A model for the NO-dependent regulation of LEE1/4/5.

<p>Solid lines indicate physical interaction between the regulator and the promoter as demonstrated by ChIP; dotted lines indicate that no physical interaction has been demonstrated between the regulator and the promoter. A: In the absence of NO, NsrR directly activates LEE1, LEE4, and LEE5 expression and indirectly represses <i>gadE</i> and therefore <i>gadX</i> expression. GadE activates <i>gadX</i> expression and acts as an indirect repressor of LEE4 and LEE5. GadX is an indirect repressor of <i>gadE</i> and LEE1 expression. B: Under NO exposure, NO binds to NsrR, which is consequently released from its target DNA. Thus, the activation of LEE1/4/5 genes by NsrR is suppressed. In parallel, <i>gadE</i> expression is restored and induces <i>gadX</i> up-regulation. In this context, GadE strongly represses LEE4 and LEE5 genes while GadX inhibits LEE1 expression.</p

Influence of NO on LEE, <i>gadX</i>, and <i>gadE</i> gene expression.

<p>A: schematic representation of the LEE showing the structural organization of the main operons. Arrows indicate the orientation of the transcription. The genes analyzed in this study are in grey boxes. B and C: EDL933 was grown for 6 h with or without NOR-4. The expression of the LEE genes (B) and of <i>gadE</i> and <i>gadX</i> (C) was analyzed by RT-qPCR. * <i>P</i><0.05, ** <i>P</i><0.01 compared to the strain grown in the absence of NOR-4; n = 3–6.</p

NsrR interacts with RNA polymerase complex.

<p>His-NsrR, His-Crp or His-Crl proteins were co-expressed in bacteria with HA-RpoA or HA-RpoS as indicated. His tagged proteins were then purified from bacterial lysates using nickel affinity. Whole extracts (1 µg) and His eluted fractions (5 µl) were probed with anti-His or anti-HA Abs.</p

Binding of GadE, GadX and NsrR on various promoter regions.

<p>The strains EDL933 Δ<i>gadE</i>-pBADMycHisA::<i>gadE</i>, Δ<i>gadX</i>-pBADHisA::<i>gadX</i>, and Δ<i>nsrR</i>-pBADMycHisA::<i>nsrR</i> were grown in the absence (black bars) or in the presence (white bars) of NOR-4. ChIP assays followed by qPCR were performed to determine the relative enrichment in DNA molecules bound to GadE (A), GadX (B), NsrR (C). Values higher than 20 (twice the values obtained for the strain EDL933 containing the empty pBADmycHisA vector) indicate protein binding to the promoters of interest. D: Bio-informatics analyses of NsrR-binding sites. Sequence logo determined from seven putative NsrR-binding sites in EDL933 (upper panel), and sequences with the best matches for the entire or one of the half sites are shown with their statistical scores (lower panel).</p

Regulation of LEE1/4/5 by GadE and GadX.

<p>The mRNA levels of <i>ler</i> (LEE1), <i>espA</i> (LEE4), and <i>tir</i> (LEE5) (A) and of <i>gadE</i> and <i>gadX</i> (B) were assessed in the strain EDL933, in the Δ<i>gadE</i>, Δ<i>gadX</i> and Δ<i>gadE</i>/<i>gadX</i> isogenic mutants, and in the complemented strains Δ<i>gadE</i>-c and Δ<i>gadX</i>-c. Bacteria were grown in the absence (black bars) or presence (white bars) of NOR-4 for 6 h. * <i>P</i><0.05, ** <i>P</i><0.01 compared to the same strain without NOR-4; § <i>P</i><0.05, §§ <i>P</i><0.01 vs. EDL933; n = 3–7.</p

Adhesion of EDL933 to intestinal epithelial cells.

<p>Hct-8 cells, pre-treated or not with a cytokine cocktail for 24 h, were co-cultured for 6 h with the EHEC strain EDL933 ± NOR-4 or L-NIL. A: Cells and bacteria were visualized after Giemsa staining; magnification, ×63. B and C: The number of bacteria adherent per Hct-8 cell was counted on 15 microscopic fields. For B, * <i>P</i><0.05, ** <i>P</i><0.01 compared to the co-cultures without NOR-4; n = 6. For C, ** <i>P</i><0.01 vs. cells not stimulated with cytokines; § <i>P</i><0.05 vs. cells treated with cytokines; n = 6.</p

Regulation of adhesion of EDL933 to human epithelial cells.

<p>HeLa cells were infected with EDL933, Δ<i>gadE</i>, Δ<i>gadX</i>, Δ<i>nsrR</i>, or with the corresponding trans-complemented strains, in the presence or absence of NOR-4. After 6 h, cells were washed and colored with May-Grünwald Giemsa (A). The number of adherent bacteria per Hela cell was determined from 50 cells (B).</p