Technical Abstract: Some strains of Pseudomonas aeruginosa produce R-type pyocins, which are high molecular weight phage tail-like protein complexes that have bactericidal activity against other Pseudomonas strains. These particles recognize and bind to bacterial surface structures via tail fibers; their primary spectrum determinant. R-type pyocins kill the cell by contracting a sheath-like structure and inserting their hollow core through the cell envelope, resulting in dissipation of the cellular membrane potential. We have re-targeted an R-type pyocin to E. coli O157:H7 by fusing a "tail spike" protein from an O157-specific phage, phiV10, to the pyocin tail fiber. The phiV10 tail spike protein recognizes and degrades the O157 lipopolysaccharide. This engineered pyocin, termed AVR2-V10, is sensitive and specific, killing 100% of diverse E. coli O157:H7 isolates but no other serotypes tested. AVR2-V10 can kill E. coli O157:H7 on beef surfaces, making it a candidate agent for eliminating this pathogen from food products. All rare AVR2-V10-resistant mutants isolated and examined have lost the ability to produce O157 antigen and are expected to have compromised virulence. In addition, E. coli O157:H7 exposed to and killed by AVR2-V10 do not release shiga toxin, as is often the case with many antibiotics, suggesting potential therapeutic applications. The demonstration that a novel R-type pyocin can be created in the laboratory by fusing a catalytic Podoviridae tail spike to a Myoviridae tail fiber is evidence that the plasticity observed among bacteriophage tail genes can, with modern molecular techniques, be exploited to produce non-natural, targeted antimicrobial agents.