The Experts below are selected from a list of 115638 Experts worldwide ranked by ideXlab platform
Kornelia Smalla - One of the best experts on this subject based on the ideXlab platform.
-
transformation of acinetobacter sp strain bd413 pfg4δnptii with transgenic Plant DNA in soil microcosms and effects of kanamycin on selection of transformants
Applied and Environmental Microbiology, 2000Co-Authors: Kaare Magne Nielsen, J D Van Elsas, Kornelia SmallaAbstract:Bacterial antibiotic resistance markers are the most fre-quently inserted genes in transgenic Plants. However, the re-sistance genes do not encode desirable traits in commerciallyused Plant varieties. Of the 15 different resistance genes incor-porated into Plants (17, 30), several encode resistance to clin-ically used antibiotics. Since Plant DNA has been shown topersist in soil over extended periods of time (8, 25, 38, 39),concerns that these transgenes may spread horizontally to bac-teria have been raised (17, 18, 22, 29). Sequence comparisonsof genes isolated from wild Plants and bacteria have indicatedthat horizontal gene transfer has occurred naturally betweenthem (13, 32). Moreover, whole-genome analyses of bacteriasuggest horizontal transfer of genetic material to be commonand a major force in bacterial evolution (14, 40).One mechanism of gene transfer that allows uptake of ge-netic material from diverged species in bacteria is naturaltransformation, which facilitates uptake of naked DNA incompetent bacteria (15). Based on this mechanism, severallaboratory studies have been conducted to elucidate the po-tential for Plant-harbored resistance determinants to be takenup by naturally occurring bacterial recipients (2, 3, 21, 28).These studies have, however, not been able to demonstrateuptake of such determinants, nor have studies of bacteria ob-tained from soil samples from field trials with transgenic Plants(8, 25). Detection of horizontal transfer in these studies reliedupon the uptake of expressed and selectable genes in thebacterial recipients grown under optimized conditions or apositive DNA hybridization signal or PCR amplification ofPlant transgenes in the bacterial fraction of soil. However,direct analyses of DNA from soil samples often fail to dem-onstrate integration of Plant transgenes into bacterial ge-nomes. Transfer of smaller DNA fragments or nonexpressedor nonselected genes would rarely be detected in these studies.Recently, uptake of transgenic Plant-harbored DNA frag-ments by bacteria based on restoration of a partially deleted(10- or 317-bp internal deletion) bacterial kanamycin (KM)resistance gene (
-
monitoring field releases of genetically modified sugar beets for persistence of transgenic Plant DNA and horizontal gene transfer
FEMS Microbiology Ecology, 1999Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:Field releases of transgenic rizomania-resistant sugar beet (Beta vulgaris) Plants were accompanied by a study of the persistence of DNA from transgenic sugar beet litter in soil and of horizontal gene transfer of Plant DNA to bacteria. The transgenic sugar beets contained the marker genes nptII and bar under the control of the bidirectional TR1/2 promoter conferring kanamycin (Km) and glufosinate ammonium resistance to the Plant. Primer systems targeting the construct allowed the specific and sensitive detection of the transgenic DNA in soil. Soil samples were analyzed by cultivation of bacteria on nonselective and Km-selective media to determine the proportion of Km-resistant bacteria and to monitor the culturable fraction for incorporation of transgenic Plant DNA. To detect the presence of transgenic DNA independently from cultivation, total soil DNA was extracted and amplified by PCR with three different primer sets specific for the transgenic DNA. Long-term persistence of transgenic DNA could be shown under field conditions (up to 2 years) and also in soil microcosms with introduced transgenic Plant DNA. No construct-specific sequences were detected by dot blot hybridizations of bacterial isolates. The experimental limitations of detecting horizontal gene transfer from Plants to bacteria under field conditions are discussed.
-
transformation of acinetobacter sp strain bd413 by transgenic sugar beet DNA
Applied and Environmental Microbiology, 1998Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:The ability of Acinetobacter sp. strain BD413(pFG4ΔnptII) to take up and integrate transgenic Plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of nptII, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, Plant DNA, and homogenates carrying the gene nptII. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA.
