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      Análisis de transferencia horizontal de genes en ensayos de biorremediación con grasas recalcitrantes Translated title: Analysing horizontal gene transfer in bioremediation trials using recalcitrant grease

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          Abstract

          El empleo de microorganismos como herramienta para la mineralización de contaminantes orgánicos es una práctica que ha tomado mucha fuerza gracias a su eficiencia y bajo costo. La transferencia horizontal vía conjugación de genes es un requerimiento básico para optimizar procesos de biorremediación, por esta razón, además de conocer la diversidad metabólica es fundamental entender las interacciones que ocurren en una comunidad bacteriana para potenciar los procesos de biorremediación en campo. En este estudio se busca evaluar el potencial de transferencia horizontal (TH), tanto in vitro como en microcosmos de suelo, de aislamientos de bacterias obtenidas de un pasivo ambiental de grasas provenientes de la explotación carbonífera del Cerrejón. Inicialmente se agruparon los aislamientos de acuerdo con sus patrones de resistencia a antibióticos: ampicilina, cloramfenicol, gentamicina, tetraciclina y kanamicina. El potencial de TH de las cepas Vlf4, Ot3, Ot6, Pgt4, Blf11 y Vlf13 fue evaluado in vitro en medio sólido Luria-Bertani (LB) donde se obtuvieron nuevos fenotipos a partir de los cruces Vlf13xOt6 y Pgt4xOt6, el nuevo fenotipo indica resistencia a los dos marcadores (ampR, kanR) y su morfología sugiere que el receptor, en los dos casos, es la cepa Ot6. Los parentales Vlf13, Pgt4 y Ot6 fueron identificados por amplificación del gen RNAr 16S como Pseudomonas sp., Pseudomonas sp. y Chryseobacterium sp., respectivamente, y los transconjugantes como Chryseobacterium sp. Posteriormente, estos dos cruces fueron sometidos a ensayos de transferencia horizontal en microcosmos de suelos, donde se hizo evidente nuevamente la presencia de TH a una menor tasa. Los resultados obtenidos indican la posibilidad de un potencial de transferencia horizontal de genes entre los aislamientos seleccionados, dando lugar a la probabilidad de formular en futuros estudios un consorcio de bacterias que demuestran tener esta ventaja adaptativa

          Translated abstract

          Using microorganisms as a tool for mineralising organic pollutants is a practice which has begun to gain strength due to its efficiency and low cost. Horizontal gene transfer (HGT) by conjugation is important for optimising bioremediation processes. The metabolic diversity of catabolic pathways must thus be studied; however, the microbial interactions occurring in bioremediation consortia must also be understood. This will lead to improving bioremediation in the field. This study was aimed at evaluating HGT potential in vitro and in soil microcosms from bacterial strains isolated from grease samples obtained from a disposal site situated in the Cerrejon coal mine. The isolates were initially grouped by selective markers: ampicillin, chloramphenicol, gentamicin, tetracycline and kanamycin. The Vlf4, Ot3, Ot6, Pgt4, Blf11 and Vlf13 strains’ HGT potential was evaluated in vitro on Luria-Bertani LB agar. New phenotypes were obtained from mating between Vlf13xOt6 and Pgt4xOt6. The new phenotype indicated resistance to both antibiotic markers and its morphology suggested that isolate Ogt6 was the receptor in both cases. Vlf13, Pgt4 and Ot6 parental strains were identified by RNAr 16S as being Pseudomonas sp. Pseudomonas sp. and Chryseobacterium sp., respectively; the transconjugants were identified as being Chryseobacterium sp. Soil microcosms were subsequently constructed with Vlf13xOt6 and Pgt4xOt6 and new phenotypes were detected at a lower rate but having the same possible receptor. These results suggested that the selected isolates had HGT potential which could lead to formulating a bacterial consortium having an adaptive advantage in future studies.

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          Most cited references17

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          Molecular Cloning : A Laboratory Manual

          <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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            TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo.

            Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids.
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              Horizontal DNA transfer between bacteria in the environment.

              In the environment horizontal DNA transfer between various bacterial species and genera takes place by transformation, transduction, but mainly by conjugation. Conjugation is responsible for the spread of genes coding for antibiotic resistance and xenobiotic degradation. Transfer events are reported in animal, rhizosphere and phylloplane ecosystems and in non polluted and polluted water and soil. Genetic exchange between Bacteria and Archaea is also observed. Evaluation of the extent of interspecies gene transfer is crucial in view of the deliberate release of a variety of unmodified and genetically modified microorganisms into the natural environments.
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                Author and article information

                Contributors
                Role: ND
                Role: ND
                Journal
                biote
                Revista Colombiana de Biotecnología
                Rev. colomb. biotecnol
                Instituto de Biotecnología, Universidad Nacional de Colombia (Bogotá )
                0123-3475
                January 2010
                : 12
                : 1
                : 22-31
                Affiliations
                [1 ] Universidad de Los Andes Venezuela
                [2 ] Universidad de Los Andes Venezuela
                Article
                S0123-34752010000100003
                25c00c0f-ac47-42ce-803a-66b56975fbac

                http://creativecommons.org/licenses/by/4.0/

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                SciELO Colombia

                Self URI (journal page): http://www.scielo.org.co/scielo.php?script=sci_serial&pid=0123-3475&lng=en
                Categories
                BIOTECHNOLOGY & APPLIED MICROBIOLOGY

                Biotechnology
                Conjugation,transconjugant,replica plating,conjugación,trasnconjugantes
                Biotechnology
                Conjugation, transconjugant, replica plating, conjugación, trasnconjugantes

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