial microorganisms.

    PubMed

    Dai, Zongjie; Nielsen, Jens

    2015-12-01

    Development of sustainable processes to produce bio-based compounds is necessary due to the severe environmental problems caused by the use of fossil resources. Metabolic engineering can facilitate the development of highly efficient cell factories to produce these compounds from renewable resources. The objective of systems biology is to gain a comprehensive and quantitative understanding of living cells and can hereby enhance our ability to characterize and predict cellular behavior. Systems biology of industrial microorganisms is therefore valuable for metabolic engineering. Here we review the application of systems biology tools for the identification of metabolic engineering targets which may lead to reduced development time for efficient cell factories. Finally, we present some perspectives of systems biology for advancing metabolic engineering further.

  5. Turbulent Fluid Acceleration Generates Clusters of Gyrotactic Microorganisms

    NASA Astrophysics Data System (ADS)

    De Lillo, Filippo; Cencini, Massimo; Durham, William M.; Barry, Michael; Stocker, Roman; Climent, Eric; Boffetta, Guido

    2014-01-01

    The motility of microorganisms is often biased by gradients in physical and chemical properties of their environment, with myriad implications on their ecology. Here we show that fluid acceleration reorients gyrotactic plankton, triggering small-scale clustering. We experimentally demonstrate this phenomenon by studying the distribution of the phytoplankton Chlamydomonas augustae within a rotating tank and find it to be in good agreement with a new, generalized model of gyrotaxis. When this model is implemented in a direct numerical simulation of turbulent flow, we find that fluid acceleration generates multifractal plankton clustering, with faster and more stable cells producing stronger clustering. By producing accumulations in high-vorticity regions, this process is fundamentally different from clustering by gravitational acceleration, expanding the range of mechanisms by which turbulent flows can impact the spatial distribution of active suspensions.

  6. Bioengineering of microorganisms for C₃ to C₅ alcohols production.

    PubMed

    Mainguet, Samuel E; Liao, James C

    2010-12-01

    Production of renewable fuels and chemicals is an absolute requirement for the sustainability of societies. This fact has been neglected during the past century as cheap and abundant, yet not renewable, sources of hydrocarbons were available. Since fossil fuel availability is decreasing, biological production of fuels and chemicals has been proposed to be a potential alternative to fossil sources. Higher alcohols (from C₃ to C₅) are useful substitutes for gasoline because of their high energy density and low hygroscopicity and are important feedstocks for other chemicals. Some Clostridia species are known to naturally ferment sugars to isopropanol and 1-butanol. However, other C₃ to C₅ alcohols are not produced in large quantities by natural microorganisms. A non-fermentative strategy to produce a broad range of higher alcohols has been devised using the ubiquitous keto acid biosynthetic pathways. This review provides a current overview of these different strategies.

  7. Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain.

    PubMed

    Gmeiner, Christoph; Spadiut, Oliver

    2015-01-01

    Recombinant protein production in microorganisms is one of the most studied areas of research in biotechnology today. In this respect the yeast Pichia pastoris is an important microbial production host due to its capability of secreting the target protein and performing posttranslational modifications. In a recent study, we described the development of a robust bioprocess for a glyco-engineered recombinant P. pastoris strain where the native α-1,6-mannosyltransfrease OCH1 was knocked out (Δoch1 strain). This strain produced the glycosylated enzyme horseradish peroxidase (HRP) with more homogeneous and shorter surface glycans than the respective benchmark strain. However, the recombinant Δoch1 strain was physiologically impaired and thus hard to cultivate. We faced cell cluster formation, cell lysis and consequent intensive foam formation. Thus, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on (1) cell physiology, (2) cell morphology, (3) cell lysis, (4) productivity and (5) product purity in a multivariate manner. However, not only process parameters might influence these characteristics, but also media supplements might have an impact. Here, we describe the effects of different heme-precursors as well as of a protease-inhibitor cocktail on the production of active HRP in therecombinant P. pastoris Δoch1strain.

