Characteristics of genes in each cluster. Number of genes in three gene classes, L1, L2 and N, which were isolated by transcriptomic analysis, was shown. Genes were sorted by chromosomal locus and function as follows: black, LEE genes; grey, non-LEE virulence-associated genes; light grey, other genes in S-loop; white, genes on K12-common loci.

Distribution of PchA and Ler binding in the LEE and EELs. Graphs show PchA and Ler binding as in Fig. 2. Upper line indicates the position from the replication origin of the chromosome. Middle row indicates G + C contents. (A) Chromosomal region around the LEE. Genes in the LEE are represented by red arrows. The five main LEE operons, LEE1 to LEE5, are represented by arrows below the open reading frames map. (B) EEL on prophage-like element Sp3. Genes encoding effector proteins are indicated by red arrows. (C) EEL on prophage-like element Sp6. (D) EEL on prophage-like element Sp9. (E) EEL on prophage-like element Sp10. (F) EEL on prophage-like element Sp14. (G) EEL on prophage-like element Sp17. (H) EEL on prophage-like element SpLE3. IS and remnant of IS are shown in light green boxes.

Hierarchical cluster analysis of gene expression changes in response to changes in Pch or Ler levels. Gene expression data from 13 microarray experiments (lines) and whole EHEC O157 Sakai 5454 genes (columns) are indicated. The microarray experiments involved 13 comparisons between EHEC O157 Sakai and derivative strains: (1) Wild type (WT) harboring pGEM-ler to WT harboring pGEM vector grown in LB. (2) The pchApchB mutant harboring pGEM-ler to the pchApchB mutant harboring pGEM-vector grown in LB. (3) The pchApchBpchC mutant harboring pGEM-ler to the pchApchBpchC mutant harboring pGEM-vector grown in LB. (4) WT harboring pGEM-pchA to WT harboring pGEM-vector grown in LB. (5) WT harboring pGEM-ler to WT harboring pGEM-vector grown in DMEM. (6) WT harboring pGEM-pchA to WT harboring pGEM-vector grown in DMEM. (7) The pchApchBpchC mutant harboring pGEM-ler to the pchApchBpchC mutant harboring pGEM-vector grown in DMEM. (8) The ler mutant harboring pGEM-pchA to the ler mutant harboring pGEM vector grown in LB. (9) The ler mutant harboring pGEM-pchA to the ler mutant harboring pGEM vector grown in DMEM. (10) The ler mutant to WT grown in LB. (11) The pchApchBpchC mutant to WT grown in LB. (12) The pchApchBpchC mutant to WT grown in DMEM. (13) The ler mutant to WT grown in DMEM. The lower panel shows gene clusters in and around the LEE genes along with a color bar indicating the gene context (green: common to K12; dark grey: EHEC O157 specific; light grey: LEE). The scale bar indicates color coding of the RNA levels. Class L1 with subclasses I and II, class L2, and class N are indicated (see text).

Distribution of PchA and Ler binding over the EHEC O157 Sakai chromosome. Graphs show PchA and Ler binding to the chromosome of the EHEC O157 Sakai strain (horizontal bar). Red and blue segments indicate S-loops (O157:H7 strain-specific regions) and backbone (regions conserved with E. coli K12), respectively. Vertical bars indicate the relative hybridization intensity of the precipitated DNA compared with DNA in the supernatant.

Correlation between Pch and/or Ler binding with transcription profiles. The binding of Pch and Ler to loci in and around genes was determined as in Fig. 2 and Fig. 3. (A) The number of effector genes assigned to class L1 and class L2. Effector genes not in either class are labeled NA (not applicable). (B) The number of genes in classes L1, L2, and N. Genes in S-loops (EHEC O157 strain-specific regions) and in the E. coli backbone (regions conserved with E. coli K-12) are identified as O157 and K12, respectively. Binding of Pch and/or Ler is denoted as follows: grey, bound by Ler and Pch; black, bound by Pch, but not Ler; dark grey, bound by Ler, but not Pch; white, no binding by Pch or Ler.