Structure of PlcR: Insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria

Abstract

Gram-positive bacteria use a wealth of extracellular signaling peptides, so-called autoinducers, to regulate gene expression according to population densities. These "quorum sensing" systems control vital processes such as virulence, sporulation, and gene transfer. Using x-ray analysis, we determined the structure of PlcR, the major virulence regulator of the Bacillus cereus group, and obtained mechanistic insights into the effects of autoinducer binding. Our structural and phylogenetic analysis further suggests that all of those quorum sensors that bind directly to their autoinducer peptide derive from a common ancestor and form a single family (the RNPP family, for Rap/NprR/PlcR/PrgX) with conserved features. As a consequence, fundamentally different processes in different bacterial genera appear regulated by essentially the same autoinducer recognition mechanism. Our results shed light on virulence control by PlcR and elucidate origin and evolution of multicellular behavior in bacteria.

Fig. 1.

Structure of PapR5:PlcR and comparison to cCF10:PrgX. Shown are ribbon presentations of PapR5:PlcR (A) and cCF10:PrgX (B) [Protein Data Bank entry 2AXZ (9)]. Chains A and B of PlcR and PrgX are colored in magenta and cyan, with their respective HTH domains in dark gray and dark blue. Helix 3 of the HTH domains that inserts into the major DNA groove is colored in red. The C-terminal extension of PrgX is colored in light gray. Ligand peptides are shown in yellow and green. Left and Right represent 90° views. (C and D) Close-up views of the peptide–protein interactions for PapR:PlcR (C) and cCF10:PrgX (D). Oligopeptides are yellow, and key residues are labeled. Unbiased 3F_o–2F_c electron density for PapR5 is shown in gray. Hydrogen bonds between cCF10 and PrgX key residues are indicated by black lines.

Fig. 2.

SAXS data and fit of models. (A) SAXS pattern for apo-PlcR (black, 1.2 mg/ml; gray, 3.6 mg/ml) and PlcR in the presence of PapR5/7 at 1.2 mg/ml (blue, PapR5; light blue, PapR7), 3.0 mg/ml (green, PapR5; light green, PapR7), and 3.6 mg/ml (dark red, PapR5; red, PapR7). Intensities are in arbitrary units (AU). Curves were offset for better visibility. (B) Three perpendicular views of the crystallographic PapR5:PlcR dimer (color scheme as in Fig. 1A) fitted into the averaged SAXS ab initio shape obtained for apo-PlcR (gray spheres). Arrows indicate HTH movements predicted for apo-PlcR. (C) Fit of calculated (line) to experimental (points) SAXS curves. Top line, crystallographic PlcR dimer to apo-PlcR; middle line, crystallographic PlcR tetramer to PapR7:PlcR at 1.2 mg/ml; bottom line, crystallographic (blue) and refined (red) PapR:PlcR dodecamer to PapR5:PlcR at 3.6 mg/ml.

Fig. 3.

PapR triggers PlcR polymerization. (A) Ninety-degree views of the crystallographic tetramers (ribbon representation) fitted into the ab initio SAXS envelope (gray) obtained for PapR:PlcR at 1.2 mg/ml. Type I and type II dimer axes are indicated. (B) Tetramer, obtained after rigid body refinement, superimposed onto the SAXS envelope obtained for PapR:PlcR at 3.0 mg/ml. (C and D) Ninety-degree views of SAXS envelopes (gray) obtained for data of PapR:PlcR at 3.6 mg/ml, superimposed on PlcR hexa-(dimers) taken from the crystal lattice (C) and after rigid body refinement (D). Spiral axes of models are indicated.

Fig. 4.

Molecular effects of peptide recognition. (A) The curvature of the PrgX TPR-like domain increases upon autoinducer binding. Cyan, apo-PrgX (Protein Data Bank entry 2AXU); magenta, cCF10:PrgX (Protein Data Bank entry 2AXZ). The pheromone (yellow) is shown in stick representation. (B) Drawing illustrating how the increased TPR domain curvature in PapR:PlcR may rearrange the HTH domains. (Lower) apo-PlcR. (Upper) PapR:PlcR.