  8. Production of bioplastics and hydrogen gas by photosynthetic microorganisms

    NASA Astrophysics Data System (ADS)

    Yasuo, Asada; Masato, Miyake; Jun, Miyake

    1998-03-01

    Our efforts have been aimed at the technological basis of photosynthetic-microbial production of materials and an energy carrier. We report here accumulation of poly-(3-hydroxybutyrate) (PHB), a raw material of biodegradable plastics and for production of hydrogen gas, and a renewable energy carrier by photosynthetic microorganisms (tentatively defined as cyanobacteria plus photosynthetic bateria, in this report). A thermophilic cyanobacterium, Synechococcus sp. MA19 that accumulates PHB at more than 20% of cell dry wt under nitrogen-starved conditions was isolated and microbiologically identified. The mechanism of PHB accumulation was studied. A mesophilic Synechococcus PCC7942 was transformed with the genes encoding PHB-synthesizing enzymes from Alcaligenes eutrophus. The transformant accumulated PHB under nitrogen-starved conditions. The optimal conditions for PHB accumulation by a photosynthetic bacterium grown on acetate were studied. Hydrogen production by photosynthetic microorganisms was studied. Cyanobacteria can produce hydrogen gas by nitrogenase or hydrogenase. Hydrogen production mediated by native hydrogenase in cyanobacteria was revealed to be in the dark anaerobic degradation of intracellular glycogen. A new system for light-dependent hydrogen production was targeted. In vitro and in vivo coupling of cyanobacterial ferredoxin with a heterologous hydrogenase was shown to produce hydrogen under light conditions. A trial for genetic trasformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum is going on. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Co-culture of Rhodobacter and Clostriumdium was applied to produce hydrogen from glucose. Conversely in the case of cyanobacteria, genetic regulation of photosynthetic proteins was intended to improve conversion efficiency in hydrogen production by the photosynthetic bacterium, Rhodobacter sphaeroides RV. A mutant acquired by

  9. Conjugational Recombination in Escherichia Coli: Genetic Analysis of Recombinant Formation in Hfr X F(-) Crosses

    PubMed Central

    Lloyd, R. G.; Buckman, C.

    1995-01-01

    The formation of recombinants during conjugation between Hfr and F(-) strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located ~500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located >300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange (s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec(+) crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to ~1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the

  10. New microorganisms and processes for MEOR. Quarterly report ending September 30, 1992

    SciTech Connect

    Sperl, G.T.; Sperl, P.L.

    1992-12-31

    Oil reservoirs naturally contain inorganic and organic materials which can be exploited through simple supplementation to support the growth of microorganisms which aid in the release of oil from the rock matrix. Other compounds which may serve as nutritional sources for microorganisms are added to reservoirs during production and operation of oil fields. These materials include sulfate, nitrate, carbonate, volatile fatty acids, nitrogen-containing corrosion inhibitors, phosphorous-containing scale inhibitors and trace elements. Our experiments show that, with minimal supplementation, growth of naturally-occurring microorganisms can be used to produce viscosifying agents to aid oil recovery. This natural microflora is also capable of removing sulfide from oil reservoirs and preventing the formation of new sulfide leading to both more oil recovery and increased value of the produced oil. The metabolic products of these microorganisms are Co{sub 2}, water, N{sub 2} and oxidized forms of sulfur, all of which are environmentally innocuous. Laboratory experiments with both defined mixtures of microorganisms as well as mixed populations both release more oil from sand pack columns.

  11. [Inactivation mechanism of microorganisms by the synergy of silver and light irradiation, and the application in household electrical appliances].

