Construction and use of an stx1 transcriptional fusion to gfp.
Shiga toxins (Stxs), also termed Vero toxins, are cytotoxic ribosome inactivating proteins that are produced by a number of gastrointestinal pathogens and that contribute to the severity of the associated diseases. In this work, we constructed and validated a transcriptional fusion of the stx1AB promoter to the gfp reporter gene. The cloned promoter region encompasses both the proximal and the distal promoter regions of stx1AB, mediating control by the host's iron-responsive Fur repressor and the Stx prophage's Q antiterminator protein, respectively. The probe was validated by demonstrating its responsiveness towards mitomycin C and EDTA, and the contribution of host and phage encoded factors could be separated by studying stx1 expression in either wild-type or isogenic lysogenic cells. Moreover, stx1AB expressing populations could be visualized by flow cytometry. The potential use of such a probe for non-destructive online detection of stx1AB expression and visualization of stx1AB expressing populations is further discussed.
Aertsen
Abram
A
Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Hevelee, Leuven, Belgium. abram.aertsen@agr.kuleuven.ac.be
Van Houdt
Rob
R
Michiels
Chris W
CW
D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D022622:Shiga Toxin 1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; D049452:Green Fluorescent Proteins