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

Abstract

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 constitutes the receptor for intimin. The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes. With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located 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 promoters and that a sequence motif located between positions -204 and -157 was needed 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 sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25 degrees C, suggesting that negative 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 (histone-like nucleoid structuring protein) on this gene.

FIG. 1

tir, cesT, and eae constitute an operon. (A) Schematic representation of the organization of the orf19, tir, cesT, and eae genes and of the transcriptional fusions constructed to study their regulation. The horizontal arrows indicate the direction of transcription. Bent-tailed arrows denote the transcriptional start sites identified in this work (see the text). The sizes (in base pairs) of the intergenic regions are shown below the thick horizontal line. Plasmid denominations are indicated in the left-hand column below. The fragments cloned into the promoterless cat gene vector pKK232-8 are denoted by solid lines, and the cat gene is indicated by an open box at the end of each fragment. The −10/SacI label indicates the presence of a mutation that replaced the putative tir −10 promoter hexamer by a SacI restriction site. The right-hand column shows the CAT activity expressed by each fusion in EPEC E2348/69 grown in DMEM at 37°C. The data obtained for EPEC B171-8 are not shown, for simplicity, but rendered the same conclusions. The CAT specific activity was determined from cells harvested at an OD₆₀₀ of 1.4. The results reported are the averages ± standard deviations of data from at least four different experiments.

FIG. 2

Primer extension analysis of the tir, orf19, and cesT promoter regions. (A) Total RNA was obtained from culture samples of strains EPEC E2348/69 wild type (lane 1), JPN15 (pEAF cured) (lane 2), EPEC E2348/69Δler (lane 3), and EPEC E2348/69Δler carrying pKORF1 (lane 4) growing in DMEM at 37°C (OD₆₀₀ = 0.8). A primer specific for the tir structural gene was used, and primer extension was performed as described in Materials and Methods. A primer extension assay using a primer specific for the 16S rRNA gene was performed as a control. (B) Primer extension analysis was performed as described for panel A but with a primer specific for the orf19 structural gene. (C) Total RNA from EPEC E2348/69 carrying pCEST (cesT-cat fusion) and a cat-specific primer were used for primer extension reactions. The upper and bottom panels show the two extended products. Lanes G, A, T, and C correspond to the DNA sequence ladder obtained with the corresponding primer. The sequences spanning the transcription start site are shown, and the transcription start sites (in bold) are marked with arrows.

FIG. 3

Nucleotide sequences of the orf19-tir (A), rorf10-orf19 (r, reverse) (B), and tir-cesT (C) intergenic regions. The transcriptional start sites (+1) are indicated by the bent-tailed open-headed arrows. The bent-tailed filled-head arrows indicate the 5′ ends of the different tir-cat fusions. The predicted −10 and −35 promoter sequences are underlined. The sequences containing motifs potentially involved in Ler activation and ammonium-dependent repression, in negative regulation by an as-yet-unidentified repressor, and in repression at high temperature are indicated by solid, broken, and dotted underlines, respectively.

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.

FIG. 5

cis-acting elements involved in tir expression. (A) Schematic representation of the tir regulatory region and tir-cat fusions. Numbering is relative to the transcription start site, which is indicated by a bent-tailed arrow. Regions containing sequences potentially involved in Ler-dependent activation (subdivided boxes) or in negative regulation (shaded boxes) are indicated. EPEC strains E2348/69 and E2348/69Δler, as well as E. coli MC4100, were transformed with different tir-cat fusions contained in plasmids pTIR394, pTIR243, pTIR204, pTIR157, pTIR122, pTIR97, pTIR80, pTIR45, and pTIR22. The resulting strains were grown in DMEM at 37°C, and the CAT specific activity was determined from samples obtained at an OD₆₀₀ of 1.4. The plots of the activities determined from samples obtained along the growth curve at other OD₆₀₀ values showed the same pattern. Values are the averages of data from at least three different experiments; error bars indicate standard deviations. (B) Total RNA was obtained from EPEC E2348/69 carrying the tir-cat fusions in pTIR204, pTIR157, pTIR80, and pTIR45, and primer extension assays were performed as described in the legend to Fig. 2. The arrow on the left indicates the transcriptional start site of the tir-cat fusions, which corresponds to that identified for the wild-type gene.

FIG. 5

cis-acting elements involved in tir expression. (A) Schematic representation of the tir regulatory region and tir-cat fusions. Numbering is relative to the transcription start site, which is indicated by a bent-tailed arrow. Regions containing sequences potentially involved in Ler-dependent activation (subdivided boxes) or in negative regulation (shaded boxes) are indicated. EPEC strains E2348/69 and E2348/69Δler, as well as E. coli MC4100, were transformed with different tir-cat fusions contained in plasmids pTIR394, pTIR243, pTIR204, pTIR157, pTIR122, pTIR97, pTIR80, pTIR45, and pTIR22. The resulting strains were grown in DMEM at 37°C, and the CAT specific activity was determined from samples obtained at an OD₆₀₀ of 1.4. The plots of the activities determined from samples obtained along the growth curve at other OD₆₀₀ values showed the same pattern. Values are the averages of data from at least three different experiments; error bars indicate standard deviations. (B) Total RNA was obtained from EPEC E2348/69 carrying the tir-cat fusions in pTIR204, pTIR157, pTIR80, and pTIR45, and primer extension assays were performed as described in the legend to Fig. 2. The arrow on the left indicates the transcriptional start site of the tir-cat fusions, which corresponds to that identified for the wild-type gene.

FIG. 6

Effect of the growth medium, the presence of ammonium, and temperature on tir expression. EPEC E2348/69 derivatives carrying the tir-cat fusions described in the legend to Fig. 5 were grown at 37°C in DMEM, DMEM plus 20 mM ammonium sulfate, or LB (A) or in DMEM at 37, 25, or 40°C (B). CAT specific activities from samples obtained at an OD₆₀₀ of 1.4 were determined and plotted. The plots of the activities determined from samples obtained along the growth curve at other OD₆₀₀ values showed the same pattern. Values are the averages of data from at least three different experiments; error bars indicate standard deviations.

FIG. 7

Regulation of orf19. (A) Expression of orf19 requires the Ler protein. The transcriptional activity directed by the orf19-cat fusion in pORF19 was tested in different strains as described in the legend to Fig. 4A. (B) Effect of the growth medium, the presence of ammonium, and temperature on orf19 expression. EPEC E2348/69 carrying the orf19-cat fusion (pORF19) was grown under different conditions to determine CAT activity, as described in the legend to Fig. 6.