    PubMed

    He, Jinshan; Moriya, Yoshifumi; Oketa, Takemi; Sasabe, Shigeru; Nishida, Hirofumi; Chen, Lesheng; Zhu, Shenmin; Zhang, Di; Omori, Hideki; Cao, Guangyi

    2008-06-01

    The inactivation efficiencies of microorganisms were found to be enhanced by using silver solution together with ultraviolet light (UV-A, 395 nm) irradiation. The inactivation efficiencies were improved remarkably especially in eukaryotic microorganism. To make clear the inactivation mechanism of microorganisms by the combination effect of silver and ultraviolet light irradiation, the resultant solution was characterized by ESR (Electron spin resonance, ESR). Scanning electron microscopy (SEM) and the methnd for measuring enzyme activity of mitochondria for eukarvotic cells were used to conjecture the mechanism, by analysis of the morphological and physiologic changes in eukaryotic cells. It is proposed that silver oxide (Ag20) can be activated by ultraviolet light irradiation and react with water molecules to produce hydroxyl radical (.OH). Hydroxyl radical could damage cell wall of eukaryotic microorganisms, and inactivate the enzyme activity of mitochondria of eukaryotic microorganism cells. Accordingly, eukaryotic microorganism cells would die. In the experiment, Staphylococcus aureus was employed as the representative of prokaryotic microorganisms, and Candida albicans and Trichophyton mentagrophytes as the representative of eukaryotic microorganisms, respectively. Moreover, the results of the technology applied to washing machine were presented and discussed. PMID:18807998

  12. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    PubMed

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  13. Sex recombination, and reproductive fitness: an experimental study using Paramecium

    SciTech Connect

    Nyberg, D.

    1982-08-01

    The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)

  14. Colony mutants of compatible nocardiae displaying variations in recombining capacity.

    PubMed

    Brownell, G H; Walsh, R S

    1972-03-01

    Colonial morphology mutants of Nocardia erythropolis were isolated following ultraviolet (UV) irradiation. The alleles rou-1/smo-1 were located by recombinant analysis and found to be linked to previously mapped characters. On the basis of recombinant class type patterns obtained from various selective characters it was postulated that the rou-1 allele may span a region of unique nucleotides in the Mat-Ce genome. Recombination frequencies of rou-1 and smo-2 bearing mutants of the Mat-Ce mating type were found to differ by over 1000 fold. Attempts to demonstrate that low recombination frequencies produced by the Smo mutants were due to Rec(-) genes were unsuccessful. No increased sensitivity to either UV or X irradiation was observed by the Smo mutants. Acriflavine treatment of either Rou or Smo colony mutants failed to accelerate reversion or to alter the recombining potentials of the mutants.

  15. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  16. DEVELOPMENT OF MICROORGANISMS WITH IMPROVED TRANSPORT AND BIOSURFACTANT ACTIVITY FOR ENHANCED OIL RECOVERY

    SciTech Connect

    M.J. McInerney; N. Youssef; T. Fincher; S.K. Maudgalya; M.J. Folmsbee; R. Knapp; D. Nagle

    2004-05-31

    Diverse microorganisms were screened for biosurfactant production and anaerobic growth at elevated salt concentrations to obtain candidates most suitable for microbial oil recovery. Seventy percent of the 205 strains tested, mostly strains of Bacillus mojavensis, Bacillus subtilis, Bacillus licheniformis, and Bacillus sonorensis, produced biosurfactants aerobically and 41% of the strains had biosurfactant activity greater than Bacillus mojavensis JF-2, the current candidate for oil recovery. Biosurfactant activity varied with the percentage of the 3-hydroxy-tetradecanoate isomers in the fatty acid portion of the biosurfactant. Changing the medium composition by incorporation of different precursors of 3-hydroxy tetradecanoate increased the activity of biosurfactant. The surface tension and critical micelle concentration of 15 different, biosurfactant-producing Bacillus strains was determined individually and in combination with other biosurfactants. Some biosurfactant mixtures were found to have synergistic effect on surface tension (e.g. surface tension was lowered from 41 to 31 mN/m in some cases) while others had a synergistic effect on CMD-1 values. We compared the transport abilities of spores from three Bacillus strains using a model porous system to study spore recovery and transport. Sand-packed columns were used to select for spores or cells with the best transport abilities through brine-saturated sand. Spores of Bacillus mojavensis strains JF-2 and ROB-2 and a natural recombinant, strain C-9, transported through sand at very high efficiencies. The earliest cells/spores that emerged from the column were re-grown, allowed to sporulate, and applied to a second column. This procedure greatly enhanced the transport of strain C-9. Spores with enhanced transport abilities can be easily obtained and that the preparation of inocula for use in MEOR is feasible. Tertiary oil recovery experiments showed that 10 to 40 mg/l of JF-2 biosurfactant in the presence of 0