Frank Gebhard - One of the best experts on this subject based on the ideXlab platform.
-
monitoring field releases of genetically modified sugar beets for persistence of transgenic Plant DNA and horizontal gene transfer
FEMS Microbiology Ecology, 1999Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:Field releases of transgenic rizomania-resistant sugar beet (Beta vulgaris) Plants were accompanied by a study of the persistence of DNA from transgenic sugar beet litter in soil and of horizontal gene transfer of Plant DNA to bacteria. The transgenic sugar beets contained the marker genes nptII and bar under the control of the bidirectional TR1/2 promoter conferring kanamycin (Km) and glufosinate ammonium resistance to the Plant. Primer systems targeting the construct allowed the specific and sensitive detection of the transgenic DNA in soil. Soil samples were analyzed by cultivation of bacteria on nonselective and Km-selective media to determine the proportion of Km-resistant bacteria and to monitor the culturable fraction for incorporation of transgenic Plant DNA. To detect the presence of transgenic DNA independently from cultivation, total soil DNA was extracted and amplified by PCR with three different primer sets specific for the transgenic DNA. Long-term persistence of transgenic DNA could be shown under field conditions (up to 2 years) and also in soil microcosms with introduced transgenic Plant DNA. No construct-specific sequences were detected by dot blot hybridizations of bacterial isolates. The experimental limitations of detecting horizontal gene transfer from Plants to bacteria under field conditions are discussed.
-
transformation of acinetobacter sp strain bd413 by transgenic sugar beet DNA
Applied and Environmental Microbiology, 1998Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:The ability of Acinetobacter sp. strain BD413(pFG4ΔnptII) to take up and integrate transgenic Plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of nptII, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, Plant DNA, and homogenates carrying the gene nptII. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA.
Jean-michel Monier - One of the best experts on this subject based on the ideXlab platform.
-
Long-term persistence and bacterial transformation potential of transplastomic Plant DNA in soil
Research in Microbiology, 2010Co-Authors: Alessandra Pontiroli, Pascal Simonet, Timothy Vogel, John Poté, Walter Wildi, Maria Ceccherini, Elizabeth Kay, Paolo Nannipieri, Jean-michel MonierAbstract:The long-term physical persistence and biological activity of transplastomic Plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified Plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, Plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the Plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic Plant DNA in soil. (C) 2010 Elsevier Masson SAS. All rights reserved.
-
Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco
Applied and Environmental Microbiology, 2009Co-Authors: Alessandra Pontiroli, Pascal Simonet, Timothy Vogel, Aurora Rizzi, Daniele Daffonchio, Jean-michel MonierAbstract:Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between Plants and bacteria. Plant DNA released during the degradation of Plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or Plant-associated bacteria could be in direct contact with Plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco Plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying Plant tissues, to develop a competence state, and to possibly acquire exogenous Plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413 (rbcL-Delta PaadA::gfp) carrying a promoterless aadA:: gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic Plant DNA. Opportunistic growth occurred on decaying Plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between Plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for Plant tissues.
-
Fate of transgenic Plant DNA in the environment
Environmental Biosafety Research, 2007Co-Authors: Alessandra Pontiroli, Pascal Simonet, Asa Frostegard, Timothy Vogel, Jean-michel MonierAbstract:This review addresses the possible ecological effects of transgenic Plants on micro-organisms in the field, hence, in the phytosphere and in the soil matrix. The important steps involved in the interaction between Plant DNA and bacteria and the factors that influence the horizontal gene transfer (HGT) process will be discussed. HGT is a process in which two partners are involved, even if indirectly. In the first section, aspects concerning bacteria, such as their physico-chemical, biological and genetic characteristics, are described. Parameters affecting transgenic DNA fate in the environment are described in the second section. Subsequently, terrestrial habitats are evaluated in terms of their capacity to favor horizontal gene transfer. Finally, we focused on several studies in order to evaluate possible perturbations of soil bacterial community composition due to cultivation of transgenic Plants in the field.
