CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA-COLI O157-H7 STRAINSEXPRESSING FIREFLY LUCIFERASE AND GREEN FLUORESCENT PROTEIN AND THEIRUSE IN SURVIVAL STUDIES
Pm. Fratamico et al., CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA-COLI O157-H7 STRAINSEXPRESSING FIREFLY LUCIFERASE AND GREEN FLUORESCENT PROTEIN AND THEIRUSE IN SURVIVAL STUDIES, Journal of food protection, 60(10), 1997, pp. 1167-1173
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.