  17. Expression and Purification of Recombinant Hemoglobin in Escherichia coli

    PubMed Central

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E.; Moriyama, Hideaki; Storz, Jay F.

    2011-01-01

    Background Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. Methodology/Principal Findings As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Conclusion/Significance Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis. PMID:21625463

  18. Producing biofuels using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  19. Local climatic adaptation in a widespread microorganism.

    PubMed

    Leducq, Jean-Baptiste; Charron, Guillaume; Samani, Pedram; Dubé, Alexandre K; Sylvester, Kayla; James, Brielle; Almeida, Pedro; Sampaio, José Paulo; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R

    2014-02-22

    Exploring the ability of organisms to locally adapt is critical for determining the outcome of rapid climate changes, yet few studies have addressed this question in microorganisms. We investigated the role of a heterogeneous climate on adaptation of North American populations of the wild yeast Saccharomyces paradoxus. We found abundant among-strain variation for fitness components across a range of temperatures, but this variation was only partially explained by climatic variation in the distribution area. Most of fitness variation was explained by the divergence of genetically distinct groups, distributed along a north-south cline, suggesting that these groups have adapted to distinct climatic conditions. Within-group fitness components were correlated with climatic conditions, illustrating that even ubiquitous microorganisms locally adapt and harbour standing genetic variation for climate-related traits. Our results suggest that global climatic changes could lead to adaptation to new conditions within groups, or changes in their geographical distributions.

  20. Characterization of Microorganisms by MALDI Mass Spectrometry

    SciTech Connect

    Petersen, Catherine E.; Valentine, Nancy B.; Wahl, Karen L.

    2008-10-02

    Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for characterization and analysis of microorganisms, specifically bacteria, is described here as a rapid screening tool. The objective of this technique is not comprehensive protein analysis of a microorganism but rather a rapid screening of the organism and the accessible protein pattern for characterization and distinction. This method is based on the ionization of the readily accessible and easily ionizable portion of the protein profile of an organism that is often characteristic of different bacterial species. The utility of this screening approach is yet to reach its full potential but could be applied to food safety, disease outbreak monitoring in hospitals, culture stock integrity and verification, microbial forensics or homeland security applications.

  1. Microorganisms detection on substrates using QCL spectroscopy

    NASA Astrophysics Data System (ADS)

    Padilla-Jiménez, Amira C.; Ortiz-Rivera, William; Castro-Suarez, John R.; Ríos-Velázquez, Carlos; Vázquez-Ayala, Iris; Hernández-Rivera, Samuel P.

    2013-05-01

    Recent investigations have focused on the improvement of rapid and accurate methods to develop spectroscopic markers of compounds constituting microorganisms that are considered biological threats. Quantum cascade lasers (QCL) systems have revolutionized many areas of research and development in defense and security applications, including his area of research. Infrared spectroscopy detection based on QCL was employed to acquire mid infrared (MIR) spectral signatures of Bacillus thuringiensis (Bt), Escherichia coli (Ec) and Staphylococcus epidermidis (Se), which were used as biological agent simulants of biothreats. The experiments were carried out in reflection mode on various substrates such as cardboard, glass, travel baggage, wood and stainless steel. Chemometrics statistical routines such as principal component analysis (PCA) regression and partial least squares-discriminant analysis (PLS-DA) were applied to the recorded MIR spectra. The results show that the infrared vibrational techniques investigated are useful for classification/detection of the target microorganisms on the types of substrates studied.

