Raw gel images.

Panels A-C show raw gel images from Figs 2A, 2C and 3 respectively. (PDF)</p

Transcription from P<i>ehxCABD</i> is inhibited by overlapping RNA polymerase binding sites.

<p><b>A. Effects of mutations in P</b><b><i>ehxCABD</i></b><b>, and overlapping RNA polymerase binding sites.</b> The graph shows LacZ activity data for <i>E. coli</i> JCB387 cells carrying different F3::<i>lacZ</i> fusions in pRW50. <b>B. i) Stimulation of P</b><b><i>ehxCABD</i></b><b> by H-NS </b><b><i>in vitro</i></b><b>.</b> The figure shows the results of an <i>in vitro</i> transcription reaction calibrated with transcripts of known size from the <i>cbpA</i> regulatory region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003589#pgen.1003589-Chintakayala1" target="_blank">[35]</a>. The 178 nt transcript initiates from P<i>ehxCABD</i> and the 108 nt RNAI transcript is an internal control. <b>ii) Stimulation of P</b><b><i>ehxCABD</i></b><b> by the -41G mutation </b><b><i>in vitro</i></b><b>.</b> The figure shows the results of an <i>in vitro</i> transcription assay comparing the wild type <i>ehxCABD</i> F3 fragment with a derivative carrying a mutation at promoter position -41.</p

H-NS is required for correct positioning of RNA polymerase at P<i>ehxCABD</i>.

<p><b>A. Footprint of RNA polymerase (σ⁷⁰ RC461-FeBABE) interactions with −10 elements in the </b><b><i>ehxCABD</i></b><b> regulatory region in the presence of H-NS.</b> The panel shows an image of <i>ehxCABD</i> DNA cleavage products separated by electrophoresis on a denaturing acrylamide gel. DNA cleavage was mediated by 640 nM RNA polymerase associated with the σ⁷⁰ RC461-FeBABE derivative that cleaves −10 hexamer sequences. Where present H-NS was pre-incubated with the DNA at concentrations of 235 nM, 470 M, 940 nM, 1645 nM or 2350 nM. The position of the <i>ehxCABD</i> promoter −10 hexamer is indicated. <b>B. Binding of H-NS to the </b><b><i>ehxCABD</i></b><b> F3 fragment.</b> The panel shows the result of a DNAse I footprint to monitor binding of H-NS to the <i>ehxCABD</i> DNA fragment. The gel is calibrated with a Maxim-Gilbert DNA sequencing reaction. H-NS was added at concentrations of 470 nM– 4700 nM. <b>C. Effect of H-NS on transcription start site selection at the </b><b><i>ehxCABD</i></b><b> regulatory region.</b> The panel shows the result of primer extension analysis using RNA extracted from strain M182 or the Δ<i>hns</i> derivative, carrying the <i>ehxCABD</i> F3 fragment cloned in pRW50, grown aerobically to mid-exponential phase (OD₆₅₀ 0.4–0.6) in LB medium. The sizes of primer extension products were determined by calibration against size standards (A, C, G and T in Lanes 1–4). The brightness and contrast have been set differently for lanes 1–4 and 5–6 so that the primer extension products can be more easily compared to the marker lanes. The image otherwise represents a single continuous gel.</p

Model for H-NS induced specificity during interactions between RNA polymerase and AT-rich gene regulatory regions.

<p>In the absence of H-NS RNA polymerase competes with itself for binding to multiple overlapping targets (left hand side of figure). In the context of native nucleoprotein RNA polymerase must instead compete with H-NS. This results in preferential recognition of the canonical RNA polymerase binding target (right hand side of figure).</p

Characterisation of the P<i>aatS</i> promoter.

