RccR gene targets.

<p>RccR gene targets.</p

RccR and HexR are highly similar and important for wheat rhizosphere colonisation.

<p><b>1A</b>: Sequence alignment for <i>rccR</i> and <i>hexR</i> from <i>P</i>. <i>fluorescens</i> SBW25. Important amino acid residues for DNA and ligand interactions are marked in blue and red respectively. <b>1B</b>: 3D homology model of the RccR protein structure. Arg-53 and -56 (blue) are the predicted DNA interaction partners in the helix-turn-helix domain. Ser-139 and -183 (red) are located in the predicted effector binding site. <b>1C</b>: Rhizosphere colonisation competition assays. The graph shows the ratio of SBW25 WT or <i>ΔrccR</i>/<i>ΔhexR</i> mutants to WT-<i>lacZ</i> colony forming units (CFU) recovered from the rhizospheres of wheat plants seven days post-inoculation. Each dot represents the ratio of CFUs recovered from an individual plant. In each case, differences between SBW25 and <i>ΔrccR</i> or <i>ΔhexR</i> strains are statistically significant (p < 0.05, Mann-Whitney U test).</p

Screening for the RccR effector.

<p><b>9A</b>: Percentage of normalized response (%Rmax) for RccR binding to the <i>rccR</i>, <b>9B</b>: <i>aceA</i> and <b>9C</b>: <i>aceE</i> consensus sequences in the presence of KDPG (effector) and PEP (negative control) at different concentrations (1-10-100 μM).</p

A model for RccR regulation of primary carbon metabolism.

<p>The figure shows a schematic representation of the metabolic pathways of glucose, glycerol, pyruvate and acetate through the Krebs cycle and the glyoxylate shunt. The protein products of the RccR gene targets are shown: PntAA/PFLU0112/B are subunits of the NAD(P) transhydrogenase membrane protein complex; PckA: phosphoenolpyruvate carboxykinase; AceE/F: pyruvate dehydrogenase subunits; Gap: glyceraldehyde-3-phosphate dehydrogenase; AceA: isocitrate lyase; GlcB: malate synthase G. RccR-regulated carbon transitions are marked in red. HexR-regulated carbon transitions are marked in blue.</p

Growth curves for SBW25 WT and <i>ΔrccR</i>, <i>ΔhexR</i>, and <i>ΔrccRΔhexR</i> mutants.

<p><b>2A</b>: Growth was measured in KB and <b>2B</b>: LB rich media as well as in <b>2C</b>: M9 0.4% glucose, <b>2D</b>: M9 0.4% glycerol, <b>2E</b>: M9 0.4% pyruvate, <b>2F</b>: M9 0.4% acetate and <b>2G</b>: M9 0.4% succinate. Marked differences in growth rate were seen between WT and <i>ΔrccR</i> in glucose (<b>C</b>) and glycerol (<b>D</b>), and between WT and <i>ΔhexR</i> mutants in pyruvate (<b>E</b>), acetate (<b>F</b>), and succinate (<b>G</b>). Experiments were repeated at least three times independently and a representative plot is shown in each case.</p

HexR controls the Entner-Doudoroff pathway in <i>P</i>. <i>fluorescens</i>.

<p><b>3A</b>: Schematic organisation of the HexR gene targets. HexR binds to a DNA consensus sequence in the intergenic regions between the <i>zwf/pgl/eda</i> operon and <i>hexR</i> genes, and between the <i>edd/glk/gltR2/gltS</i> operon and the <i>gap-1</i> gene. HexR negatively regulates expression of these gene targets, but not of itself. <b>3B</b>: The HexR regulon. HexR gene targets are involved in the glucose phosphorylative and Entner-Doudoroff pathways in <i>P</i>. <i>fluorescens</i>. Glk: glucokinase; Zwf: glucose 6-P dehydrogenase; Pgl: 6-phosphogluconolactonase; Edd: 6-phosphogluconate dehydratase; Gap-1: glyceraldehyde 3-phosphate dehydrogenase; the blue and light blue stars indicate activation of the glucose transport system, which is positively regulated by the transcriptional regulators GltR2 and GltS. <b>3C</b>: <i>zwf</i>, <i>edd</i>, and <i>gap</i> gene expression in glucose, <b>3D</b>: in glycerol, <b>3E</b>: in pyruvate and <b>3F</b>: in acetate in the <i>hexR</i> mutant background relative to WT (qRT-PCR data). <b>3G</b>: <i>hexR</i> promoter activity in SBW25 Δ<i>hexR</i> relative to WT, determined by β-gal assays tested in glucose, glycerol, pyruvate and acetate conditions. <b>3H</b>: SBW25 <i>hexR</i> gene expression determined by qRT-PCR after media exchange and 30 min growth in glucose, pyruvate or Root Solution (RS; media without carbon sources, used as a negative control).</p

