Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides

Abstract

The transcriptional regulator PlcR and its cognate cell-cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR-PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli.

Figure 1.

Theoretical fragmentation of PapRI and vMALDI-MS/MS mass spectrum. (A) Theoretical fragmentation of the peptide ADLPFEF (PapR₇I). (B) vMALDI-MS/MS mass spectrum. Peaks in red correspond to b ion fragmentation expected masses, and peaks in blue to y ion fragmentation.

Figure 2.

Molecular model of PapR₇I bound to PlcRI. (A) Atomic model of PlcRI:PapR₇I. The structure was modelled based on PDB entry 2QFC. The two protomers of the PlcR dimer are coloured in red and cyan. Helices are represented by tubes, within the transparent molecular surface. PapR₇I is shown as a stick model. (B) PlcR is shown as molecular surface and PapR₇I as a stick model. Surfaces are coloured: blue, positively charged atoms; red, negatively charged atoms; green, hydrophobic atoms; salmon, polar oxygens; marine, polar nitrogens; yellow, sulphur. Underlined atom labels correspond to PlcR residues.

Figure 3.

Affinity of PlcR:PapR complexes. Anisotropy binding titrations of the fluorescein-labelled pentapeptide LPFEF (Fl- LPFEF) with PlcR, in the absence (closed circles) and in the presence of the competitor non-labelled peptides LPFEF (closed squares), PFEF (closed diamonds), ADLPFEF (closed triangles), VGADLPFEF (stars) and non-specific peptide (open squares). The concentration of Fl- LPFEF was 1.5 nM and that of the competitor peptides 121 µM. The K_d values shown correspond to analysis of the competition profiles using the model explained in Materials and methods section.

Figure 4.

Comparison of the activity of the four PlcRs in association with their cognate penta- and hepta-peptides. Synthetic penta- or hepta- peptides of PapRI (LPFEF, ADLPFEF), PapRII (MPFEF, SDMPFEF), PapRIII (VPFEF, NEVPFEF) and PapRIV (LPFEH, SDLPFEH) (2 µM) were added to a culture of 407 plcA′-lacZ ΔplcR-papR at stationary phase (OD₆₀₀ 3 ± 0.3), complemented with PlcRI (pHT304ΩplcRI), PlcRII (pHT304ΩplcRII), PlcRIII (pHT304ΩplcRIII) and PlcRIV (pHT304ΩplcRIV), respectively. β-Galactosidase assays were performed 1 h after peptide addition. Vertical bars: SEM.

Figure 5.

Cross-talk between the four PlcR groups. Synthetic heptapeptides PapRI (ADLPFEF), PapRII (SDMPFEF), PapRIII (NEVPFEF) or PapRIV (SDLPFEH) (2 µM) were added to a stationary phase culture of 407 plcA′-lacZ ΔplcR-papR (OD₆₀₀ 3 ± 0.3) complemented with PlcRI, PlcRII, PlcRIII and PlcRIV, respectively. β-Galactosidase assays were performed 1 h after peptide addition. Vertical bars: SEM.

Figure 6.

Model structures for the complexes formed by PlcRI–IV and their cognate PapR heptameric peptides. Underlined atom labels correspond to PlcR residues. (A) Molecular detail of PlcRI (ribbon representation) in a complex with PapRI (stick model). (B) PlcRII in a complex with PapRII. (C) PlcRIII in a complex with PapRIII. (D) PlcRIV in a complex with PapRIV. The arrow indicates the L246 residue.

Figure 7.

Activation of modified PlcR proteins. Synthetic heptapeptides of PapRI (ADLPFEF) (2 µM) were added to a culture of 407 plcA′-lacZ ΔplcR–papR (OD₆₀₀ 3 ± 0.3) at stationary phase complemented with PlcRK87A, PlcRK89A, PlcRK87A-K89A and PlcRY275A, respectively. The PlcR–PapR complex activity was measured by β-galactosidase assays 1 h after peptide addition. Vertical bars: SEM.

Figure 8.

Sequence alignment of seven representative PlcR sequences. Sequence alignment of seven representative PlcRs was performed with JalView, using the Blosum62 colouring scheme. The strain names are indicated in parentheses and the roman numbers refer to the PlcR groups. Based on 3D-structure, HTH domain and TRP domains are indicated in yellow and in blue, respectively. The α-helices from the HTH domain are in green. The first and the last α-helices from TPR domains are in light yellow and in orange, respectively. Asterisks represent PlcR residues implicated in PapR binding. Position 278 is coloured in red.

Figure 9.

Activation of chimeric PlcR proteins. Synthetic heptapeptides of PapRI (ADLPFEF), PapRII (SDMPFEF), PapRIII (NEVPFEF) or PapRIV (SDLPFEH) (2 µM) were added to a culture of 407 plcA′-lacZ ΔplcR–papR (OD₆₀₀ 3 ± 0.3) at stationary phase complemented with PlcRI, PlcRI′stop (A), PlcRI′II and PlcRA278K (B), PlcRI′III (C) and PlcRI′IV (D), respectively. The PlcR–PapR complex activity was measured by β-galactosidase assays 1 h after peptide addition. Vertical bars: SEM.