  2. UV inactivation of pathogenic and indicator microorganisms

    SciTech Connect

    Chang, J.C.; Ossoff, S.F.; Lobe, D.C.; Dorfman, M.H.; Dumais, C.M.; Qualls, R.G.; Johnson, J.D.

    1985-06-01

    Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.

  3. Assessment of microorganisms from Indonesian Oil Fields

    SciTech Connect

    Kadarwati, S.; Udiharto, M.; Rahman, M.; Jasjfi, E.; Legowo, E.H.

    1995-12-31

    Petroleum resources have been the mainstay of the national development in Indonesia. However, resources are being depleted after over a century of exploitation, while the demand continues to grow with the rapid economic development of the country. In facing the problem, EOR has been applied in Indonesia, such as the steamflooding project in Duri field, but a more energy efficient technology would be preferable. Therefore, MEOR has been recommended as a promising solution. Our study, aimed at finding indigenous microorganisms which can be developed for application in MEOR, has isolated microbes from some oil fields of Indonesia. These microorganisms have been identified, their activities studied, and the effects of their metabolisms examined. This paper describes the research carried out by LEMIGAS in this respect, giving details on the methods of sampling, incubation, identification, and activation of the microbes as well as tests on the effects of their metabolites, with particular attention to those with potential for application in MEOR.

  4. Local climatic adaptation in a widespread microorganism

    PubMed Central

    Leducq, Jean-Baptiste; Charron, Guillaume; Samani, Pedram; Dubé, Alexandre K.; Sylvester, Kayla; James, Brielle; Almeida, Pedro; Sampaio, José Paulo; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R.

    2014-01-01

    Exploring the ability of organisms to locally adapt is critical for determining the outcome of rapid climate changes, yet few studies have addressed this question in microorganisms. We investigated the role of a heterogeneous climate on adaptation of North American populations of the wild yeast Saccharomyces paradoxus. We found abundant among-strain variation for fitness components across a range of temperatures, but this variation was only partially explained by climatic variation in the distribution area. Most of fitness variation was explained by the divergence of genetically distinct groups, distributed along a north–south cline, suggesting that these groups have adapted to distinct climatic conditions. Within-group fitness components were correlated with climatic conditions, illustrating that even ubiquitous microorganisms locally adapt and harbour standing genetic variation for climate-related traits. Our results suggest that global climatic changes could lead to adaptation to new conditions within groups, or changes in their geographical distributions. PMID:24403328

  5. UV inactivation of pathogenic and indicator microorganisms.

    PubMed Central

    Chang, J C; Ossoff, S F; Lobe, D C; Dorfman, M H; Dumais, C M; Qualls, R G; Johnson, J D

    1985-01-01

    Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts. PMID:2990336

  6. Construction and characterization of a recombinant invertebrate iridovirus.

    PubMed

    Ozgen, Arzu; Muratoglu, Hacer; Demirbag, Zihni; Vlak, Just M; van Oers, Monique M; Nalcacioglu, Remziye

    2014-08-30

    Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol.

  7. The transfer of viable microorganisms between planets.

    PubMed

    Davies, P C

    1996-01-01

    There is increasing acceptance that catastrophic cosmic impacts have played an important role in shaping the history of terrestrial life. Large asteroid and cometary impacts are also capable of displacing substantial quantities of planetary surface material into space. The discovery of Martian rocks on Earth suggests that viable microorganisms within such ejecta could be exchanged between planets. If this conjecture is correct, it will have profound implications for the origin and evolution of life in the solar system.

