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.

FIG. 1.

(A and A′) PCR-based control of the presence of the plastid aadA marker gene in the genome of Acinetobacter sp. strain BD413(pBAB2) transformants. Primers FGPaad1172 and FGPaad1554 (2) are complementary to part of the aadA gene and amplify a 382-bp-long DNA fragment. Lanes M contained 1-kb size marker ladders. Lanes A to D, controls with the templates sterile H₂ O (A), pLEP01plasmid DNA (B), plastid plant DNA (C), and recipient Acinetobacter sp. strain BD413(pBAB2) DNA (D). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. (B and B′) PCR-based control of the presence of the plastid-specific chimeric construction in the genome from Acinetobacter sp. strain BD413(pBAB2) transformants. Primer chloro1, which is complementary to part of the rbcL gene (a site for homologous recombination), was used in combination with FGPaad1554 (2), which is complementary to the part of the aadA gene, to amplify a 1.9-kbp-long DNA fragment. Lanes M contained 1-kb size marker ladders. Lanes A to D, controls with the templates sterile H₂ O (A), pLEP01 plasmid DNA (B), plastid plant DNA (C), and recipient Acinetobacter sp. strain BD413(pBAB2) DNA (D). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. (C and C′) Restriction fragment length polymorphism (RFLP) (enzyme RsaI) of PCR products resulting from positive amplifications with the primers chloro1 and FGPaad1554, which target the plastid chimeric construction. Lanes M contained 1-kb size marker ladders. Lanes B and C, controls with templates pLEP01 plasmid DNA (B) and plastid plant DNA (C). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. Lane N is a marker ladder containing a 123-bp marker. (D and D′) RFLP (restriction enzyme HaeIII) of PCR products resulting of amplifications with primers pA (5′-AGAGTTTGATCCTGGCTCAG-3′) and pH (5′-AAGGAGGTGATCCAGCGCCA-3′), which target the ribosomal genes. Lanes M contained 1-kb size marker ladders. Lanes A and B, controls with the templates sterile H₂O (A) and recipient Acinetobacter sp. strain BD413(pBAB2) DNA (B). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. The similarity of the patterns between the recipient strain and the spectinomycin-resistant clones indicate that they are all the same bacterium, Acinetobacter sp. strain BD413.

FIG. 1.

(A and A′) PCR-based control of the presence of the plastid aadA marker gene in the genome of Acinetobacter sp. strain BD413(pBAB2) transformants. Primers FGPaad1172 and FGPaad1554 (2) are complementary to part of the aadA gene and amplify a 382-bp-long DNA fragment. Lanes M contained 1-kb size marker ladders. Lanes A to D, controls with the templates sterile H₂ O (A), pLEP01plasmid DNA (B), plastid plant DNA (C), and recipient Acinetobacter sp. strain BD413(pBAB2) DNA (D). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. (B and B′) PCR-based control of the presence of the plastid-specific chimeric construction in the genome from Acinetobacter sp. strain BD413(pBAB2) transformants. Primer chloro1, which is complementary to part of the rbcL gene (a site for homologous recombination), was used in combination with FGPaad1554 (2), which is complementary to the part of the aadA gene, to amplify a 1.9-kbp-long DNA fragment. Lanes M contained 1-kb size marker ladders. Lanes A to D, controls with the templates sterile H₂ O (A), pLEP01 plasmid DNA (B), plastid plant DNA (C), and recipient Acinetobacter sp. strain BD413(pBAB2) DNA (D). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. (C and C′) Restriction fragment length polymorphism (RFLP) (enzyme RsaI) of PCR products resulting from positive amplifications with the primers chloro1 and FGPaad1554, which target the plastid chimeric construction. Lanes M contained 1-kb size marker ladders. Lanes B and C, controls with templates pLEP01 plasmid DNA (B) and plastid plant DNA (C). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. Lane N is a marker ladder containing a 123-bp marker. (D and D′) RFLP (restriction enzyme HaeIII) of PCR products resulting of amplifications with primers pA (5′-AGAGTTTGATCCTGGCTCAG-3′) and pH (5′-AAGGAGGTGATCCAGCGCCA-3′), which target the ribosomal genes. Lanes M contained 1-kb size marker ladders. Lanes A and B, controls with the templates sterile H₂O (A) and recipient Acinetobacter sp. strain BD413(pBAB2) DNA (B). Lanes 1 to 31, PCR products resulting from amplifications with DNA from Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in planta. Lane T, PCR products resulting from amplifications with DNA from one of the numerous Acinetobacter sp. strain BD413(pBAB2) spectinomycin-resistant clones after a gene transfer occurred in vitro. The similarity of the patterns between the recipient strain and the spectinomycin-resistant clones indicate that they are all the same bacterium, Acinetobacter sp. strain BD413.

FIG. 2.

Hybridization-based control of the presence of the plastid aadA marker gene in the genome of Acinetobacter sp. strain BD413(pBAB2) transformants. The oligoprobe consisted of the oligonucleotide FGPaad1554, which is complementary to part of the aadA gene. Dot blot hybridizations (standard protocol, hybridization temperature of 41°C) were conducted on plasmids extracted from transformants (Qiagen extraction protocol). Clone A2, positive-control plasmid pLEP01 (30 ng); clone A5, positive-control plastid plant DNA (250 ng); clone F8, negative-control plasmid pBAB2 (30 ng). Clones B1 to B8, C1 to C8, D1 to D8, and E1 to E7 correspond to hybridization on plasmid DNA from transformants of Acinetobacter sp.