More Information on HGT from Biotech Crops to Intestinal Bacteria

In an effort to assess the potential for enteric bacteria to be transformed with ingested DNA, Scott et al. (2000) conducted a series of experiments that utilized the Green Fluorescent Protein (GFP) experimental system. They first constructed a bacterial plasmid integration vector that included a GFP expression cassette and a 450 base pair region of homology to genomic sequences in a variety of bacteria. This construct should increase transformation frequencies to artificially high levels when compared to transgenic DNA sequences because the extensive amount of DNA sequence homology greatly favors integration through homologous recombination. Not surprisingly, this plasmid could transform strains of Lactococcus, Enterococcus and Streptococcus when the researchers used optimized in vitro methods to transform the bacteria. However, when the GFP-expressing Lactococcus was cultured in a simulated human gut environment inoculated with human fecal flora, the transformed Lactococcus exhibited impaired survival relative to non-transformed enteric bacteria. Therefore, HGT was not successful because the gene did not persist in the population even though it was taken up and integrated into the bacterial host genome.

The possibility of plasmid DNA being incorporated via a normal biological process into endogenous gut bacteria is minimized due to the non-conjugative nature of typical plasmids used in recombinant DNA laboratories (Hamer, 1977) and the low frequency with which unaided transformation (uptake of DNA) occurs. Furthermore, beyond the difficulty of unaided transformation is the general lack of stable incorporation of exogenous DNA into host genomes (Behr et al., 1989). Maniatis et al. (1982) calculated the probability of transferring plasmids into natural bacteria in the gut environment to be less than one in one million. However, the transfer of biotech plant genes should be significantly less. The Maniatis et al. (1982) calculation assumes the plasmid is free rather than incorporated into the plant genome. Movement of transgenes from ingested plant material to gut bacteria would require the transgene construct to be precisely removed from the plant genome and incorporated into the bacterial genome.

Mitten et al. (1996) evaluated the potential transfer of a transgene from ingested transgenic plant material to gut bacteria. They estimate a transformation frequency of 1 in 750 billion bacterial cells. More details on this work are discussed later in the section that focuses on HGT and antibiotic resistance markers.

A series of research projects, commissioned by the British Food Standards Agency (FSA), also concluded that HGT of DNA from food derived through biotechnology to human gut bacteria is extremely unlikely (FSA; project codes FSG01007, G010008G01010 and G01011). Study G010008, later published in the journal Nature Biotechnology (Netherwood et al., 2004), included human volunteers. Seven ileostomists were given a single meal containing soy flower from glyphosate resistant soy and the presence of the transgene was monitored over time in the intestine (small bowel). In another experiment of the same study, healthy volunteers, with an intact intestinal tract, were given the same meal and the fate of the transgene was monitored in the feces. After having passed the complete intestinal tract, no transgene was detectable by PCR. However, the transgene was detected in the stoma from the ileostomists. When intestinal microflora of the ileostomists, sampled before the start of the experiment, was cultivated, a small portion of the transgene was detectable at very low levels by PCR in samples from three of the seven volunteers. The authors concluded that gene transfer from transgenic soybean appears to have occurred before the experiment. The fact that attempts to isolate the bacteria supposedly harboring the transgene fragment failed, is explained with the uncultivable nature of those bacteria. In their conclusions, Netherwood et al. (2004) state the gene transfer events in their study would be highly unlikely to pose a risk to human health. This is because a non-functional gene fragment was transferred and because it was transferred to just a few of the millions of intestinal bacteria.

Again, it must be emphasized that enteric bacteria are exposed continuously to an extremely wide spectrum of DNA fragments. The component-sequences of transgenes are all sequences to which enteric bacteria have been exposed through non-biotech foods or in accompanying microflora. Given the high numbers of foreign bacteria and viruses in this intestinal mixture at any one time, and the combinatorial genetic "mixing-and-matching" that normally occurs among prokaryotes and between prokaryotes and viruses, there is likely little new that is being added to this DNA mix from biotechnology. This led Schubbert et al. (1994) to state:

"This barrage of linear DNA fragments, i.e. of recombinationally highly active DNA fragments, in nature should mitigate any concerns that one might have had in the past about biological consequences of experiments carried out with recombinant DNA over the course of the past two decennia."