Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli

To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane co nstitutes the receptor for intimin. The tir gene is encoded by the locus fo r enterocyte effacement (LEE), where it is flanked upstream by orf19 and do wnstream by the cesT and eae genes. With the use of a series of cat transcr iptional fusions and primer extension analysis, we confirmed that tir, cesT , and eae form the LEES operon, which is under the control of a promoter lo cated upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promot ers and that a sequence motif located between positions -204 and -157 was n eeded for the Ler-dependent activation of the tir operon. Sequence elements located between positions -204 and -97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions, Upon deletion of the upstream seq uences, the tir promoter was fully active even in the absence of Ler, indic ating that tir expression is subject to a repression mechanism that is coun teracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25 degreesC, suggesting that nega tive regulation also occurs at or downstream of the promoter. Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histo ne-like nucleoid structuring protein) on this gene.