Hostname: page-component-78c5997874-j824f Total loading time: 0 Render date: 2024-11-19T04:32:50.775Z Has data issue: false hasContentIssue false
  • English
  • Français

Leaf-feeding larvae of Manduca sexta (Insecta, Lepidoptera) drastically reduce copy numbers of aadA antibiotic resistance genes from transplastomic tobacco but maintain intact aadA genes in their feces

Published online by Cambridge University Press:  26 October 2007

Nicole Brinkmann  and
Christoph C. Tebbe
Nicole Brinkmann
Affiliation:
Institute of Agroecology, Federal Agricultural Research Centre (FAL), Bundesallee 50, 38116 Braunschweig, Germany
Christoph C. Tebbe
Affiliation:
Institute of Agroecology, Federal Agricultural Research Centre (FAL), Bundesallee 50, 38116 Braunschweig, Germany

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.

The objective of this study was to evaluate the effect of insect larval feeding on the fate and genetic transformability of recombinant DNA from a transplastomic plant. Leaves of tobacco plants with an aadA antibiotic resistance gene inserted into their chloroplast genome were incubated with larvae of the tobacco hornworm Manduca sexta (Lepidoptera). The specifically designed Acinetobacter strain BD413 pBAB2 was chosen to analyze the functional integrity of the aadA transgene for natural transformation after gut passages. No gene transfer was detected after simultaneous feeding of leaves and the Acinetobacter BD413 pBAB2 as a recipient, even though 15% of ingested Acinetobacter BD413 cells could be recovered as viable cells from feces 6 h after feeding. Results with real-time PCR indicated that an average of 98.2 to 99.99% of the aadA gene was degraded during the gut passage, but the range in the number of aadA genes in feces of larvae fed with transplastomic leaves was enormous, varying from 5 × 106 to 1 × 109 copies.g-1. DNA extracted from feces of larvae fed with transplastomic leaves was still able to transform externally added competent Acinetobacter BD413 pBAB2in vitro. Transformation frequencies with concentrated feces DNA were in the same range as those found with leaves (10-4–10-6 transformants per recipient) or purified plasmid DNA (10-3–10-7). The presence of functionally intact DNA was also qualitatively observed after incubation of 30 mg freshly shed feces directly with competent Acinetobacter BD413 pBAB2, demonstrating that aadA genes in feces have a potential to undergo further horizontal gene transfer under environmental conditions.

Keywords

transplastomic plantsaadA genenatural transformationAcinetobacterinsect larvae
Type
Research Article
Information
Environmental Biosafety Research , Volume 6 , Issue 1-2: Thematic Issue on Horizontal Gene Transfer , January 2007 , pp. 121 - 133
Copyright
© ISBR, EDP Sciences, 2007

