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Secretion of effector proteins in enteropathogenic Escherichia coli (EPEC) is mediated by a specialized type III secretion system whose components are encoded in the LEE1, LEE2 and LEE3 operons. Using cat transcriptional fusions and primer extension analysis, we determined that the LEE2 and LEE3 operons are expressed from two overlapping divergent promoters, whose expression is negatively regulated by flanking common upstream and downstream silencing regulatory sequences (SRS1 and SRS2). In the absence of either SRS1 or SRS2, expression of the LEE2 and LEE3 operons became independent of Ler, a positive regulatory protein encoded by the first gene of the LEE1 operon. Similarly, in the absence of the histone-like protein H-NS, expression from both promoters became Ler independent even if both SRSs were present. In addition, the efficient expression of both the LEE2 and the LEE3 promoters required PerC (BfpW), a protein coded by the third gene of the per (bfpTVW) locus, but only in the presence of the EAF plasmid. Our deletion analysis also showed that the negative regulation observed in the presence of ammonium or at temperatures above 37°C (e.g. 40°C) required the SRSs or elements located therein. In contrast, the negative regulation observed in LB or at temperatures below 37°C (e.g. 25°C) was still observed even in the absence of both SRSs and seems to act only on the promoters. Together, these results suggest that Ler acts as an antirepressor protein that overcomes the H-NS-mediated silencing on the LEE2/LEE3 divergent promoter region, which is probably caused by the formation of a repressing H-NS–nucleoprotein complex.