<p><b>A.</b> Primer extension analysis of the <i>aatS</i> transcript. Lanes 1–4 on the gel are arbitrary size standards, used for calibration, generated by sequencing of M13mp18 phage DNA. Lane 5 shows the primer extension product generated using RNA from wildtype M182 cells carrying the <i>aatS</i>1::<i>lacZ</i> fusion. Lane 6 shows the primer extension product generated using RNA from M182<i>Δcrp</i> cells carrying the <i>aatS</i>1::<i>lacZ</i> fusion. The transcription start site is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157016#pone.0157016.g001" target="_blank">Fig 1B</a>. <b>B.</b> β-galactosidase activity determined using lysates of M182 wildtype or M182Δ<i>crp</i> cells carrying P<i>aatS</i> cloned upstream of <i>lacZ</i> in plasmid pRW50. Values shown are percentages of activity observed in strain M182 (92 Miller units). We obtained 7 and 3 Miller units of activity from lysates of M182 or M182Δ<i>crp</i> carrying promoterless pRW50. Error bars represent the standard deviation of three independent experiments. <b>C.</b> Multi-round i<i>n vitro</i> transcription assay using P<i>aatS</i>. The <i>aatS</i>1 DNA fragment was cloned into pSR upstream of a <i>λoop</i> terminator. Purified, supercoiled pSR plasmid was incubated with purified CRP at 37°C, and the reaction started by the addition of 400 nM σ⁷⁰- RNA polymerase holoenzyme. CRP concentrations are; 0 nM, 200 nM, or 400 nM. The 108 nt RNAI transcript from the pSR replication origin, and the 169 nt transcript from P<i>aatS</i>, are indicated. The gel is calibrated with an arbitrary G+A DNA sequencing reaction as a size standard.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Identification of the<i>ehxCABD</i> promoter.

<p><b>A. i) Schematic representation of the </b><b><i>ehxCABD</i></b><b> operon and gene regulatory region.</b> The figure shows genes (as block arrows) within the <i>ehxCABD</i> operon (orange). The adjacent open reading frame is coloured blue. The <i>ehxCABD</i> regulatory region fragments used in this study are shown as solid black lines labelled F1 through F3. The 248 bp F1 fragment contains regulatory DNA upstream of, and including, the <i>ehxC</i> start codon. The F3 and F2 fragments are equivalent to upstream and downstream parts of the F1 fragment respectively. <b>ii) Plasmid maps for pRW50 (containing a LacZ reporter) and pLux (containing a Luciferase reporter)</b> that were used to test the ability of the F1–F3 fragments to drive transcription. <b>B. Promoter activity of different </b><b><i>ehxCABD</i></b><b> regulatory DNA fragments.</b> The panel shows a summary of data from β-galactosidase and Luciferase assays using the different pRW50 and pLux constructs in <i>E. coli</i> strains JCB387 and O157:H7 respectively. The data are expressed as a percentage of the signal obtained for the F1 fragment. <b>C. Location of the </b><b><i>ehxCABD</i></b><b> transcription start site.</b> The gel shows products from an mRNA primer extension analysis of the F3 fragment (Lane 5). The gel was calibrated using arbitrary size standards (A, C, G and T in Lanes 1–4). <b>D. Location of the </b><b><i>ehxCABD</i></b><b> promoter.</b> The panel shows the base sequence of the non-template strand. The transcript start sites identified in panel A are highlighted in green with the most abundant start site labelled as “+1”. The proposed extended −10 and −35 hexamer elements of the <i>ehxCABD</i> promoter are also in green as well as being underlined. Two sequences that resemble a promoter −10 element are boxed by a dashed red line. The positions of mutations designed to disrupt the various RNA polymerase binding elements are also shown. The -41G mutation disrupts the highly conserved “T” that occurs in the first position of −10 elements. Concomitantly, the genuine P<i>ehxCABD</i> −35 hexamer remains intact. We made more conservative changes to disrupt the downstream −10 like sequence. This element is embedded within the region of P<i>ehxCABD</i> that participates in open complex formation. Thus, we made several A to T transversions to remove the problematic −10 like sequence whilst maintaining the AT-content of the DNA. We reasoned that this would be least disruptive to DNA opening during transcription initiation. However, we cannot rule out the possibility of small structural changes to P<i>ehxCABD</i>.</p

Co-association of RNA polymerase and H-NS with the<i>ehxCABD</i> gene regulatory region.

