Expression of red-shifted green fluorescent protein by Escherichia coli O157:H7 as a marker for the detection of cells on fresh produce.
Escherichia coli O157:H7 was transformed with a plasmid vector red-shifted green fluorescence protein (pEGFP) to express red-shifted green fluorescence protein (EGFP) from Aequorea victoria. The EGFP expression among total cells and nonviable cells was determined at the cellular level by microscopic observation of immunostained and membrane-impermeable, dye-stained cultures, respectively. E. coli O157:H7 retained pEGFP during frozen storage at -80 degrees C. The percentage of EGFP expression was improved by repeated subculturing, reaching 83.4 +/- 0.1%, although the fluorescence intensity varied among cells. A low percentage of EGFP-expressing cells was nonviable. The percentage of EGFP decreased when the culture plate was kept at 4 degrees C, suggesting that some cells lost pEGFP during refrigeration. The storage of the culture suspension in sterile deionized water at 4 degrees C for 24 h reduced the percentage of EGFP expression, indicating that some EGFP was denatured. The application of EGFP as a marker for E. coli O157:H7 on green leaf lettuce, cauliflower, and tomato was evaluated using confocal scanning laser microscopy. EGFP-transformed cells were readily visible under confocal scanning laser microscopy on all produce types. The numbers of E. coli O157:H7 cells detected with EGFP were equivalent to those detected with immunostaining for green leaf lettuce and cauliflower but less for tomato. E. coli O157:H7 attached preferentially to damaged tissues of green leaf lettuce and tomato over intact tissue surfaces and to flowerets of cauliflower than to stem surfaces. EGFP can serve as a marker to characterize E. coli O157:H7 attachment on green leaf lettuce and cauliflower but may not be suitable on tomato.
Takeuchi
K
K
Center for Food Safety and Quality Enhancement, Department of Food Science and Technology, University of Georgia, Athens 30602-2106, USA.
Frank
J F
JF
D001937:Brassica / Q000382:microbiology; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D005511:Food Handling; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins; D006651:Histocytochemistry; D007202:Indicators and Reagents / Q000737:chemistry; D018545:Lactuca / Q000382:microbiology; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D018551:Solanum lycopersicum / Q000382:microbiology; D018613:Microscopy, Confocal; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D014169:Transformation, Bacterial; D014675:Vegetables / Q000382:microbiology*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins