The peptide pheromone-inducible conjugation system of Enterococcus faecalis plasmid pCF10: cell-cell signalling, gene transfer, complexity and evolution

Abstract

Expression of a large set of gene products required for conjugative transfer of the antibiotic resistance plasmid pCF10 is controlled by cell-cell communication between plasmid-free recipient cells and plasmid-carrying donor cells using a peptide mating pheromone cCF10. Most of the recent experimental analysis of this system has focused on the molecular events involved in initiation of the pheromone response in the donor cells, and on the mechanisms by which the donor cells control self-induction by endogenously produced pheromone. Recently, studies of the molecular machinery of conjugation encoded by the pheromone-inducible genes have been initiated. In addition, the system may serve as a useful bacterial model for addressing the evolution of biological complexity.

Figure 1

Map of pCF10. The figure shows the approximate positions of a number of genes involved in replication, pheromone sensing and control, and conjugative DNA transfer as described in the text. The curved inner lines depict contiguous segments of pCF10 encoding the biological functions indicated. As described in the text, each of these segments probably originated from a different ancestral source. With the exception of prgX, all of the genes from prgW–pcfH are transcribed in the clockwise direction. The transposon Tn925 encodes the tetracycline resistance determinant tetM.

Figure 2

Two models for the role of signalling peptides in expression of pCF10 conjugation functions. The single arrows indicate positive control, the inverted arrows indicate negative control and the double arrows indicate polypeptide synthesis from a plasmid (iCF10, Asc10) or chromosomal (cCF10) gene. Original model: initial studies suggested a simple model whereby a single unidirectional pheromone signal from recipient cells to donors caused induction of the transfer functions, including cell aggregation mediated by Asc10. Current model: further analysis of the system has shown that both donors and recipients can produce pheromone, and that the molar ratio of chromosomally encoded pheromone (cCF10) to plasmid-encoded inhibitor (iCF10) determines the induction state of the donor cell. In a monoculture of donor cells growing in laboratory medium, the balance of these two peptides keeps the transfer system off. The balance can be shifted in favour of pheromone, either by production of pheromone by recipient cells in close proximity (upper left) or by interaction of inhibitor with plasma components (upper right) when the bacteria are growing in the bloodstream of a mammalian host.

Figure 3

DNA looping model for PrgX. (a) Uninduced donor cell. PrgX molecules bound to each DNA target site interact (the model shown here has each site occupied by a PrgX dimer; the two dimers interact to form a tetramer) to form a DNA loop that stabilizes DNA/protein interactions and increases occupancy of both binding sites. RNA polymerase access to the prgQ promoter, which overlaps the lower binding site, is restricted. This reduces prgQ mRNA synthesis. (b) Pheromone-induced donor cell. Pheromone interaction with PrgX breaks up tetramers, opening the loop, decreasing occupancy of the binding sites and allowing for increased polymerase access to the prgQ promoter. This increases production of prgQ mRNA.

Figure 4

Structural consequences of pheromone and inhibitor binding to PrgX. Extensive intermolecular interactions between amino acid residues in the N-terminal and central domains of PrgX allow the protein to form a stable dimer in all structures examined. In the absence of exogenous peptides, as well as in the presence of iCF10, the PrgX C-terminus assumes a conformation that promotes interaction between pairs of dimers to form stable tetramers; this structure favours formation of DNA loops as shown in figure 3. When complexed with cCF10, the C-terminal arm changes the structure and rotates such that the protein–protein interactions favouring tetramer formation are weakened significantly. Both peptides occupy the same binding cleft formed by a series of parallel and anti-parallel helical domains in the central part of the protein and, in both cases, residues from the C-terminal region interact with the bound peptide. However, iCF10 interacts with different C-terminal PrgX residues than is the case for cCF10.