<p><b>A. ChIP analysis of RNA polymerase and H-NS binding at the </b><b><i>ehxCABD</i></b><b> promoter.</b> The figure illustrates the result of a ChIP experiment designed to monitor the binding of H-NS and RNA polymerase to the <i>ehxCABD</i> F3 promoter fragment. The image shows a gel on which PCR products, generated with primers designed to detect P<i>ehxCABD</i>, <i>yabN</i> or <i>lacZ</i>, were analysed. The source of the DNA template (i.e. total cellular DNA or DNA from an immunoprecipitation) is shown above the gel image and the different PCR products are labelled to the right of the image. The mock immunoprecipitation contained no antibody. <b>B. EMSA analysis of H-NS and RNA polymerase binding at the </b><b><i>ehxCABD</i></b><b> promoter.</b> The results of an Electrophoretic Mobility Shift Assay (EMSA) are shown. The <i>ehxCABD</i> F3 DNA fragment (Lane 1) was incubated with 480 nM RNA polymerase (Lane 2), 2350 nM H-NS (Lane 3) or 4700 nM H-NS (Lane 5). The positions of the various H-NS-DNA and RNA polymerase-DNA complexes are indicated. Lanes 4 and 6 show complexes formed in the presence of 480 nM RNA polymerase and either 2350 nM or 4700 nM H-NS respectively. The bands highlighted by boxes were extracted and the presence of both H-NS and RNA polymerase proteins in the band was confirmed.</p

RNA polymerase binds multiple sites in the<i>ehxCABD</i> gene regulatory region.

<p><b>A. i) Footprint of RNA polymerase (σ⁷⁰ RC461-FeBABE) interactions with −10 elements in the </b><b><i>ehxCABD</i></b><b> regulatory region.</b> The gel shows DNA cleavage products resulting from incubation of the <i>ehxCABD</i> promoter F3 fragment with RNA polymerase containing σ⁷⁰ RC461-FeBABE (640 nM). Note that σ⁷⁰ RC461-FeBABE results in specific cleavage of promoter −10 elements. Cleavage of the P<i>ehxCABD</i> −10 element is indicated by a green box. Additional sites at which the DNA is cleaved are highlighted by red stars. The gel was calibrated with a G+A sequencing ladder (Lane 1). <b>ii) KMnO₄ reactivity pattern of the </b><b><i>ehxCABD</i></b><b> promoter in the presence and absence of RNA polymerase.</b> The panel shows DNA cleavage products resulting from KMnO₄ treatment of a complex formed between RNA polymerase (320 nM) and the <i>ehxCABD</i> F3 fragment. Thus, the sites of DNA cleavage correspond to DNA unwinding by RNA polymerase at −10 hexamers. The P<i>ehxCABD</i> −10 element is indicated by a green box. Additional sites at which the DNA is cleaved are highlighted by yellow stars. <b>B. i) Footprint of RNA polymerase (σ⁷⁰ RC461-FeBABE) interactions with −10 elements in the </b><b><i>cbpA</i></b><b> regulatory region.</b> The image shows an identical set of reactions to those illustrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003589#pgen-1003589-g004" target="_blank">Figure 4Ai</a> except that a DNA fragment containing the <i>cbpA</i> P6 promoter was used. The <i>cbpA</i> P6 −10 hexamer is highlighted by a green box. <b>ii) KMnO₄ reactivity pattern of the </b><b><i>cbpA</i></b><b> P6 promoter in the presence and absence of RNA polymerase.</b> The image shows an identical set of reactions to those illustrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003589#pgen-1003589-g004" target="_blank">Figure 4Aii</a> except that a DNA fragment containing the <i>cbpA</i> P6 promoter was used. The <i>cbpA</i> P6 −10 hexamer is highlighted by a green box.</p

The <i>aatPABC</i> operon of ETEC H10407.

<p>Schematic of the <i>aatPABC</i> operon and adjacent <i>tnpA</i> gene. The two DNA strands are shown as black lines. Known genes are shown as black arrows and the predicted <i>aatS</i> gene as a grey arrow. Gene names are shown in italic and gene function in parenthesis. The position of a putative CRP binding site is indicated by striped ovals. <b>A.</b> Sequence if the <i>tnpA</i>-<i>aatC</i> intergenic region. The CRP site is highlighted as a striped rectangle with the two half sites highlighted bold. The start codon of the <i>aatS</i> open reading frame is highlighted with a grey rectangle. The transcription start site, as determined by mRNA primer extension is denoted “+1” and indicated by a bent arrow. Distances upstream (-) and downstream (+) of this start site are numbered. The -35 and -10 hexamers are boxed, and the ribosome binding site (RBS) is underlined.</p