Fig. 1. Structure and transcription of the LEE4 operon.

A. Scheme depicting the structure of LEE4. ORFs are shown as grey boxes and intergenic regions as black lines. The two promoters previously proposed to drive sepL and espADB transcription in EHEC are indicated (black triangles) (Beltrametti et al, 1999). The arrows represent the primers (fLF and rLF) employed in RT-PCR in (B). The dashed line represents the probe used in Northern blot assays in (C).

B. Agarose gel electrophoresis of reverse transcription (RT) PCR products. EHEC strain Sakai was grown in LB and DMEM 1% glucose cultures. Cells were collected at mid-exponential phase (Mid-exp) and late exponential (Late-exp) for RNA extraction. Reverse transcription was performed with (+) or without (-) reverse transcriptase and PCR was performed with sepL and espF primers (fLF and rLF, respectively) (Table I). gDNA indicates that genomic DNA was used as a substrate for PCR with the same primers employed for RT-PCR. (M) λ DNA/HindIII marker.

C. Northern blot assay with a probe for espA. RNA was extracted from cultures grown in DMEM 1% glucose at different OD₆₀₀ readings. T1 to T6 correspond to the time points when samples were collected and are shown in Figure 2B. The bottom panel shows rRNAs of the analyzed samples.