RccR controls expression of pyruvate metabolism, gluconeogenesis and the glyoxylate shunt.

<p><b>5A-C</b>: RccR gene target expression determined by qRT-PCR. Data are shown for SBW25 <i>ΔrccR</i> relative to WT in <b>5A</b>: glucose media, <b>5B</b>: glycerol media, <b>5C</b>: pyruvate media, and <b>5D</b>: acetate media. <b>5E</b>: <i>rccR</i> promoter activity determined by β-gal assay in glucose, glycerol, pyruvate and acetate media conditions. Data are shown for the SBW25 <i>ΔrccR</i> background relative to WT. <b>5F</b>: SBW25 <i>rccR</i> gene expression determined by qRT-PCR after media exchange and 30 min growth in glucose, glycerol, pyruvate, acetate or Root Solution (RS; media without carbon sources, used as a negative control).</p

RccR binds the DNA consensus binding site of its targets.

<p><b>7A</b>: SPR experiments measuring the biomolecular interactions between the RccR protein and indicated DNA consensus sequences. Percentage of normalized response (%Rmax) of RccR (1μM and 0.1 μM concentrations) binding the consensus sequences found by MEME and manual sequence analysis alongside a random sequence DNA control. %Rmax indicates the experimental RccR binding values (Response registered from the SPR machine) normalized on the maximal response (R_max) that can be potentially reached when all ligand binding sites (DNA) are occupied by the analyte (RccR protein). <b>7B</b>: Sensorgrams (up) and fitting (down) curves showing RccR affinity to <i>aceE</i>, <i>aceA</i> and <i>rccR</i> consensus sequences.</p

Mapped reads from the RccR ChIP-seq experiment.

<p><b>4A-H</b>: Locations of genes and operons of interest are shown below each peak. Blue arrows indicate the direction of gene transcription and <i>PFLU</i> gene numbers are indicated in each case. Relative scales are indicated for each panel as well as the gene position in the SBW25 genome. Green and red peaks denote the SBW25 WT datasets, while blue and black show data for the <i>ΔrccR</i> mutant strain. Green and black lines indicate bacterial growth in glycerol, while red and blue indicate bacterial growth in pyruvate.</p

RccR binds the 28bp and the 15bp binding sites.

<p><b>8A</b>: DNaseI footprinting panel of RccR on <i>rccR</i>, <i>aceA</i>, <i>aceE</i> promoters. Radiolabelled promoter probes were incubated with increasing concentrations of purified RccR-His (0, 10, 20, 40, 80, 160 nM of RccR-His from left to right in each panel) before DNaseI digestion and DNA purification. Recovered DNA fragments were subjected to electrophoretic separation along with a Maxam and Gilbert G+A sequence reaction ladder (leftmost lane of each autoradiograph). On the left of each autoradiograph, a schematic representation of the genomic region is reported, with symbols as follows: block arrow represents the coding sequence, bent arrow represents the transcriptional start site identified in this study (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006839#pgen.1006839.s003" target="_blank">S3 Fig</a>), while black box indicates the -10 promoter element. Protected regions are highlighted by a black box on the right of each autoradiograph, while DNaseI hypersensitive sites are evidenced by black arrowheads. <b>8B</b>: mapping of the RccR binding sites on the <i>rccR</i>, <i>aceA</i> and <i>aceE</i> promoter regions. Arrowheads denote hypersensitive sites, protected regions are included in open boxes, and conserved pseudopalindromic sequences are highlighted in light grey. Bent arrow indicates the transcriptional start site identified in this study (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006839#pgen.1006839.s003" target="_blank">S3 Fig</a>) and the first transcribed nucleotide is in bold.</p