References

Bell, RA, Joachim, FG (1976) Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Annals Entomol. Soc. America 69: 365–373 CrossRef
Bennett, PM, Livesey CT, Nathwani D, Reeves DS, Saunders JR, Wise R (2004) An assessment of the risks associated with the use of antibiotic resistance genes in genetically modified plants: report of the Working Party of the British Society for Antimicrob. Chemother. J. Antimicrob. Chemother. 53: 418–431 CrossRef
Berenbaum MR (1980) Adaptive significance of the midgut pH in larval lepidoptera. Am. Nat. 115: 138–146
Bertolla F, Simonet P (1999) Horizontal gene transfers in the environment: natural transformation as a putative process for gene transfers between transgenic plants and microorganisms. Res. Microb. 150: 375–384
Bertolla, F, Frostegard, A, Brito, B, Nesme, X, Simonet, P (1999) During infection of its host, the plant pathogen Ralstonia solanacearum naturally develops a state of compentence and exchanges genetic material. Mol. Plant Microbe Interact. 12: 467–472 CrossRef
Chambers, PA, Duggan, PS, Heritage, J, Forbes, JM (2002) The fate of antibiotic resistance marker genes in transgenic plant feed material fed to chickens. J. Antimicrob. Chemother. 49: 161–164 CrossRef
Coddington EJ, Chamberlin ME (1999) Acid/base transport across the midgut of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 45: 493–500
Daniell, H, Khan, MS, Allison, L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7: 84–91 CrossRef
de Vries J, Heine M, Harms K, Wackernagel W (2003) Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp. Appl. Environ. Microbiol. 69: 4455–4462
Deni J, Message B, Chioccioli M, Tepfer D (2005) Unsuccessful search for DNA transfer from transgenic plants to bacteria in the intestine of the tobacco horn worm Manduca sexta. Transgenic Res. 14: 207–215
Dow JAT (1984) Extremely high pH in biological systems: a model for carbonate transport. American J. Physiol. 246: 633–635
Dow JAT (1992) pH gradients in lepidopteran midgut. J. Experimental Biol. 172: 355–375
Dröge M, Pühler A, Selbitschka W (1998) Horizontal gene transfer as a biosafety issue: A natural phenomenon of public concern. J. Biotechnol. 64: 75–90
Duggan, PS, Chambers, PA, Heritage, J, Forbes, JM (2000) Survival of free DNA encoding antibiotic resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage effluent. FEMS Microbiol. Lett. 191: 71–77 CrossRef
Duggan PS, Chambers PA, Heritage J, Forbes JM (2003) Fate of genetically modified maize DNA in the oral cavity and rumen of sheep. British J. Nutrition 89: 159–166
Evans HE (1985) The tobacco hornworm. In The Pleasures of Entomology, Smithsonian Instiution, pp 145–156
Forsman, A, Ushameckis, D, Bindra, A, Yun, Z, Blomberg, J (2003) Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract. Mol. Genet. Genomics 270: 362–368 CrossRef
Gebhard F, Smalla K (1998) Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol. 64: 1550–1554
Giordana B, Leonardi MG, Casartelli M, Consonni P, Parenti P (1998) K+-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions. Am. J. Physiol.-Reg. I 274: 1361–1371
Heritage J (2004) The fate of transgenes in the human gut. Nature Biotechnol. 22: 170–172
Heritage J (2005) Transgenes for tea? Trends Biotechnol. 23: 17–21
Jelenic S (2003) Controversy associated with the common component of most transgenic plants – kanamycin resistance marker gene. J. Faculty Food Technol. Biotechnol. 41: 183–190
Kay, E, Vogel, TM, Bertolla, F, Nalin, R, Simonet, P (2002) In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria. Appl. Environ. Microbiol. 68: 3345–3351 CrossRef
Kharazmi M, Sczesny S, Blaut M, Hammes WP, Hertel C (2003) Marker rescue studies of the transfer of recombinant DNA to Streptococcus gordonii in vitro, in foods and gnotobiotic rats. Appl. Environ. Microbiol. 69: 6121–6127
Lorenz, MG, Wackernagel, W (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58: 563–602
Magrini V, Creighton C, White D, Hartzell PL, Youderian P (1998) The aadA gene of plasmid R100 confers resistance to spectinomycin and streptomycin in Myxococcus xanthus. J. Bacteriol. 180: 6757–6760
Martin-Orue, SM, O'Donnell, AG, Arino, J, Netherwood, T, Gilbert, HJ, Mathers, JC (2002) Degradation of transgenic DNA from genetically modified soya and maize in human intestinal simulations. British J. Nutrition 87: 533–542 CrossRef
McAllan, AB (1980) The degradation of nucleic acids in and the removal of breakdown products from the small-intestines of steers. British J. Nutrition 44: 99–112 CrossRef
Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA, Flint HJ (1999) Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl. Environ. Microbiol. 65: 6–10
Moffett D, Cummings S (1994) Transepithelial potential and alkalization in an in situ preparation of tobacco hornworm (Manduca sexta) midgut. J. Experimental Biol. 194: 341–345
Netherwood T, Martin-Orue SM, O'Donnell AG (2004) Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nature Biotechnol. 22: 204–209
Nielsen KM, Bones AM, Smalla K, van Elsas JD (1998) Horizontal gene transfer from transgenic plants to terrestrial bacteria - a rare event? FEMS Microbiol. Rev. 22: 79–103
Nielsen KM, van Elsas JD, Smalla K (2000) Transformation of Acinetobacter sp. strain BD413 (pFG4 $\rm \Delta $ nptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants. Appl. Environ. Microbiol. 66: 1237–1242
Paget E, Lebrun M, Freyssinet G, Simonet P (1998) The fate of recombinant plant DNA in soil. European J. Soil Biol. 34: 81–88
Palka-Santini M, Schwarz-Herzke B, Hosel M, Renz D, Auerochs S, Brondke H, Doerfler W (2003) The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins. Mol. Gen. Genomics 270: 201–215
Palmen R, Vosman B, Buijsman P, Breek CKD, Hellingwerf KJ (1993) Physiological characterization of natural transformation in Acinetobacter calcoaceticus. J. Gen. Microbiol. 139: 295–305
Raw JD (1983) Biochemistry, Harper and Row Publishers, New York, Inc, p 358
Sandvang D (1999) Novel streptomycin and spectinomycin resistance gene as a gene cassette within a class 1 integron isolated from Escherichia coli. Antimicrob. Agents Chemotherapy 43: 3036–3038
Schubbert R, Hohlweg U, Renz D, Doerfler W (1998) On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus. Mol. Gen. Genet. 259: 569–576
Schubbert R, Renz D, Schmitz B, Doerfler W (1997) Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. USA 94: 961–966
Snyder MJ, Walding JK, Feyereisen R (1994) Metabolic fate of the allelochemical nicotine in the tobacco hornworm Manduca sexta. Insect Biochem. Mol. Biol. 24: 837–846
Tebbe, CC, Vahjen, W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59: 2657–2665
Waterhouse DF (1949) The hydrogen ion concentration in the alimentary canal of larval and adult Lepidoptera. Australian J. Sci. Res. Ser. B 132 428–437
Wilcks A, van Hoek AHAM, Joosten RG, Jacobsen BBL, Aarts HJM (2004) Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation. Food Chem. Toxicol. 42: 493–502
Wink M, Theile V (2002) Alkaloid tolerance in Manduca sexta and phylogenetically related sphingids (Lepidoptera: Sphingidae). Chemoecology 12: 29–46
Yuan YJ, Lu ZX, Wu N, Huang LJ, Lü FX, Bie XM (2005) Isolation and preliminary characterization of a novel nicotine-degrading bacterium, Ochrobactrum intermedium DN2. Int. Biodeter. Biodegr. 56: 45–50