Bio-informatic analyses of PhoB-binding sites.

<p>Sequence logos determined from 12 putative PhoB-binding sites in EDL933 are indicated (upper panel). Potential consensus sequences identified in the promoter regions of LEE 1, LEE 2 operons and upstream <i>stx2AB</i> genes are shown with their statistical scores and genomic positions (lower panel). Pho box prediction probabilities were determined using the matrix frequencies of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094285#pone.0094285.s006" target="_blank">Table S4</a> that were uploaded into the Gibbs software algorithm. <a href="http://ccmbweb.ccv.brown.edu/gibbs/gibbs.html" target="_blank">http://ccmbweb.ccv.brown.edu/gibbs/gibbs.html</a>.</p

Effect of Pi and PhoB on <i>stx2</i> gene expression and toxin production.

<p>Low Pi and PhoB increased transcription of <i>stx2</i> and its toxin production and of BP933W genes except for repressor gene <i>cI</i> that was repressed. <b>A</b>. Heat map of the expression levels of BP933W genes between wild-type EDL933 strain grown in low or high Pi conditions and between wild-type and Δ<i>phoB</i> strain grown in low Pi conditions. <b>B.</b> The expression levels of the BP933W genes <i>cI, cro</i> and <i>stx2AB</i> were analyzed by RT-qPCR in the wild-type strain and the Δ<i>phoB</i> mutant grown in low or high Pi media as indicated. The wild-type strain induced by mitomycin C (WT+MitC) was used as a positive control <b>C.</b> Fluorescence of the wild-type EDL933 strain carrying a chromosomal fusion reporting <i>stx2</i> transcription level grown in Pi+ or Pi- conditions. <b>D.</b> The production of Stx2 measured by ELISA in extra-cellular protein (ECP) and whole cell protein (WCP) fractions of the EDL933 wild-type strain grown in Pi+ or Pi- conditions and in the Δ<i>phoB</i> mutant and its complemented derivative. Asterisks represent the significant ANOVA <i>P value</i> (*<0.05, **<0.01, ***<0.001).</p

Global analysis of differentially expressed genes in response to Pi starvation or phoB inactivation in EDL933 strain.

<p><b>A.</b> Classification of genes whose expression levels were altered in Pi-dependent and in PhoB-dependent manners. Left and right circles indicate the differentially expressed genes of wild-type and Δ<i>phoB</i>-mutant strains with expression levels that were altered over 2-fold under Pi limitation. Venn diagram: Group 1 includes 936 PhoB-independent genes that are differentially expressed under Pi limitation in the wild-type strain, but that did not change in the Δ<i>phoB</i> mutant. Group 3 includes 472 PhoB-dependent genes differentially expressed in the Δ<i>phoB</i> mutant but did not change under Pi-limitation in the wild-type strain. Group 2 included 131 PhoB-dependent Pi response genes that are differentially expressed under Pi limitation in the wild-type strain and between the wild-type and the Δ<i>phoB</i> strains. <b>B.</b> Functional classification of genes with altered expression in strain EDL933 grown in Pi-limited conditions compared to cells grown in Pi-rich conditions (Pi-dependent (white bars)) and EDL933 incubated in Pi-limited conditions compared to Δ<i>phoB</i> mutant cells grown in the Pi-limited conditions (PhoB-dependent (gray bars)).</p

Genes among upregulated and downregulated PhoB-dependent Pi response genes.

<p>Genes among upregulated and downregulated PhoB-dependent Pi response genes.</p

PhoB binds in vitro to LEE1, LEE2 and <i>stx2</i> promoter regions.

<p>A. Schematic representation of LEE and lambdoid prophage BP933W DNA regions. Arrows indicate the orientation of transcription. The blue lines/arrows indicate the probes used for EMSA assays. The square symbol indicates the predicted Pho box. B. Increasing amounts of GST-purified recombinant PhoB^CA were used in the EMSA assay to shift the 6-FAM labeled DNA probes amplified from LEE1 (−253 to +64 bp), LEE2 (−228 to +81 bp), LEE3 (−109 to −259 bp) and <i>stx2AB</i> (−402 to −3 bp) promoter regions.</p

<i>E. coli</i> strains and plasmids used in this study.

a<p>Km^r, kanamycin-resistant; Cm^r, chloramphenicol-resistant; Gm^r, gentamicin-resistant; Ap^r; ampicillin-resistant</p

Low Pi conditions and deletion of <i>phoB</i> gene increased LEE gene expression and T3SS secretion of EspB in EDL933 strain.

<p>A. A heat map of the expression levels of LEE genes between wild-type strain grown in low or high Pi concentrations, and between wild-type and Δ<i>phoB</i> strain grown in low Pi condition. Expression values were determined from the variance analysis by the EB (Wright & Simon) algorithm and are represented colorimetrically, with red representing up regulation (ratio of +2.3) and blue representing downregulation (ratio of -1) on a log₂ scale. The data are represented as the means from three biological replicates. B. Expression level of LEE genes <i>ler, sepZ, escV, tir</i> and <i>espB</i> was analyzed by RT-qPCR in the wild-type, the Δ<i>phoB</i> mutant and its complement (Δ<i>phoB</i>+C) and the Δ<i>pst</i> mutant grown in low or high Pi media as indicated. C. Western-blot using anti-EspB antibody against the supernatant of the wild-type, the Δ<i>phoB</i> mutant and its complement and the Δ<i>pst</i> mutant grown in low or high Pi media as indicated. The Δ<i>escN</i> mutant grown in DMEM was used as a negative control. One μg/mL of Gro-El was added as a protein precipitation control. Asterisks represent the significant ANOVA <i>p</i> value (*<0.05, **<0.01, ***<0.001). ns: Not significant.</p

Regulation of adhesion of EDL933 to human epithelial cells by the Pho regulon.

<p>HeLa cells were infected with EDL933 and indicated mutants. After 6-type and Δ<i>phoB</i> strains (<b>A</b> and <b>B</b>). The adhesion decreased in the Δ<i>pst</i> mutant (<i>P</i> <0.001) to levels similar to that of the Δ<i>escN</i> mutant which lacks functional TTSS (<b>C</b> and <b>D</b>). The attachment phenotype was restored in the <i>trans</i> complemented Δ<i>pst</i> mutant (<b>E</b>). The number of adherent bacteria per HeLa cell was determined from 25 cells (<b>F</b>). Magnification 64 ×.</p