Horizontal Gene Transfer of E. coli by direct DNA uptake from extracellular liquid phase

The "fate of naked DNA" or "environmental DNA (eDNA)" from genetically engineered (GE) biomasses in relation to possible horizontal gene transfer (HGT) is an emerging area of biosafety research. Using a broad-host plasmid (pAM5409) as a mimic of eDNA containing a yellow fluorescent protein gene (yfp) and conferring spectinomycin/streptomycin (Sp/Sm) resistance, we incubated each of the E. coli strains DH5, DH10B and K12 with the plasmid eDNA to evaluate HGT through direct uptake of plasmid DNA from extracellular liquid phase. According to experimental observations, the HGT can occur within 1 day of donor (eDNA) and recipient cells liquid incubation in E. coli strains DH5 and DH10B, but not in K12 which has its intact genes encoding restriction enzymes and endonucleases. HGT occurrences were observed by analyzing colony forming units (CFU) expression of yellow fluorescent protein (YFP). The HGT frequency by direct uptake of plasmid DNA from the liquid phase was determined to be 1.2 x 10-8 per cell day for both DH5 and DH10B. The frequency of HGT was slightly higher than that of spontaneous mutation for natural antibiotic resistance development that was estimated to be 4.8 x 10-9 per cell day. Extension of the liquid eDNA and recipient cells incubation time from 1 day to 3 and 5 days did not increase the HGT frequency under the current experimental conditions. Despite K12 failing to form CFUs following exposure to eDNA, this does not imply K12 did not uptake eDNA; eDNA acquired could have been fragmented by K12 restriction enzymes into non-functional oligomers incapable of HGT.