FIG. 4.
Expression of tir requires the Ler protein. (A) The transcriptional activity directed by the tir-cat fusion in pTIR394 and the bfpA-cat fusion contained in pCAT232 was tested in EPEC strains E2348/69 (wild type), JPN15 (an EAF-minus derivative), and Δler (a ler in-frame deletion mutant of E2348/69) carrying or not carrying pKORF1 (ler+), as well as in E. coli K-12 strain MC4100 (wild type) carrying or not carrying either pKORF1 (ler+) or pCS-TVW (per/bfpTVW+). The CAT specific activity was determined from cells grown in DMEM at 37°C and harvested at an OD600 of 1.4. The data are the averages of results from at least three different experiments. Error bars indicate standard deviations. (B) Primer extension analysis of the tir-cat fusion in pTIR394 was carried out with total RNA extracted from samples obtained at an OD600 of 0.8 from the cultures described above. The primer extension reactions were performed as described in the legend to Fig. 2, using a primer specific for cat. The arrow on the left indicates the transcriptional start site of tir-cat.