  8. Control of microorganisms in flowing nutrient solutions.

    PubMed

    Evans, R D

    1994-11-01

    Controlling microorganisms in flowing nutrient solutions involves different techniques when targeting the nutrient solution, hardware surfaces in contact with the solution, or the active root zone. This review presents basic principles and applications of a number of treatment techniques, including disinfection by chemicals, ultrafiltration, ultrasonics, and heat treatment, with emphasis on UV irradiation and ozone treatment. Procedures for control of specific pathogens by nutrient solution conditioning also are reviewed.

  9. MICROORGANISMS DIE-OFF RATES IN URBAN STORMWATER RUNOFF 2007

    EPA Science Inventory

    Stormwater best management practices (BMPs) are often considered effective tools to mitigate the effects of stormwater pollutants before they are discharged to receiving waters. However, BMP performance for microorganisms removal is not well documented. Microorganisms die-off in...

  10. MICROORGANISMS DIE-OFF RATES IN URBAN STORMWATER RUNOFF 2007

    EPA Science Inventory

    Stormwater best management practices (BMPs) are often considered effective tools to mitigate the effects of stormwater pollutants before they are discharged to receiving waters. However, BMP performance for microorganisms removal is not well documented. Microorganisms die-off in ...

  11. MODELING THE FATE OF MICROORGANISMS IN WATER, WASTEWATER, AND SOIL

    EPA Science Inventory

    The natural environment is filled with microorganisms, most of which are natural residents and colonize various ecological niches. These microorganisms either live independently within the environment, or live in association with various host organisms. There also are places and ...

  12. Investigation to identify paint coatings resistive to microorganism growth

    NASA Technical Reports Server (NTRS)

    Cooper, C. W.; Kemp, H. T.

    1971-01-01

    All selected coatings contain nutrients that support microbial growth and survival. Incorporation of microbiocidal agents into coatings more susceptible to attack is recommended for improved inhibition of microorganism growth and for increased protection against deterioration of coatings by microorganisms.

  13. Metal resistance in acidophilic microorganisms and its significance for biotechnologies.

    PubMed

    Dopson, Mark; Holmes, David S

    2014-10-01

    Extremely acidophilic microorganisms have an optimal pH of <3 and are found in all three domains of life. As metals are more soluble at acid pH, acidophiles are often challenged by very high metal concentrations. Acidophiles are metal-tolerant by both intrinsic, passive mechanisms as well as active systems. Passive mechanisms include an internal positive membrane potential that creates a chemiosmotic gradient against which metal cations must move, as well as the formation of metal sulfate complexes reducing the concentration of the free metal ion. Active systems include efflux proteins that pump metals out of the cytoplasm and conversion of the metal to a less toxic form. Acidophiles are exploited in a number of biotechnologies including biomining for sulfide mineral dissolution, biosulfidogenesis to produce sulfide that can selectively precipitate metals from process streams, treatment of acid mine drainage, and bioremediation of acidic metal-contaminated milieux. This review describes how acidophilic microorganisms tolerate extremely high metal concentrations in biotechnological processes and identifies areas of future work that hold promise for improving the efficiency of these applications.

  14. Microorganisms in stormwater; a summary of recent investigations

    USGS Publications Warehouse

    Mallard, Gail E.

    1980-01-01

    All storm runoff contains a variety of bacteria, including total coliform, fecal coliform, and fecal streptococci, which are derived from the land over which the water flows. Most total coliform are native soil organisms, whereas the fecal coliform and fecal streptococci originate from the feces of wild and domestic animals. Urban runoff has been reported to contain pathogenic organisms, but this probably presents little direct threat to human health because the runoff is not ingested. Runoff water can, however, have other negative effects such as contamination of surface water, which may result in beach closures, or contamination of shellfish. This type of contamination is generally of short duration because indicator bacteria and pathogens die out rapidly in the aquatic environment. Similarly, bacteria and viruses deposited on soil by stormwater are inactivated by drying, competition from soil microflora, and a variety of other processes. Every storm producing runoff is unique in the number and type of microorganisms because these vary from site to site, from storm to storm, and during the course of the storm. Stormwater to be examined for microorganisms must be collected in sterile containers and processed immediately. (USGS)

