Biology subjects

Hols, P.

Publications and source records attributed to Hols, P..

5 recordsLinked to original sources

bioRxiv2026

A streamlined ABC extruder-repressor module drives multi-bacteriocin resistance in streptococci

Bacteria inhabiting competitive microbial environments must rapidly detect and neutralize antimicrobial peptides (AMPs) produced by rivals. The activation of defense pathways relies on dedicated sensors and complex regulatory cascades. Here, we uncover a streamlined, membrane-embedded mechanism in Gram-positive bacteria that directly links detection to transcriptional control. We show that a YtrA-family transcriptional repressor is regulated through an unconventional direct physical interaction with its cognate ABC transporter. In Streptococcus salivarius, the MbrAB efflux pump sequesters the YtrA-like repressor MbrR through a competitive binding mechanism. Upon bacteriocin sensing, MbrR shifts from promoter-proximal DNA repression sites to membrane-associated sequestration via interaction with MbrA, thereby activating the bacteriocin defense system. ATP binding by MbrA facilitates MbrR recruitment, whereas ATP hydrolysis promotes its release, providing a dynamic, flux-responsive feedback loop finely tuned to environmental threat. Structural modeling, synteny, and conservation analyses reveal that this transporter-mediated sequestration mechanism is highly conserved across Gram-positive bacteria, suggesting a widespread and efficient strategy for AMP resistance that bypasses the need for classical sensor kinases. These findings expand the known repertoire of bacterial sensing and resistance systems and provide new insight into how Gram-positive bacteria swiftly adapt to interbacterial antagonism. IMPORTANCEBacteria in crowded microbial communities face constant chemical warfare, yet how they sense and counteract antimicrobial peptides (AMPs) remains incompletely understood. Here we uncover a minimalist sensing-response module in streptococci in which YtrA-family transcriptional repressors are directly regulated by their partner ABC transporters. In Streptococcus salivarius, the MbrAB transporter detects incoming bacteriocins and physically sequesters the MbrR repressor, triggering rapid induction of a multi-gene defense program. ATP binding drives MbrR capture, while hydrolysis resets the system, providing a fast, energy-coupled switch that bypasses canonical two-component signaling. Comparative genomics shows that this transporter-repressor circuit is highly conserved across Bacillota, pointing to a broadly distributed and efficient strategy for AMP resistance. These findings reveal a direct physical link between membrane transport and transcriptional control, redefining how Gram-positive bacteria can sense and respond to microbial threats. ONE SENTENCE SUMMARYWe identify a conserved transporter-repressor module in streptococci that directly couples bacteriocin detection to transcriptional activation, revealing a minimalist and rapid mechanism for antimicrobial peptide resistance.

microbiology↗

bioRxiv2025

Chemical dysbiosis byproducts trigger predation via alternative activation of a peptide quorum sensor in salivarius streptococci

Cell-to-cell communication in Gram-positive bacteria is predominantly orchestrated by cytoplasmic sensors of the RRNPPA family. To date, all characterized members of this family are activated by small, unmodified peptide pheromones that mediate bacterial signaling. In the human commensal Streptococcus salivarius, the RRNPPA sensor ComR controls both competence (DNA transformation) and predation (bacteriocin production). Here, we reveal that ComR can be dually activated by its cognate peptide (XIP) and a distinct class of small organic molecules. A targeted screen of [~]200 organic compounds identified hydroxyphenylacetic acid (HPAA), a bacterial dysbiosis byproduct accumulating in human fluids, as a potent inducer of ComR. Using in vivo and in vitro approaches, we demonstrated that HPAA and structurally related carboxylic acids derived from bulky hydrophobic amino acids bind the pheromone-accommodating pocket, leading to ComR activation. Strikingly, while XIP-mediated activation is transient and regulates both competence and predation, HPAA induces a sustained, predation-oriented response. Furthermore, we showed that Porphyromonas gingivalis, an oral pathogen, produces sufficient (H)PAA quantity to trigger bacteriocin production in S. salivarius, revealing a previously unrecognized chemical interplay between oral microbiota members. These findings highlight the remarkable versatility of cytoplasmic sensors to integrate diverse environmental cues, shedding new light on bacterial peptide-based communication and microbial homeostasis in the human microbiome. IMPORTANCEBacterial communication through quorum sensing (QS) is crucial for coordinating key physiological processes. While QS in Gram-positive bacteria has been predominantly associated with peptide pheromones, our study uncovers an undescribed alternative signaling mechanism. We demonstrate that ComR, a cytoplasmic receptor of the RRNPPA family, can be activated by its canonical peptide signal and by small organic molecules derived from the anaerobic breakdown of hydrophobic amino acids. This alternative activation pathway enhances the ability of S. salivarius to respond to microbial dysbiosis, maintaining ecological balance in the digestive tract. Beyond revealing a novel layer of bacterial communication, our findings suggest that many RRNPPA-family receptors previously considered "orphan" may respond to yet-undiscovered chemical signals. This work expands our understanding of bacterial sensing and opens new avenues for modulating microbial interactions in health and disease.

