In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria
Answers
Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.
The tremendous adaptation potential of prokaryotes is mainly related to their ability to exchange genes by specific mechanisms such as conjugation, transduction, and transformation (22). The efficiency of such mechanisms during bacterial incorporation of genes from transgenic plants, and particularly those encoding antibiotic resistance, is difficult to assess (15), although the occurrence of such transfers under natural soil conditions would remain rare (3). In soil, in spite of the persistence of plant DNA, there would be relatively few naturally transformable bacteria (13, 17) and these prokaryotes would rarely find the required conditions to develop competence (4), thus apparently significantly reducing the probability of gene transfer. For those bacteria that have developed specific symbiotic or pathogenic relationships with plants, conditions for gene transfer could be favorable, as shown with the plant pathogen Ralstonia solanacearum (2). This bacterium multiplied in its host plant, disorganized tissues, and colonized the plant via the vascular tissue, leading to the development of a competence stage of active transformability in planta (2). Although bacteria-bacteria gene transfer occurred between R. solanacearum in planta, no gene transfer from the transgenic plant to the R. solanacearum was detected. This lack of detection was not necessarily due to the lack of transfer but was possibly due in part to the low transformation efficiency of R. solanacearum and the dilution of the transgene by the entire plant genome (3). The ratio between target versus non-target DNA sequences on which homologous recombination can occur was very low, thereby possibly preventing the integration mechanism necessary to produce a measurable number of transformants.
While the potential of plant environments to mediate plant-bacteria gene exchange has not been established, two recent events have increased the likelihood. Genetic engineering of the chloroplast genome led to a new generation of transgenic plants (21). These transplastomic plants were designed to prevent dissemination of the transgene via pollen and to increase the number of gene copies in the transgenic plant (8). Transplastomic plants harbor around 10,000 copies of the transgene per cell (1, 7), which can be compared to less than 10 for traditional and nuclear-modified transgenic plants.
The second event was the discovery that plants can also be colonized by soil bacteria which exhibit much higher transformation frequencies than R. solanacearum. This is the case for the opportunistic soil bacterium Acinetobacter sp. strain BD413, which was found to actively colonize R. solanacearum-infected plants and therein develop a competence state (12). Thus, given the development of transplastomic plants, we focused our efforts on determining whether Acinetobacter sp. could incorporate DNA sequences from pl
Answer:
Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.
The tremendous adaptation potential of prokaryotes is mainly related to their ability to exchange genes by specific mechanisms such as conjugation, transduction, and transformation (22). The efficiency of such mechanisms during bacterial incorporation of genes from transgenic plants, and particularly those encoding antibiotic resistance, is difficult to assess (15), although the occurrence of such transfers under natural soil conditions would remain rare (3). In soil, in spite of the persistence of plant DNA, there would be relatively few naturally transformable bacteria (13, 17) and these prokaryotes would rarely find the required conditions to develop competence (4), thus apparently significantly reducing the probability of gene transfer. For those bacteria that have developed specific symbiotic or pathogenic relationships with plants, conditions for gene transfer could be favorable, as shown with the plant pathogen Ralstonia solanacearum (2). This bacterium multiplied in its host plant, disorganized tissues, and colonized the plant via the vascular tissue, leading to the development of a competence stage of active transformability in planta (2). Although bacteria-bacteria gene transfer occurred between R. solanacearum in planta, no gene transfer from the transgenic plant to the R. solanacearum was detected. This lack of detection was not necessarily due to the lack of transfer but was possibly due in part to the low transformation efficiency of R. solanacearum and the dilution of the transgene by the entire plant genome (3). The ratio between target versus non-target DNA sequences on which homologous recombination can occur was very low, thereby possibly preventing the integration mechanism necessary to produce a measurable number of transformants.
While the potential of plant environments to mediate plant-bacteria gene exchange has not been established, two recent events have increased the likelihood. Genetic engineering of the chloroplast genome led to a new generation of transgenic plants (21). These transplastomic plants were designed to prevent dissemination of the transgene via pollen and to increase the number of gene copies in the transgenic plant (8). Transplastomic plants harbor around 10,000 copies of the transgene per cell (1, 7), which can be compared to less than 10 for traditional and nuclear-modified transgenic plants.
The second event was the discovery that plants can also be colonized by soil bacteria which exhibit much higher transformation frequencies than R. solanacearum. This is the case for the opportunistic soil bacterium Acinetobacter sp. strain BD413, which was found to actively colonize R. solanacearum-infected plants and therein develop a competence state (12). Thus, given the development of transplastomic plants, we focused our efforts on determining whether Acinetobacter sp. could incorporate DNA sequences from pl