Mutational analysis of the locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic Escherichia coli

Abstract

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) comprises a cluster of operons encoding a type III secretion system and related proteins, all of which are essential for bacterial colonization of the host intestines. The LEE1 operon encodes Ler, which positively regulates many EPEC and EHEC virulence genes located in the LEE region and elsewhere in the chromosome. In addition, Ler is a specific autorepressor of LEE1 transcription. To better understand the function of Ler, we screened for Ler mutants defective in autorepression. We isolated 18 different point mutations in Ler, rendering it defective in autorepression and in DNA binding. Among these mutants were those defective in positive regulation as well as in autorepression, dominant-negative mutants, and a mutant deficient in oligomerization. Importantly, a group of Ler autorepression mutants complemented an EPEC ler deletion mutant for transcription activation in a dosage-dependent manner, suggesting that Ler and possibly other autorepressors have an intrinsic compensatory mechanism that enables them to sustain mutations. In addition, the phenotypes of the different mutants identified by the screen define a novel domain in Ler that is required for oligomerization.

FIG. 1.

Identification and initial characterization of 18 ler mutants in autorepression. (A) The pGY1 plasmid, harboring a ler transcriptional fusion to gfp under the control of the native ler promoter (P_LEE1), was introduced into a mutS mismatch-repair-deficient strain. The mutated plasmids were recovered and introduced into an EPEC ler::kan strain, and mutants in autorepression were identified as fluorescent colonies. Eighteen mutants were further analyzed. These mutations (B, marked in red) are scattered throughout the Ler amino acid sequence. The predicted coiled-coil and DNA binding motifs are in yellow and light blue, respectively. The core DNA binding domain is underlined. (C) The 18 mutants were reconstructed in pGY1 and the expression of green fluorescent protein, representing the transcription from P_LEE1, was measured at late exponential-growth phase. (D) Protein levels of the Ler mutants were analyzed using immunoblot analysis with an anti-Ler antibody.

FIG. 2.

DNA binding by the Ler autorepression-defective mutants. The binding of Ler mutants to the LEE1 and LEE2 regulatory regions and to the yccC (etk) coding region (used as a negative control) was tested using an ELDIA. Whole bacterial extracts, generated from EPEC ler::kan containing plasmids encoding the different Ler mutations (pGY1 and pGY1-derived plasmids) were used. As a positive control, we used pGY1 expressing native Ler (Ler WT), and as a negative control, we used a strain containing the vector, which is not expressing Ler (Vector). The mutations of the different Ler proteins are indicated below the columns. DNA binding is expressed as a percentage of the maximum binding. Protein levels in the extracts used for the experiments were analyzed by immunoblot analysis using the anti-Ler antibody, shown below the graph. Similar results were obtained in three independent experiments.

FIG. 3.

Ler forms homooligomers. (A) The pDF5 plasmid, from which two forms of Ler are expressed (the native Ler and Ler fused at its N terminus to a 16-amino-acid peptide containing His₆ [MRGSHHHHHHPRRLFI]-Ler) was introduced into E. coli W3110. The expressed His₆-Ler was purified by metal affinity chromatography, and proteins retained on the column were eluted and analyzed by SDS-PAGE and Coomassie staining (right lane). The left lane contains size markers, and the different sizes are indicated. Two highly purified protein bands are located in the right lane near the 16-kDa marker. (B) Proteins from two lanes of a similar gel were transferred onto a nitrocellulose membrane, and the membrane was cut along one of the lanes. The left half of the membrane was developed by Coomassie staining and thus appears darker. The right half of the membrane was used for immunoblot analysis and developed with anti-His₆ antibody. Only the upper band reacted with the anti-His₆ antibody.

FIG. 4.

Oligomerization of the Ler autorepression-defective mutants and truncation analysis. (A) Oligomerization of the Ler autorepression-defective mutants was assessed by introducing the plasmids encoding them into an EPEC strain chromosomally encoding Ler-His₆. Ler-His₆ and associated proteins were pulled down from cleared extracts with Talon beads, and proteins associated with Ler-His₆ were eluted with 8 M urea. Under these conditions, Ler-His₆ remained attached to the beads. The eluted samples were further treated with fresh Talon beads to remove any residual Ler-His₆ and were then analyzed by SDS-PAGE, followed by an immunoblot analysis with anti-Ler antibodies. pGY1 expressing native Ler (wild-type) was used as a positive control, and the strain expressing Ler-His₆ was used as a negative control. The different mutations are indicated below the corresponding lanes. (B) Schematic representation of the N50Stop and G65Stop truncated Ler variants. (C) EPEC wild-type (top panel) or EPEC ler::kan (bottom panel) containing the plasmids pIR1 (vector), pGY1 expressing wild-type ler (wild-type), and pGY1-derived plasmids expressing a truncated Ler containing only the first 50 amino acids [ler(N50stop)] or only the first 65 amino acids [ler(G65stop)]. To test the ability of the different plasmids to restore or block EspB secretion (the latter is via dominant negativity), the supernatant of bacterial cultures of these strains grown in LEE-inducing conditions was subjected to immunoblot analysis with anti-EspB antibody.

