Characterization of SepL of Enterohemorrhagic Escherichia coli

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 2 5 and 2 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 Mn 2 1 . 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 EDL933 D 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. labs Germany). Database searches and amino acid sequence anal- ysis were performed using the BLASTP 1 BEAUTY (4, 52), TBLASTN (46), PSORT (39), and TMPred (22) algorithms. b -Galactosidase assays. A fragment comprising the sepL promoter region was generated by PCR using the primers 1627-lac1 and 1627-lac2, which incorpo- rated additional Bam HI restriction sites. The PCR fragment was then digested with Bam HI and translationally fused to the lacZ gene of the promoter probe vector pUJ9TT (24), thereby generating plasmid pUJ2. To perform b -galactosi-dase assays, bacteria were grown until they reached the exponential growth phase and cultures were reinoculated to an optical density at 600 nm (OD 600 ) of 0.1 into the appropriate medium. To test the inﬂuence of oligoelements on gene expression, bacteria were grown in M9-glucose medium supplemented with either MgSO 4 (1, 7, and 30 mM), MgCl 2 (1, 7, and 30 mM), MnSO 4 (0.0033, 0.33, and 3.3 mM), CaCl 2 (0.01, 0.1, and 1 mM), FeSO 4 (0.25, 25, and 250 m M), or Fe(NO 3 ) 3 (1 mM). NH 4 Cl was added to nitrogen-free M9 medium at concen- trations of 0.5, 2, and 10 mM. Osmotic regulation was tested in minimal M9-glucose medium by the addition of NaCl or sucrose at ﬁnal concentrations of 10 or 430 mM. Samples were taken at different time intervals, the OD 600 was determined, and aliquots were removed and centrifuged at 8,000 3 g to recover bacterial pellets. The b -galactosidase assay was performed with the b -GAL reporter gene assay chemiluminescent kit (Boehringer GmbH, Mannheim, Ger-many) according to the supplier’s instructions, except that lysis was performed by resuspending bacteria in 500 m l of the supplied lysis solution supplemented with chloroform (20 m l) and 0.1% sodium dodecyl sulfate (SDS) (20 m l) and incu- bating them subsequently for 30 min at room temperature. The samples were measured using a Victor 1420 multilabel counter ﬂuorometer (EG&G WAL- LAC, Turku, Finland), and the results were normalized for the number of bacterial cells used. Nonspeciﬁc effects on b -galactosidase activity resulting from the composition of the media were ruled out by using adequate protein stan-dards. Primer extension analysis. Overnight bacterial cultures were grown in LB medium at 37°C for 30 or 60 min, and total RNA was extracted with the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the supplier’s instructions. To perform the primer extension analysis, the primer ORF1-PE mapping 34 bp downstream of the ATG start of sepL was end labeled with [ g - 32 P]dATP at 37°C for 40 min. The labeled primer was hybridized with 25 m g of RNA at 50°C for 20 min and extended wit h 1 U ofavian myeloblastosis virus reverse transcriptase (Promega, Madison, Wis.) at 42°C for 40 min (43). The primer extension products were analyzed on a sequencing gel using ladders generated with the same primer and the Deaza G/A T7 Sequencing Mixes Kit (Pharmacia Biotech, Pisca-taway, N.J.) as a reference.