PlcRa, a new quorum-sensing regulator from Bacillus cereus, plays a role in oxidative stress responses and cysteine metabolism in stationary phase

Abstract

We characterized a new quorum-sensing regulator, PlcRa, which is present in various members of the B. cereus group and identified a signaling heptapeptide for PlcRa activity: PapRa(7). We demonstrated that PlcRa is a 3D structural paralog of PlcR using sequence analysis and homology modeling. A comparison of the transcriptomes at the onset of stationary phase of a ΔplcRa mutant and the wild-type B. cereus ATCC 14579 strain showed that 68 genes were upregulated and 49 genes were downregulated in the ΔplcRa mutant strain (>3-fold change). Genes involved in the cysteine metabolism (putative CymR regulon) were downregulated in the ΔplcRa mutant strain. We focused on the gene with the largest difference in expression level between the two conditions, which encoded -AbrB2- a new regulator of the AbrB family. We demonstrated that purified PlcRa bound specifically to the abrB2 promoter in the presence of synthetic PapRa(7), in an electrophoretic mobility shift assay. We further showed that the AbrB2 regulator controlled the expression of the yrrT operon involved in methionine to cysteine conversion. We found that the ΔplcRa mutant strain was more sensitive to hydrogen peroxide- and disulfide-induced stresses than the wild type. When cystine was added to the culture of the ΔplcRa mutant, challenged with hydrogen peroxide, growth inhibition was abolished. In conclusion, we identified a new RNPP transcriptional regulator in B. cereus that activated the oxidative stress response and cysteine metabolism in transition state cells.

Figure 1. Homology modeling of PlcRa.

A. Alignment of the sequences of B. thuringiensis 407 Cry^- PlcR chain A (pdb code: 2QFC_A) and ATCC 14579 B. cereus 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.

Figure 2. In silico analysis of PapRa_7, a new putative signal peptide.

A. Sequence alignment of B. thuringiensis 407 Cry- PapR and B. cereus ATCC 14579 PapRa. The putative signal sequences are coloured in red and PapRa₇ (CSIPYEY) and PapR₇ (ADLPFEF) heptapeptides are highlighted in blue. B. In silico 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 Figure 1 for TPRs location). The residues N205 and K208 mediate peptide main chain binding by hydrogen bonds.

Figure 3. Analysis of plcRa expression.

A. Kinetics of plcRa gene expression. Specific β-galactosidase activity (U/mg protein) of strain B. cereus ATCC 14579 harboring the transcriptional P_plcRa’-lacZ fusion. Time zero corresponds to the onset of the stationary growth phase, and t _n is the number of hours before (–) or after time zero. The cells were grown at 37°C in LB medium. Error bars are shown. B. Determination of the transcriptional start site of plcRa. The 5′ RACE-PCR method was used to identify the transcriptional start site of plcRa. The start site (+1, in bold typeface) and the −10 and −35 putative promoter elements from the vegetative sigma factor are shown in bold typeface and underlined. The putative ribosome-binding site sequence and putative start codon of plcRa are shown in bold typeface and underlined.

Figure 4. PlcRa activates abrB2 gene expression early in stationary phase.

β-galactosidase specific activity (U/mg protein) of the wild-type (black circles), ΔplcRa (white triangles) and complemented ΔplcRa (white diamonds) strains harboring the transcriptional P_abrB2’-lacZ fusion, in LB. Errors bars are shown.

Figure 5. Electrophoretic mobility shift assay to determine conditions of PlcRa binding to abrB2 promoter region.

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.

Figure 6. Addition of synthetic PapRa₇ or overexpression of papRa enhanced abrB2 gene expression in a PlcRa-dependent manner.

A. Expression of the P_abrB2’-lacZ fusion in the wild-type and in the ΔplcRa mutant strains in the presence of synthetic PapRa₇. The cells were grown at 37°C in LB medium and PapRa₇ was added at t _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_abrB2’-lacZ transcriptional fusion in the wild-type strain carrying pHT1618P_xyl’-papRa. The cells were grown at 37°C in HCT medium in the presence or absence of 10 mM xylose. Xylose was added at t ₋₁ as indicated by a white arrow.

Figure 7. AbrB2 controls the expression of yrrT operon, involved in methionine to cysteine conversion.

β-galactosidase specific activity (U/mg protein) of wild-type (black circles), ΔplcRa (black triangle) and ΔabrB2 (black diamonds) strains harboring both pHT304_yrrT’-lacZ and pHT1618KΩP_xyl-abrB2 plasmids, in HCT. See legends figure 5 for growth conditions. White symbols indicate cultures in the presence of xylose_.

Figure 8. Sensitivity to peroxide and disulfide stress of a B. cereus plcRa mutant.

We assessed the viability of wild-type (WT), ΔplcRa (plcRa) and complemented ΔplcRa (plcRa+) strains in early stationary phase. Early stationary-phase cells grown in LB medium (∼ OD 3, ∼ t _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.**: P<0.01. C. The addition of cystine strongly improved the peroxide stress resistance of the plcRa mutant. We assessed the growth inhibition of wild-type and ΔplcRa 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 (∼ t _−0.3). White symbols indicate cultures treated with H₂O₂. t 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.