An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation

Abstract

Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicinresistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 10 9 exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 10 3 cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal Acinetobacter DNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown.

An overview of general features of risk assessments of genetically modified crops

“…Although some DNA survived in laboratory-created environments that simulated human or animal gastrointestinal tracts, complementary research has concluded that the likelihood of functioning DNA being taken up by bacteria in the human or animal gut is extremely low and will be dependent upon rDNA and the chromosomes of any naturally competent bacteria in the gut sharing regions of identical sequences (Flint et al 2002;Nordgård et al 2007). Similar recent studies with insects (Brinkmann and Tebbe 2007; Ray et al 2007) also support this conclusion. Thus, breakdown of DNA in the gut, combined with the breakdown of the DNA due to food processing, reduces the risk of dissemination (Van den Eede et al 2004).…”

Horizontal Gene Exchange in Environmental Microbiota

“…The occurrence of conjugal plasmid transfer and transduction was also observed in the flea and house fly gut (Hinnebusch et al, 2002; Petridis et al, 2006; Akhtar et al, 2009). An assessment of natural transformability of bacteria in the insect gut, however, failed to detect any transformation event, even with the use of A. baylyi strain BD413 as a recipient (Ray et al, 2007). …”

Acinetobacter baylyi ADP1: Transforming the choice of model organism

“…The spread of transgenic plant DNA has been observed from decaying leaves, by simulated rainwater, in field plots, from roots, and via pollen (21,22). While efforts to detect HGT to A. baylyi in the gastrointestinal tracts of insects and animals were unsuccessful, digested transgenic plant tissue in insect fecal matter remains capable of transforming A. baylyi (23)(24)(25)(26). These studies inform continued assessment of HGT and provide insights into ways to design genetically modified organisms to decrease the risk of transgene dissemination.…”