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.
Center for Food Safety and Quality Enhancement, Department of Food Science and Technology, University of Georgia, Athens 30602-2106, USA.
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Frank
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