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

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

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

EHEC counts and HPA, 1,3-PD and glycerol quantification in RF-Glyc80.

<p>The strain FCH6 Rif^R (≈ 10⁴ CFU/mL) was co-incubated with <i>L</i>. <i>reuteri</i> LB1-7 (≈ 10⁷ CFU/mL) in RF samples supplemented with 80 mM glycerol under anaerobiosis. At each time point the strain FCH6 Rif^R was enumerated and accumulation of HPA and 1,3-PD, and disappearance of glycerol were monitored. Bars represent the SEM of three independent experiments. Gly: glycerol.</p

Kinetics of EHEC growth or disappearance and HPA production in LB broth.

<p>(A) The strain FCH6 Rif^R (≈ 10⁴ CFU/mL) was co-incubated with <i>L</i>. <i>reuteri</i> LB1-7 (≈ 10⁷ CFU/mL) in LB broth supplemented or not with different concentration of glycerol. The strain FCH6 Rif^R was then enumerated after 24 hours of incubation under anaerobiosis. Bacterial growth curves are expressed as a single representation of three independent experiments. (B) The strain FCH6 Rif^R was co-incubated with <i>L</i>. <i>reuteri</i> in LB broth supplemented with 10 mM glycerol under anaerobiosis. At each time point the strain FCH6 Rif^R was enumerated and accumulation of HPA was quantified. The bacterial growth curve is expressed as a single representation of three independent experiments. Bars represent the SEM of three independent experiments.</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

Survival of EHEC co-incubated with <i>L</i>. <i>reuteri</i> strains in RF samples supplemented or not with glycerol.

<p>The strain FCH6 Rif^R was co-inoculated with ≈ 10⁷ CFU/mL of <i>L</i>. <i>reuteri</i> LB1-7 (HPA producer) or 100–23 (negative control) in RF samples under anaerobiosis for 24 hours. The strain FCH6 Rif^R inoculated alone in RF samples was used as control. RF samples were supplemented or not with glycerol at different concentrations. Bars represent the SEM of three independent experiments. Asterisks indicate statistical significance (***: P<0.001).</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

Concentration of glycerol and microbial metabolites.

<p>Concentration of glycerol and microbial metabolites.</p

Growth or survival of EHEC in rectum contents after incubation in RF.

<p>The strain FCH6 Rif^R was first incubated in filter-sterilized RF (FS-RF) samples alone or inoculated with ≈ 10⁷ CFU/mL of <i>L</i>. <i>reuteri</i> LB1-7 supplemented with 80 mM glycerol under anaerobiosis for 24 hours. The bacterial pellet was then inoculated into Rec samples and incubated under anaerobiosis. RF = 0 represents inoculation of EHEC in FS-RF samples; Rec t = 0 corresponds to RF t = 24h i.e. number of EHEC surviving the incubation in FS-RF during 24 hours; Rec t = 6h and Rec t = 24h correspond to EHEC survival in Rec samples after 6 and 24 hours of incubation respectively. Bars represent the SEM of three independent experiments. Effect of <i>L</i>. <i>reuteri</i> + Glyc80 is significant *, P<0.05; ***, P<0.001.</p