  15. Exposure to airborne microorganisms and endotoxin in herb processing plants.

    PubMed

    Dutkiewicz, J; Krysińska-Traczyk, E; Skórska, C; Sitkowska, J; Prazmo, Z; Golec, M

    2001-01-01

    Microbiological air sampling was performed in two herb processing plants located in eastern Poland. Air samples for determination of the levels of bacteria, fungi, dust and endotoxin were collected at 14 sites during cleaning, cutting, grinding, sieving, sorting and packing of 11 kinds of herbs (nettle, caraway, birch, celandine, marjoram, mint, peppermint, sage, St. John's wort, calamus, yarrow), used for production of medications, cosmetics and spices. It was found that processing of herbs was associated with a very high pollution of the air with bacteria, fungi, dust and endotoxin. The numbers of microorganisms (bacteria and fungi) in the air of herb processing plants ranged within 40.6-627.4 x 10(3) cfu/m3 (mean +/- S.D = 231.4 +/- 181.0 x 10(3) cfu/m3). The greatest concentrations were noted at the initial stages of production cycle, during cleaning, cutting and grinding of herbs. The numbers of airborne microorganisms were also significantly (p<0.0001) related to the kind of processed herb, being the greatest at processing marjoram, nettle, yarrow and mint. The values of the respirable fraction of airborne microflora in the examined facilities varied within a fairly wide range and were between 14.7-67.7%. The dominant microorganisms in the air of herb processing plants were mesophilic bacteria, among which endospore-forming bacilli (Bacillus spp.) and actinomycetes of the species Streptomyces albus were most numerous. Among Gram-negative bacteria, the most common was endotoxin-producing species Alcaligenes faecalis. Altogether, 37 species or genera of bacteria and 23 species or genera of fungi were identified in the air of herb processing plants, of these, 11 and 10 species or genera respectively were reported as having allergenic and/or immunotoxic properties. The concentrations of dust and bacterial endotoxin in the air of herb processing plants were large with extremely high levels at some sampling sites. The concentrations of airborne dust ranged within 3

  16. Exposure to airborne microorganisms and endotoxin in herb processing plants.

    PubMed

    Dutkiewicz, J; Krysińska-Traczyk, E; Skórska, C; Sitkowska, J; Prazmo, Z; Golec, M

    2001-01-01

    Microbiological air sampling was performed in two herb processing plants located in eastern Poland. Air samples for determination of the levels of bacteria, fungi, dust and endotoxin were collected at 14 sites during cleaning, cutting, grinding, sieving, sorting and packing of 11 kinds of herbs (nettle, caraway, birch, celandine, marjoram, mint, peppermint, sage, St. John's wort, calamus, yarrow), used for production of medications, cosmetics and spices. It was found that processing of herbs was associated with a very high pollution of the air with bacteria, fungi, dust and endotoxin. The numbers of microorganisms (bacteria and fungi) in the air of herb processing plants ranged within 40.6-627.4 x 10(3) cfu/m3 (mean +/- S.D = 231.4 +/- 181.0 x 10(3) cfu/m3). The greatest concentrations were noted at the initial stages of production cycle, during cleaning, cutting and grinding of herbs. The numbers of airborne microorganisms were also significantly (p<0.0001) related to the kind of processed herb, being the greatest at processing marjoram, nettle, yarrow and mint. The values of the respirable fraction of airborne microflora in the examined facilities varied within a fairly wide range and were between 14.7-67.7%. The dominant microorganisms in the air of herb processing plants were mesophilic bacteria, among which endospore-forming bacilli (Bacillus spp.) and actinomycetes of the species Streptomyces albus were most numerous. Among Gram-negative bacteria, the most common was endotoxin-producing species Alcaligenes faecalis. Altogether, 37 species or genera of bacteria and 23 species or genera of fungi were identified in the air of herb processing plants, of these, 11 and 10 species or genera respectively were reported as having allergenic and/or immunotoxic properties. The concentrations of dust and bacterial endotoxin in the air of herb processing plants were large with extremely high levels at some sampling sites. The concentrations of airborne dust ranged within 3

  17. Protein Languages Differ Depending on Microorganism Lifestyle

    PubMed Central

    Grzymski, Joseph J.; Marsh, Adam G.

