Colicinogenic maize: Transgenic analysis and effectiveness against pathogenic Escherichia coli O157:H7

Escherichia coli O157:H7 is a highly virulent food-borne pathogen that asymptomatically inhabits the digestive tract of cattle. Contamination occurs during meat slaughtering and processing, and manure contamination of water. Annually, this pathogen causes numerous outbreaks of food-borne illness. A transgenic maize line was developed that expressed a protein, colicin E7, previously shown to inhibit E. coli O157:H7. To verify expression in plant cells the full length colicin E7 gene driven by CaMV35S promoter was transformed into Hill maize callus by biolistic bombardment resulting in 77 independent events. 84% of the samples contained the colicin E7 gene and 48% of these samples transcribed colicin E7. An activity assay verified that callus protein extract reduced approximately 50% of diluted E. coli O157:H7 cultures, confirming expression of the colicin E7 protein. Two groups of maize lines were developed by biolistic bombardment; one contained the colicin E7 gene driven by the CaMV35S promoter, designated 265. The second contained the colicin E7 plus the immunity E7 gene driven by the CaMV35S promoter, designated 266. The immunity E7 gene produces a protein that binds to colicin E7 protecting the producing organism. Nine events were developed for the 265 group representing 86 plants and eleven events were developed for the 266 group representing 66 plants. PCR and Southern blotting results indicated integration of the colicin E7 gene into the maize genome, with unique insertion patterns ranging from two to ten copies. Approximately 50% of the 265 plants and nearly 80% of the 266 plants were positive for colicin E7 transcription. Western analysis using colicin E7-specific antibodies determined 27 positive plants for the two groups, 14 from the 265 group and 13 from the 266 group. Protein extracts of seven maize plants significantly inhibited E. coli O157:H7 cultures (p<0.05). These results document the development of novel transgenic maize lines that may be capable of producing colicin E7; further research will determine their effectiveness to inhibit E. coli O15:H7 in the digestive tract of cattle.