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Journal of Bacteriology, November 2000, p. 6490-6498, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of SepL of Enterohemorrhagic Escherichia coli

Andreas U. Kresse,1 Fabrizio Beltrametti,1 Astrid Müller,1 Frank Ebel,2 and Carlos A. Guzmán1,*

Vaccine Research Group, Department of Microbial Pathogenesis and Vaccine Research, Division of Microbiology, GBF-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany,1 and Unité de Génétique Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France2

Received 16 June 2000/Accepted 30 July 2000

The sepL gene is expressed in the locus of enterocyte effacement and therefore 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 sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions -5 and -36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn2+. Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933Delta sepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.

* Corresponding author. Mailing address: Vaccine Research Group, Department of Microbial Pathogenesis and Vaccine Research, Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: 49-531-6181558. Fax: 49-531-6181411. E-mail: cag{at}gbf.de.

Journal of Bacteriology, November 2000, p. 6490-6498, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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