Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli.
A novel chromosome-plasmid hybrid bioluminescent reporter system (C-P reporter system) utilizing Photorhabdus luminescens luxCDABE genes has been constructed to monitor the expression of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) in enterohemorrhagic Escherichia coli (EHEC) in real time. The luxCDABE genes of P. luminescens have been cloned and divided into a luxCDAB cassette and a luxE gene. A promoter-less luxE gene introduced downstream from stx1 and from stx2 on EHEC chromosomes in single copies, and other luxCDAB genes were expressed on a multicopy number expression plasmid into the same cells. These Stx1- and Stx2-bioluminescent reporter strains expressed bioluminescence into bacteria cells when the expression of the promoter-less luxE gene was expressed in response to the promoter activity of stx1 and stx2, respectively. The expression levels of bioluminescence were identical to the production levels of Stx1 and Stx2 in the Stx1- and Stx2-bioluminescent reporter strains, and these strains produced both Stxs at the same respective levels as those of the parent EHEC strains. Using these reporter strains, we examined the profiles of Stx1 and Stx2 expression in EHEC. We found that production of both Stx1 and Stx2 in EHEC was enhanced upon contact with intestinal epithelial cells and within macrophages. However, the expression profiles between Stx1 and Stx2 in EHEC were different from each other under these conditions. Thus, these results suggested that this C-P reporter system is useful for determining the gene expression profile of bacteria.
Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba, 260-8670, Japan. tshimizu@faculty.chiba-u.jp
Takeshi
T
Shimizu
Yuko
Y
Ohta
Hiroyasu
H
Tsutsuki
Masatoshi
M
Noda
D001422:Bacterial Adhesion; D018938:Caco-2 Cells; D002876:Chromosomes, Bacterial / Q000378:metabolism; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008264:Macrophages / Q000382:microbiology; D020637:Photorhabdus / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*
D008164:Luminescent Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2