CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA-COLI O157-H7 STRAINSEXPRESSING FIREFLY LUCIFERASE AND GREEN FLUORESCENT PROTEIN AND THEIRUSE IN SURVIVAL STUDIES

The firefly (Photinus pyralis) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) ge ne on plasmid vector pGFP were introduced into strains of Escherichia coli 0157:H7. The recombinant E. coli strains were indistinguishable f rom their parent strains in biochemical and immunological assays and i n a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strain s. At 37 degrees C all of the recombinant strains maintained the vecto rs and expressed luciferase and the green fluorescent protein when gro wn both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombina nts containing pGFP emitted bright green fluorescence when excited wit h UV light and the addition of any other proteins, substrates, or cofa ctors was not required. The green fluorescent protein-expressing E. co li 0157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organi sm declined to undetectable levels in 24 days at refrigeration tempera ture while in orange juice the strains survived with only small decrea ses in number during the 24-day sampling period. These recombinant E. coli 0157:H7 strains, containing readily identifiable and stable marke rs, could be useful as positive controls in microbial assays as well a s in studies monitoring bacterial survival and the behavior of E. coli 0157:H7 in foods and in a food processing environment.