Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure–function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a ‘test-bed’ assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides. Together, our results not only provide a model for XIP recognition and specificity, but also allow for the prediction of novel XIP peptides that induce ComR activity. Bacteria transmit chemical signals to each other in a process known as quorum sensing. This adaptation is central to pathogenesis as it allows bacteria to coordinate a group response, such as when to form a biofilm, secrete virulence factors, or uptake new DNA. Quorum sensing in many species of Gram-positive bacteria utilizes secreted short peptide signals that are sensed by protein receptors that alter gene expression. The ComR-ComS receptor-signal pair is a regulator of competence for genetic transformation that is broadly conserved in a majority of Streptococcus species, including those pathogenic in humans and animals. Despite this conservation, we observe sequence diversity among ComRS orthologs that raises questions relating to sensory specificity and the molecular mechanism of peptide recognition. To address this, we directly tested the possibility for signaling cross-talk and identified three general categories of ComR receptors displaying strict, intermediate, and promiscuous responses to heterologous peptides. To elucidate the molecular mechanism of receptor specificity we determined an X-ray crystal structure of ComR from S. suis. We observe a conserved face of the ligand pocket important for peptide binding and a variable face that functions in peptide specificity. Finally, basic criteria necessary for peptide responses were used to redesign active peptides from inactive templates.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci. Bacterial cell-cell communication systems are based on the secretion of signal molecules. These quorum-sensing systems allow bacteria to coordinate genes expression according to the density of their local population. In Gram-positive bacteria, intracellular quorum sensors regulated by re-internalized signal peptides control the expression of genes involved in essential bacterial processes such as horizontal gene transfer, biofilm formation, sporulation or virulence. In most streptococci, including pathogenic species, the ComR regulator and its cognate signal peptide ComS activate competence for natural DNA transformation, a major mechanism for horizontal gene transfer and antibiotic resistance acquisition. To elucidate the molecular mechanism of ComR activation, we performed the structure-function analysis of the ComRS system from S. thermophilus. We solved the crystal structures of the apo form of ComR and of the complex with XIP, the mature form of ComS, and DNA. We showed that peptide binding shifts the protein from an inactive monomeric state, characterized by the sequestration of the DNA-binding domain, to an active dimer. This idiosyncratic mechanism was confirmed by in vitro interaction measurements and in vivo transcription assays using ComR mutants and XIP variants. These results may be of special interest for the future design of new antimicrobials targeting antibiotic-resistant pathogens.

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.