Enterococcal Sex Pheromones: Evolutionary Pathways to Complex, Two-Signal Systems

Abstract

Gram-positive bacteria carry out intercellular communication using secreted peptides. Important examples of this type of communication are the enterococcal sex pheromone systems, in which the transfer of conjugative plasmids is controlled by intercellular signaling among populations of donors and recipients. This review focuses on the pheromone response system of the conjugative plasmid pCF10. The peptide pheromones regulating pCF10 transfer act by modulating the ability of the PrgX transcription factor to repress the transcription of an operon encoding conjugation functions. Many Gram-positive bacteria regulate important processes, including the production of virulence factors, biofilm formation, sporulation, and genetic exchange using peptide-mediated signaling systems. The key master regulators of these systems comprise the RRNPP (RggRap/NprR/PlcR/PrgX) family of intracellular peptide receptors; these regulators show conserved structures. While many RRNPP systems include a core module of two linked genes encoding the regulatory protein and its cognate signaling peptide, the enterococcal sex pheromone plasmids have evolved to a complex system that also recognizes a second host-encoded signaling peptide. Additional regulatory genes not found in most RRNPP systems also modulate signal production and signal import in the enterococcal pheromone plasmids. This review summarizes several structural studies that cumulatively demonstrate that the ability of three pCF10 regulatory proteins to recognize the same 7-amino-acid pheromone peptide arose by convergent evolution of unrelated proteins from different families. We also focus on the selective pressures and structure/function constraints that have driven the evolution of pCF10 from a simple, single-peptide system resembling current RRNPPs in other bacteria to the current complex inducible plasmid transfer system.

FIG 1

Diagram of the signaling circuits in the E. faecalis pCF10 conjugation system (adapted from Annual Review of Genetics [19]). Recipient and donor have similar chromosomes, but the donor also carries pCF10. The plasmid confers a response to the chromosomally encoded peptide C, which induces conjugation. The plasmid encodes the antagonistic peptide I, which inhibits C competitively. Two constitutively expressed pCF10 gene products, PrgZ and PrgY, function in pheromone import and in reduction of the amount of active C excreted by plasmid-carrying cells, respectively, as detailed in the text. Imported C interacts with PrgX (not shown) in the cytoplasm to induce a conjugation response. Pheromone induction of donor cells results in the synthesis of conjugation-related gene products, including surface adhesin proteins, type 4 secretion proteins (T4SS), and DNA transfer proteins (DTR).

FIG 2

Genetic organization of pheromone-inducible conjugation genes found on enterococcal plasmids (approximate size of the entire region indicated at the top). This map depicts the prg genes of pCF10 with single-letter designations, but similar gene content and organization are found on other well-studied plasmids, such as pAD1 and pPD1 (17). The left portion of the map shows conserved genes involved in pheromone sensing, and the relative locations of the genes of the pheromone-inducible prgQ operon encoding the I peptide, surface adhesin gene module (ABUC), downstream type IV secretion system (T4SS) genes, and conjugative DNA transfer genes (Dtr) are shown. The prgQ gene encodes the production of I, whereas an ∼1-kb segment between prgQ and prgA encodes two small open reading frames (ORFs) and small RNAs (sRNAs) that regulate the expression of downstream genes posttranscriptionally (65). The sizes of the individual genes are not drawn to scale. I, the putative origin of the system as a surface protein module negatively regulated by quorum sensing through the X/Q cassette; this gene pair resembles RRNPP systems recently identified in numerous Gram-positive pathogens (21, 31). II shows how the system became more complex as it acquired the ability to enable its host cell to recognize C as an indicator of close proximity of plasmid-free recipients (mate sensing). At the mechanistic level, the C peptide competes with I, which functions as a classic quorum-sensing signal of donor density (self-sensing) (64). Evolution of the ability to differentially respond to these two antagonistic peptides was accompanied by the acquisition of genes encoding an oligopeptide binding protein, PrgZ, which binds both C and I with high affinity and increases their import via the Opp ABC transporter (37, 38), and PrgY, a predicted membrane peptidase that reduces the production of endogenous C by the host cell (36). III depicts the acquisition of T4SS and Dtr genes conferring conjugative transfer ability. There is high conservation of the regions indicated by I and II among many pheromone plasmids, suggesting that they all arose from a common ancestor, but step III likely occurred multiple times to link different conjugation gene cassettes to the pheromone-inducible aggregation module.

FIG 3

Comparison of the peptide binding of PrgZ (gray) complexed with C (a) and PrgX (dark red) complexed with C (b) or I (c). Each upper subfigure shows the full protein structure in a cartoon representation, with the bound ligand in spheres, C in teal, and I in green. The lower enlarged representations at the bottom show the ligand (as sticks) with interacting protein residues. As can be seen by comparing panels b and c, the C peptide has a very different structure when bound to PrgZ compared with its structure when bound to PrgX.

FIG 4

Predicted structure of PrgY. The extracellular part of PrgY, here shown as a cartoon representation, was modeled using Phyre2 and colored from the N terminus (blue) toward the C-terminal end of the model (yellow). The C-terminal domain, which could not be modeled, is predicted to contain 4 transmembrane helices, shown here as rectangles in a membrane. The predicted active site, based on the homology of PrgY to the Tiki metalloproteases (46), is shown within the dashed line.