    2014-01-01

    Few quantitative measures of genome architecture or organization exist to support assumptions of differences between microorganisms that are broadly defined as being free-living or pathogenic. General principles about complete proteomes exist for codon usage, amino acid biases and essential or core genes. Genome-wide shifts in amino acid usage between free-living and pathogenic microorganisms result in fundamental differences in the complexity of their respective proteomes that are size and gene content independent. These differences are evident across broad phylogenetic groups–a result of environmental factors and population genetic forces rather than phylogenetic distance. A novel comparative analysis of amino acid usage–utilizing linguistic analyses of word frequency in language and text–identified a global pattern of higher peptide word repetition in 376 free-living versus 421 pathogen genomes across broad ranges of genome size, G+C content and phylogenetic ancestry. This imprint of repetitive word usage indicates free-living microorganisms have a bias for repetitive sequence usage compared to pathogens. These findings quantify fundamental differences in microbial genomes relative to life-history function. PMID:24828817

  18. Prokaryotic silicon utilizing microorganisms in the biosphere

    NASA Astrophysics Data System (ADS)

    Gupta, D.; Das, S.

    2012-12-01

    Although a little study has been done to determine the silicon utilizing prokaryotes, our previous experiments indicated that almost all Gram-positive bacteria are silicon utilizing; one of them, Streptococci survived exposure on the lunar surface for a long period in experiment done by others. Our initial experiments with these Gram positive microorganisms showed that there were limited growths of these microorganisms on carbon free silicate medium probably with the help of some carry over carbon and nitrogen during cultivation procedures. However, increase in growth rate after repeated subcultures could not be explained at present. The main groups of prokaryotes which were found silicon utilizing microorganisms were Mycobacterium, Bacillus, Nocardia, Streptomyces, Staphylococcus, Streptococcus, Lactobacillus, and Clostridium. In a another previous study by us when silicon level was studied in such grown up cells on carbon "free" silicate medium by electron prove microanalyser, it was found that silicon in cells grown on carbon "free" silicate medium was much higher (24.9%) than those grown on conventional carbon based medium (0.84%). However, these initial findings are encouraging for our future application of this group of organisms on extraterrestrial surfaces for artificial micro-ecosystem formation. It was found that when electropositive elements are less in extraterrestrial situation, then polymerization of silicon-oxygen profusion may occur easily, particularly in carbon and nitrogen paucity in the rocky worlds of the Universe.

  19. Identification of a Large Pool of Microorganisms with an Array of Porphyrin Based Gas Sensors.

    PubMed

    Zetola, Nicola M; Modongo, Chawangwa; Mathlagela, Keikantse; Sepako, Enoch; Matsiri, Ogopotse; Tamuhla, Tsaone; Mbongwe, Bontle; Martinelli, Eugenio; Sirugo, Giorgio; Paolesse, Roberto; Di Natale, Corrado

    2016-04-01

    The association between volatile compounds (VCs) and microorganisms, as demonstrated by several studies, may offer the ground for a rapid identification of pathogens. To this regard, chemical sensors are a key enabling technology for the exploitation of this opportunity. In this study, we investigated the performance of an array of porphyrin-coated quartz microbalance gas sensors in the identification of a panel of 12 bacteria and fungi. The porphyrins were metal complexes and the free base of a functionalized tetraphenylporphyrin. Our results show that the sensor array distinguishes the VC patterns produced by microorganisms in vitro. Besides being individually identified, bacteria are also sorted into Gram-positive and Gram-negative.

  20. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.