Lack of detectable DNA uptake by bacterial gut isolates grown in vitro and by Acinetobacter baylyi colonizing rodents in vivo

¹ Norwegian Institute of Gene Ecology, Science Park, 9294 Tromsø, Norway
² Microbe Division, Japan Collection of Microorganisms, RIKEN Bioresource Center, Wako, Saitama 351-0198, Japan
³ Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden
⁴ Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway
⁵ Department of Pharmacy, Faculty of Medicine, University of Tromsø, 9037 Tromsø, Norway

Corresponding author: knielsen@farmasi.uit.no

Abstract

Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA containing a kanamycin resistance gene (nptII). Strain BD413 was found in various gut locations in germ-free mice at 10³-10⁵ CFU per gram GIT content 24–48 h after administration. However, subsequent DNA exposure of the colonized mice did not result in detectable bacterial transformants, with a detection limit of 1 transformant per 10³-10⁵ bacteria. Further attempts to increase the likelihood of detection by introducing weak positive selection with kanamycin of putative transformants arising in vivo during a 4-week-long feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) fed either purified DNA (50 µg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of germfree mice was also highly inhibitory to transformation of A. baylyi, indicating that microbially-produced nucleases are not responsible for the sharp 500- to 1 000 000-fold reduction of transformation frequencies seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was examined for competence expression in vitro, without yielding any transformants. In conclusion, model choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can be measured experimentally in the GIT. Our observations suggest the contents of the GIT shield or adsorb DNA, preventing detectable exposure of feed-derived DNA fragments to competent bacteria.