-
Detection of potential transgenic Plant DNA recipients among soil bacteria
Environmental Biosafety Research, 2007Co-Authors: Jean-michel Monier, Pascal Simonet, Elizabeth Kay, Dominique Bernillon, Aurélie Faugier, Oleksandra Rybalka, Yves Dessaux, Timothy VogelAbstract:The likelihood of gene transfer from transgenic Plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) Plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize Plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased Plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco Plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco Plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic Plant DNA recipients might be present in soil bacterial communities.
Pascal Simonet - One of the best experts on this subject based on the ideXlab platform.
-
Long-term persistence and bacterial transformation potential of transplastomic Plant DNA in soil
Research in Microbiology, 2010Co-Authors: Alessandra Pontiroli, Pascal Simonet, Timothy Vogel, John Poté, Walter Wildi, Maria Ceccherini, Elizabeth Kay, Paolo Nannipieri, Jean-michel MonierAbstract:The long-term physical persistence and biological activity of transplastomic Plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified Plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, Plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the Plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic Plant DNA in soil. (C) 2010 Elsevier Masson SAS. All rights reserved.
-
Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco
Applied and Environmental Microbiology, 2009Co-Authors: Alessandra Pontiroli, Pascal Simonet, Timothy Vogel, Aurora Rizzi, Daniele Daffonchio, Jean-michel MonierAbstract:Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between Plants and bacteria. Plant DNA released during the degradation of Plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or Plant-associated bacteria could be in direct contact with Plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco Plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying Plant tissues, to develop a competence state, and to possibly acquire exogenous Plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413 (rbcL-Delta PaadA::gfp) carrying a promoterless aadA:: gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic Plant DNA. Opportunistic growth occurred on decaying Plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between Plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for Plant tissues.
-
Extracellular Plant DNA in Geneva groundwater and traditional artesian drinking water fountains
Chemosphere, 2009Co-Authors: John Poté, Pascal Simonet, Walter Rosselli, Walter Wildi, Patrick Mavingui, Elisabeth Navarro, Timothy M VogelAbstract:DNA, as the signature of life, has been extensively studied in a wide range of environments. While DNA analysis has become central to work on natural gene exchange, forensic analyses, soil bioremediation, genetically modified organisms, exobiology, and palaeontology, fundamental questions about DNA resistance to degradation remain. This paper investigated on the presence of Plant DNA in groundwater and artesian fountain (groundwater-fed) samples, which relates to the movement and persistence of DNA in the environment. The study was performed in the groundwater and in the fountains, which are considered as a traditional artesian drinking water in Geneva Champagne Basin. DNA from water samples was extracted, analysed and quantified. Plant gene sequences were detected using PCR amplification based on 18S rRNA gene primers specific for eukaryotes. Physicochemical parameters of water samples including temperature, pH, conductivity, organic matter, dissolved organic carbon (DOC) and total organic carbon (TOC) were measured throughout the study. The results revealed that important quantities of Plant DNA can be found in the groundwater. PCR amplification based on 18S rDNA, cloning, RFLP analysis and sequencing demonstrated the presence of Plant DNA including Vitis rupestris, Vitis berlandieri, Polygonum sp. Soltis, Boopis graminea, and Sinapis alba in the water samples. Our observations support the notion of Plant DNA release, long-term persistence and movement in the unsaturated medium as well as in groundwater aquifers.
-
Fate of transgenic Plant DNA in the environment
Environmental Biosafety Research, 2007Co-Authors: Alessandra Pontiroli, Pascal Simonet, Asa Frostegard, Timothy Vogel, Jean-michel MonierAbstract:This review addresses the possible ecological effects of transgenic Plants on micro-organisms in the field, hence, in the phytosphere and in the soil matrix. The important steps involved in the interaction between Plant DNA and bacteria and the factors that influence the horizontal gene transfer (HGT) process will be discussed. HGT is a process in which two partners are involved, even if indirectly. In the first section, aspects concerning bacteria, such as their physico-chemical, biological and genetic characteristics, are described. Parameters affecting transgenic DNA fate in the environment are described in the second section. Subsequently, terrestrial habitats are evaluated in terms of their capacity to favor horizontal gene transfer. Finally, we focused on several studies in order to evaluate possible perturbations of soil bacterial community composition due to cultivation of transgenic Plants in the field.