FIG. 5.

Complementation experiments with the Ler autorepression defective mutants. Plasmids expressing each of the Ler mutants (pGY1 or a pGY1 derivative) were introduced into EPEC ler::kan. The capacity of the mutated Ler to restore the expression of Tir and EspB (expressed from the promoters P_LEE5 and P_LEE4, respectively) (A), EspB secretion (B), and the formation of actin pedestals (C) was determined. (A) Expression of Tir and EspB was tested using immunoblot analysis with antibodies raised against these proteins. (B) To test for EspB secretion, the different strains were used to infect HeLa cells for 3 h. The growth medium was recovered, cleared, and analyzed by immunoblot analysis using anti-EspB antibodies. The ler mutations in the complementing plasmids are indicated below the lanes. The names of the LEE-encoded proteins are indicated on the left-hand side of the blots, and the specific operon carrying each gene is in parentheses. The EspB protein in the bacterial lysate (in panel A) is the upper band out of the seen doublet. The origin of the lower band that cross-reacted with the anti-EspB antibody is not known, and it was seen also in strains deleted of the espB gene (data not shown). (C) To test for the formation of actin pedestals, EPEC ler::kan containing different complementing plasmids was used to infect HeLa cells for 3 h. The infected cells were fixed and actin filaments were stained with rhodamine-phalloidin. A few representative examples are shown, including an uncomplemented ler::kan mutant (no plasmid), a ler::kan mutant complemented with pGY1 (wild-type), and mutants complemented with pGY1-ler(L23R) (L23R) or with pGY1-ler(G89D) (G89D). Phase-contrast images of the infected cells are shown in the first panel, GFP produced by the bacteria in the second panel (green), and actin in the third panel (red). The fourth panel shows overlays of the red and green images. Arrows indicate some actin pedestals.

FIG. 6.

Identification of dominant-negative Ler autorepression-defective mutants. pGY1 or a pGY1-derivative plasmid expressing each of the Ler mutants was introduced into wild-type EPEC. The expression of Tir and EspB (natively expressed from the Ler-activated promoters P_LEE5 and P_LEE4, respectively) was determined using immunoblot analysis with antibodies raised against these proteins. The ler mutations are indicated below the lanes. The names of the LEE-encoded proteins are indicated on the left-hand side of the blots and the specific operon carrying each gene is written in parentheses.

FIG. 7.

Increased dosage of the CID mutants is required for their function. (A) A schematic representation of the compensatory mechanism: an autorepressor represses its own transcription (from the Pautorep promoter) and activates transcription from other promoters (Pact). A mutant autorepressor can either retain a partial function like the CID mutants or no function. A CID mutation (middle panel) alleviates autorepression, causing an increase in the concentration of the mutated regulator, which in turn compensates for its reduced DNA binding affinity, enabling it to activate the transcription from Pact. This situation demonstrates the compensatory mechanism embedded in autorepressed regulatory systems. When a mutation completely abolishes the regulator's function (see the “Null mutant” bottom panel), even a high concentration cannot compensate for the mutation. (B) The L23R, G82E, A98V, and G102R CID mutants were cloned under the regulation of the Ptac promoter, from which their expression level could be controlled (plasmids pGY2742, pGY2743, pGY2744, and pGY2745, respectively). Wild-type Ler and LerG89D (a DN mutant) were also subjected to the same analysis (plasmids pGY2746 and pGY3576, respectively). The plasmids were electroporated into EPEC ler::kan, and the strains were grown with increasing concentrations of IPTG (0, 1, 5, 10, 20, and 50 μM). The secretion of EspB, representing the expression and assembly of a functional TTSS, was tested by subjecting the supernatant of the cultures to an immunoblot analysis using anti-EspB antibody. (C) The expression level of Ler was determined by subjecting the pellet of the cultures to an immunoblot analysis using anti-Ler antibody (only two or three IPTG concentrations for each Ler mutant are shown).

FIG. 8.

A schematic of Ler domains. Based on the comparison to H-NS together with our results, we suggest the following model for Ler domains: an N terminus that functions either as a high-order oligomerization domain or as a domain that interacts with other proteins, a central domain that functions as a homooligomerization domain, and a C-terminal DNA binding domain. The three domains are essential for DNA binding and transcription regulation.