Ler is a negative autoregulator of the LEE1 operon in enteropathogenic Escherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes.

FIG. 1.

Schematic representation of the LEE1 regulatory region and of the gfp transcriptional fusions used to determine the minimal regulatory region of LEE1. The transcriptional start site of the LEE1 operon is marked +1, and the translational start site of Ler is marked +174. The plasmids containing the gfp fusions are indicated. The values to the right of each fusion indicate the fluorescence intensity, emitted by EPEC bacteria containing the corresponding fusion. These bacteria were grown at 37°C in DMEM to an OD₆₀₀ of 0.3. Fluorescence intensity was measured from 10,000 bacteria by flow cytometry, and a mean result for three independent experiments is presented with the standard deviation shown in parentheses.

FIG. 2.

Specific repression of the LEE1 promoter by Ler overexpression. (A) Plasmids pTU312, pSA11, and pSA8, carrying gfp under the regulation of the LEE1 promoter (PLEE1), the tac promoter (Ptac), or the oxyS promoter (PoxyS), respectively, were transformed into EPEC bacteria containing (+) or lacking (−) a compatible plasmid (pTU14), expressing Ler from Ptac. Cultures were grown to an OD₆₀₀ of 0.13, and IPTG (1 mM) was added for about 2.5 h to induce Ptac. The PoxyS promoter was activated by adding hydrogen peroxide (1 mM) to cultures at an OD₆₀₀ of 0.27 for 15 min. The level of gfp expression from 10,000 bacteria was determined by flow cytometry, and mean results for three independent experiments are shown. Vertical lines on the bars indicate standard errors. (B) Effect of ectopically expressed Ler on the expression of a chromosomal PLEE1-lacZ transcriptional fusion in E. coli K-12 MC4100. Ptac-ler was induced by IPTG as described for panel A. The expression levels are the mean values for three experiments, with vertical lines indicating the standard errors. (C) EPEC bacteria encoding chromosomal Ler-6His (EPEC ler-6his) containing (+) or lacking (−) pTU14 (expressing ler) were grown to an OD₆₀₀ of 0.35. The bacteria were then harvested and lysed. A portion of the crude extract was subjected to SDS-polyacrylamide gel electrophoresis and Western analysis using anti-Ler, anti-DnaK, anti-Tir, or anti-EscJ antibodies. 6His-Ler was precipitated from the remaining crude extract by use of Talon cobalt beads, eluted, and analyzed by Western blotting, using anti-Ler antibody. The antibodies, used for the development of the blots, are indicated above each blot, and arrows indicate the corresponding proteins. Molecular weights (in thousands) are indicated. The specificity of the anti-Ler antibody was verified. The antibody did not react with a band corresponding to Ler in an extract of the EPEC ler::kan mutant, and preimmune antiserum did not react with a band corresponding to Ler in an extract of wild-type EPEC (data not shown).

FIG. 3.

(A) Schematic representation of the two bicistronic operons in pIR1Ler and pIR1Ler(L29R). (B and C) Expression of gfp by the EPEC ler::kan mutant (B) or E. coli K-12 W3110 (C), containing pIR1 (dotted line), pIR1Ler (thin line), or pIR1Ler(L29R) (thick line). The experiments shown in panels B and C were repeated several times, and the results of typical experiments are shown. In the inset in panel B, the levels of Ler and Ler(L29R) in the EPEC ler::kan mutant containing pIR1Ler and pIR1Ler(L29R) are shown by use of a Western blot developed with anti-Ler antibody. (D) Stability of Ler and Ler(L29R). EPEC ler::kan mutant bacteria containing either pIR1Ler or pIR1Ler(L29R) were grown in DMEM, at 37°C, to an OD₆₀₀ of 0.3. Chloramphenicol was then added to the culture (time zero), and levels of Ler and Ler(L29R) at different time points posttreatment were determined by Western blotting using anti-Ler antibody. To achieve similar initial amounts of Ler and Ler(L29R), extracts containing Ler(L29R) were diluted fivefold before the gel was loaded.

FIG. 4.

In vitro binding of Ler to the promoter region of LEE1 and LEE2-LEE3. (A) A gel mobility shift assay was carried out using purified Ler and three different DNA fragments: LEE1 fragment −173+11, containing the LEE1 regulatory region; LEE1 fragment +26+270, a negative control DNA; and LEE2-LEE3 fragment +117−288 containing the regulatory region of the LEE2-LEE3 promoters (see Materials and Methods for details). The DNAs were mixed with twofold-increasing concentrations of Ler. The numbers above the lanes indicate nanomolar Ler concentrations. (B) The DNA-protein interaction with LEE1 fragment −173+11 and LEE2-LEE3 fragment +117−288 was quantified by phosphorimaging, and the results were plotted. The corresponding fragment is indicated above each graph. (C) DNase I footprinting analysis of LEE1 fragment −173+11. DNA was mixed and incubated with the indicated amounts of Ler or IHF prior to the DNase I treatment. The vertical line on the left side indicates the DNA region protected by Ler. Vertical lines on the right side indicate the DNA regions protected by IHF and the −35 sequence. The corresponding nucleotide sequence is shown in the lanes labeled G and A.

FIG. 5.

Binding of Ler and Ler(L29R) to the LEE1 regulatory region in vivo. (A) EPEC wild-type (wt) and EPEC ler::kan mutant bacteria were grown in DMEM at 37°C to an OD₆₀₀ of 0.35. The cultures were then used for ChIP analysis with anti-Ler antibody. Serial 2-fold dilutions of immunoprecipitated DNA (lanes 1 to 5) or 10-fold dilutions of the total input DNA (lanes 6 to 9) were used as templates for PCR. The reactions utilized primers specific to sequences located upstream of the LEE1 promoter (LEE1 regulatory region), primers specific to a region downstream of the LEE1 regulatory region, and primers specific to sequences located outside the LEE (see Materials and Methods for details). The PCR products were separated on 5% polyacrylamide gels. (B). E. coli K-12 W3110 bacteria containing pIR1Ler or pIR1Ler(L29R) were grown and subjected to ChIP analysis, as described above. The 1:5 (lane 1), 1:25 (lane 2), and 1:125 (lane 3) dilutions of immunoprecipitated DNA were subjected to PCR using primers specific to the LEE1 regulatory region. The PCR products were separated on 5% polyacrylamide gels.

FIG. 6.

Effect of autorepression on the stability of the LEE1 promoter. Images of gfp expression via the PLEE1 by EPEC ler::kan mutant bacteria producing Ler or Ler(L29R) are shown. The level of gfp expression within individual bacteria (n = 600) was determined. The distribution of gfp expression levels is plotted below each image for EPEC ler::kan/pIR1Ler (black bars) and EPEC ler::kan/pIR1Ler(L29R) (gray bars). The calculated coefficient of variation (CV; standard deviation divided by the mean) is indicated.