AbrB2 controls the expression of <i>yrrT</i> operon, involved in methionine to cysteine conversion.

<p> β-galactosidase specific activity (U/mg protein) of wild-type (black circles), Δ<i>plcRa</i> (black triangle) and Δ<i>abrB2</i> (black diamonds) strains harboring both pHT304<i>_yrrT’-lacZ</i> and pHT1618KΩP<i>_xyl</i>-<i>abrB2</i> plasmids, in HCT. See legends <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051047#pone-0051047-g005" target="_blank">figure 5</a> for growth conditions. White symbols indicate cultures in the presence of xylose_.</p

Homology modeling of PlcRa.

<p>A. Alignment of the sequences of <i>B. thuringiensis</i> 407 Cry^- PlcR chain A (pdb code: 2QFC_A) and ATCC 14579 <i>B. cereus</i> PlcRa. The highly conserved residues are indicated in red in blue boxes, the strictly conserved residues are indicated in white in red boxes. Helices were the only secondary structural elements found and are displayed with the predicted domains above the sequences. The numbers indicate positions relative to the PlcR sequence. B. Homology modeling of the PlcRa homodimer from the target structure of PlcR (pdb code: 2QFC). Chains A and B of PlcR are shown in gray; the modeled chains A and B of PlcRa are shown in blue and cyan, respectively. Below: distribution of the domains in each monomer of PlcRa: the HTH at the N-terminus followed by the linker helix, with the five TPR motifs at the C-terminus.</p

Sensitivity to peroxide and disulfide stress of a <i>B. cereus plcRa</i> mutant.

<p>We assessed the viability of wild-type (WT), Δ<i>plcRa</i> (plcRa) and complemented Δ<i>plcRa</i> (plcRa+) strains in early stationary phase. Early stationary-phase cells grown in LB medium (∼ OD 3, ∼ <i>t</i>_0.4) were treated for 10 minutes with 1 mM H₂O₂ (A) or for 40 minutes with 10 mM diamide (B) in LB and plated on LB. The results shown are the mean values for survival, expressed as a %, with standard deviations, and are representative of three independent experiments.**: <i>P</i><0.01. C. The addition of cystine strongly improved the peroxide stress resistance of the <i>plcRa</i> mutant. We assessed the growth inhibition of wild-type and Δ<i>plcRa</i> strains in early stationary phase. Growth curves of the wild-type strain (black circles) and the mutant (black triangles) in LB medium without (solid line) or with 1 mM cystine (dashed line). Hydrogen peroxide (0.4 mM) was added at an OD of 2 (∼ <i>t</i>_−0.3). White symbols indicate cultures treated with H₂O₂. <i>t</i>₀ is indicated by a black arrow and hydrogen peroxide addition by a white arrow. This experiment was carried out four times and the results of one representative experiment are shown.</p

Strains used.

<p>Strains used.</p

Sulphur metabolism genes expressed more strongly in the wild-type strain than in the <i>B. cereus plcRa</i> mutant strain^a.

<p>a. Genes with at least a three-fold difference in expression are shown. In the case of operons (probable or demonstrated) ratios below 3 were considered acceptable.</p><p>b. Locus tag in type strain ATCC 14579.</p><p>c. The gene names indicated correspond to <i>B. subtilis</i> homologs, with the exception of <i>dps2</i> (BA5290), which corresponds to a homologous gene in <i>B. anthracis</i>.</p><p>d. Cultures for RNA extraction were collected one hour (<i>t</i>₁) or two hours (<i>t</i>₂) after the entry into stationary phase.</p

Electrophoretic mobility shift assay to determine conditions of PlcRa binding to <i>abrB2</i> promoter region.

<p>Fragment was generated by PCR amplification and end labeled with biotine. A constant amount of probe (5 fmol) was incubated at room temperature with the indicated concentrations of PlcRa without (A) or with PapRa₇ (B, C) at these concentrations: 0.2 µM (well 1, 2), 2 µM (well 3) and 20 µM (well 4) final concentration. C. The EMSA was carried out in the presence of 500-fold excess (wells 1–4) of the same unlabeled PCR-amplified DNA. Samples were run on 6% non-denaturing polyacrilamide gels.</p

Genes expressed more strongly in the wild-type strain than in the <i>B. cereus plcRa</i> mutant strain^a.

<p>a,b,c,d. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051047#pone-0051047-t002" target="_blank">Table 2</a> for legends.</p

<i>In silico</i> analysis of PapRa_7, a new putative signal peptide.

<p>A. Sequence alignment of <i>B. thuringiensis</i> 407 Cry- PapR and <i>B. cereus</i> ATCC 14579 PapRa. The putative signal sequences are coloured in red and PapRa₇ (CSIPYEY) and PapR₇ (ADLPFEF) heptapeptides are highlighted in blue. B. <i>In silico</i> docking of PapRa₇ in PlcRa TPR pocket. Close 90° view of the interaction of CSIPYEY with residues K89 (TPR1 motif), Q161 (TPR2 motif), N205 (TPR3 motif), K208 (TPR3 motif) & Y280 (TPR5 motif) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051047#pone-0051047-g001" target="_blank">Figure 1</a> for TPRs location). The residues N205 and K208 mediate peptide main chain binding by hydrogen bonds.</p

Addition of synthetic PapRa₇ or overexpression of <i>papRa</i> enhanced <i>abrB2</i> gene expression in a PlcRa-dependent manner.

<p>A. Expression of the P<i>_abrB2</i>’-<i>lacZ</i> fusion in the wild-type and in the Δ<i>plcRa</i> mutant strains in the presence of synthetic PapRa₇. The cells were grown at 37°C in LB medium and PapRa₇ was added at <i>t</i>_0.2 (onset of stationary phase) at different concentrations: 2 µM or 4 µM or 20 µM. Dashed lines correspond to LB cultures with PapRa_7, and thick line corresponds to LB culture without PapRa₇. B. Expression of the P<i>_abrB2</i>’-<i>lacZ</i> transcriptional fusion in the wild-type strain carrying pHT1618P_xyl’-<i>papRa</i>. The cells were grown at 37°C in HCT medium in the presence or absence of 10 mM xylose. Xylose was added at <i>t</i>₋₁ as indicated by a white arrow.</p

Prion Protein and Shadoo Are Involved in Overlapping Embryonic Pathways and Trophoblastic Development

<div><p>The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various <em>Prnp</em> transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the <em>Prnp</em>-knockout-<em>Sprn</em>-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various <em>Prnp</em> and <em>Sprn</em> genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in <em>Eutherians</em> and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.</p> </div