In Situ Transfer of Antibiotic Resistance Genes from Transgenic (Transplastomic) Tobacco Plants to Bacteria

Abstract

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 plantbacteria gene exchange has not been established, two recent events have increased the likelihood. Genetic engineering of the chlorop...

Antibiotic-resistant soil bacteria in transgenic plant fields

“…This includes the long-term DNA persistence in soil, the heterogeneous soil structure favoring contact between DNA and bacteria, the prokaryotic origin of the plant transgene sequences that represent a specific risk for a facilitated integration in a bacterial genome by HGT as demonstrated under laboratory (41), and greenhouse conditions (39). In addition, in the Bt176 event that we investigated here, the bacterial promoter was introduced concomitantly with the antibiotic resistance gene that would facilitate its expression in a potential recipient.…”

“…Transgene transfer from plants to bacteria has been detected under greenhouse conditions with specifically selected donor and recipient organisms (39). Despite these experiments that simulated the environment, plant bacteria HGT events remain undetected under field conditions.…”

Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene

“…There are reports that bacteriophage and plasmid DNA, when fed to mice at very high levels, can later be detected in their cells (Schubbert et al 1998), but no data exist to demonstrate that plant DNA can be transferred into and be stably maintained or expressed in mammalian cells. There are some experimental data indicating the transfer of plant DNA into bacteria under laboratory conditions but only if homologous recombination is facilitated (Kay et al 2002). However, there is no evidence that the transgenic markers presently in use pose a health risk to humans or domestic animals.…”

Transplastomic plants for innovations in agriculture. A review

“…The overwhelming majority of experimental data on elucidating horizontal gene transfer with transplastomic plants comes from a circle around scientists affiliated with the University of Lyon in France (Kay et al 2002;Demanèche et al 2011). In a first series of experiments, transplastomic tobacco plants were colonized by naturally competent Acinetobacter baylyi cells that contained a plasmid without or with (pBAB2) tobacco chloroplast sequences incorporated to facilitate homologous recombination (Kay et al 2002).…”