Characterization of sept of enterohemorrhagic Escherichia coli

The sept gene is expressed in the locus of enterocyte effacement and theref ore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sept gene of the enterohemorrhagic Escherichi a coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sept is transcribed monocistronically and independently from the esp operon located downstream, which codes for the s ecreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sept start c odon. The analysis of the upstream regions led to the identification of can onical promoter sequences between positions -5 and -36. Translational fusio ns using lacZ as a reporter gene demonstrated that sept is activated in the exponential growth phase by stimuli that are characteristic for the intest inal niche, e.g., a temperature of 37 degreesC, a nutrient-rich environment , high osmolarity, and the presence of Mn2+. Protein localization studies s howed that Sept was present in the cytoplasm and associated with the bacter ial membrane fraction. To analyze the functional role of the Sept protein d uring infection of eukaryotic cells, an in-frame deletion mutant was genera ted. This sept mutant was strongly impaired in its ability to attach to HeL a cells and induce a local accumulation of actin. These defects were partia lly restored by providing the sept gene in trans. The EDL933 Delta sepL mut ant also exhibited an impaired secretion but not biosynthesis of Esp protei ns, which was fully complemented by providing sept in trans. These results demonstrate the crucial role played by Sept in the biological cycle of EHEC .