-
Detection of potential transgenic Plant DNA recipients among soil bacteria
Environmental Biosafety Research, 2007Co-Authors: Jean-michel Monier, Pascal Simonet, Elizabeth Kay, Dominique Bernillon, Aurélie Faugier, Oleksandra Rybalka, Yves Dessaux, Timothy VogelAbstract:The likelihood of gene transfer from transgenic Plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) Plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize Plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased Plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco Plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco Plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic Plant DNA recipients might be present in soil bacterial communities.
Timothy Vogel - One of the best experts on this subject based on the ideXlab platform.
-
Long-term persistence and bacterial transformation potential of transplastomic Plant DNA in soil
Research in Microbiology, 2010Co-Authors: Alessandra Pontiroli, Pascal Simonet, Timothy Vogel, John Poté, Walter Wildi, Maria Ceccherini, Elizabeth Kay, Paolo Nannipieri, Jean-michel MonierAbstract:The long-term physical persistence and biological activity of transplastomic Plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified Plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, Plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the Plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic Plant DNA in soil. (C) 2010 Elsevier Masson SAS. All rights reserved.
-
Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco
Applied and Environmental Microbiology, 2009Co-Authors: Alessandra Pontiroli, Pascal Simonet, Timothy Vogel, Aurora Rizzi, Daniele Daffonchio, Jean-michel MonierAbstract:Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between Plants and bacteria. Plant DNA released during the degradation of Plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or Plant-associated bacteria could be in direct contact with Plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco Plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying Plant tissues, to develop a competence state, and to possibly acquire exogenous Plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413 (rbcL-Delta PaadA::gfp) carrying a promoterless aadA:: gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic Plant DNA. Opportunistic growth occurred on decaying Plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between Plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for Plant tissues.
-
Fate of transgenic Plant DNA in the environment
Environmental Biosafety Research, 2007Co-Authors: Alessandra Pontiroli, Pascal Simonet, Asa Frostegard, Timothy Vogel, Jean-michel MonierAbstract:This review addresses the possible ecological effects of transgenic Plants on micro-organisms in the field, hence, in the phytosphere and in the soil matrix. The important steps involved in the interaction between Plant DNA and bacteria and the factors that influence the horizontal gene transfer (HGT) process will be discussed. HGT is a process in which two partners are involved, even if indirectly. In the first section, aspects concerning bacteria, such as their physico-chemical, biological and genetic characteristics, are described. Parameters affecting transgenic DNA fate in the environment are described in the second section. Subsequently, terrestrial habitats are evaluated in terms of their capacity to favor horizontal gene transfer. Finally, we focused on several studies in order to evaluate possible perturbations of soil bacterial community composition due to cultivation of transgenic Plants in the field.
-
Detection of potential transgenic Plant DNA recipients among soil bacteria
Environmental Biosafety Research, 2007Co-Authors: Jean-michel Monier, Pascal Simonet, Elizabeth Kay, Dominique Bernillon, Aurélie Faugier, Oleksandra Rybalka, Yves Dessaux, Timothy VogelAbstract:The likelihood of gene transfer from transgenic Plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) Plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize Plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased Plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco Plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco Plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic Plant DNA recipients might be present in soil bacterial communities.
-
Degradation and Transformability of DNA from Transgenic Leaves
Applied and Environmental Microbiology, 2003Co-Authors: M. Ceccherini, Timothy Vogel, J. Pote, E. Kay, V. Van, J. Marechal, G. Pietramellara, P. Nannipieri, Pascal SimonetAbstract:The fate of transplastomic (chloroplast genome contains the transgene) tobacco Plant DNA in Planta was studied when the Plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the Plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted Plant DNA), and the frequency that recipient bacteria can be transformed by Plant DNA were used to evaluate the quality and quantity of Plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying Plant tissues. Our results indicate that while most of the DNA will be degraded inside Plant cells, sufficient DNA persists to be released into the soil.