microbiology↗

bioRxiv2024

Uncovering the class II-bacteriocin predatiome in salivarius streptococci

Facing the surge of antibiotic resistance, the medical field has a critical need for alternatives to treat bacterial infections. Among these, the use of bacteriocins, ribosomally-synthesized antimicrobial peptides produced by bacteria, is considered to be a promising route. In the human commensal Streptococcus salivarius, the production of unmodified class II bacteriocins is directly controlled at the transcriptional level by the quorum-sensing ComRS system. Here, we used an integrated approach combining bioinformatics and synthetic biology to identify novel bacteriocins from salivarius streptococci active against human pathogens. We developed a bioinformatic pipeline that combines conservation of DNA motifs for genetic regulation and features of bacteriocin primary sequences to uncover cryptic class II bacteriocins. Notably, we discovered more than 50 novel bacteriocin candidates clustered into 21 groups from 100 genomes of S. salivarius. Strain-based analysis of bacteriocin cocktails revealed an important diversity restricted to seven distinct loci, probably resulting from bacteriocin intra- and inter- species exchanges. Using in vitro or in vivo production and synthetic biology tools, we showed that most of them are active against a panel of Gram-positive bacteria, including clinically- relevant pathogens. Overall, this work provides a new search-to-test generic pipeline for the discovery of novel bacteriocins in Gram-positive bacteria that could be used in cocktails for broad applications in the food and biomedical fields. IMPORTANCETo survive in highly challenging environments, streptococci have evolved a competence- predation coupling mechanism for genome plasticity. This developmental process is highly regulated at the transcriptional level, masking the predation killing effects in usual laboratory conditions. Here, we present a general strategy that combines bioinformatics and synthetic biology to unveil class II bacteriocins in streptococci. Its implementation to the beneficial species Streptococcus salivarius revealed around 40 class II salivaricin cocktails explained by the plasticity of seven independent loci. Notably, the salivaricin predatiome includes a subtle blend of fratricins, sobrinicins, and broad-spectrum bacteriocins with overlapping activities against a wide spectrum of low-GC Gram-positive bacteria, including notorious pathogens. Furthermore, most of those bacteriocins are predicted to be variants of a common -hairpin structure, indicating that their mode of action evolved convergently. Finally, the discovery of ca. 50 novel bacteriocins offers perspectives for the rational assembly of potent cocktails active against pathogenic staphylococci, streptococci or enterococci.

microbiology↗

bioRxiv2024

Unveiling the regulatory network controlling natural transformation in lactococci

Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we discovered that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium. IMPORTANCELactic acid bacteria (LAB) play important roles in our daily lives as members of our microbiota or as starters of dairy products. Understanding the natural horizontal gene transfer mechanisms that shape their genomes will allow us to better control and understand their evolution over time. The DNA transformation machinery is found in all beneficial LAB species. With the exception of streptococci, however, the conditions of its activation remain unknown. In this study, the physiological conditions that activate competence for DNA transformation in Lactococcus lactis, the most important lactococcal species, were identified. We also unveiled the guardians of the master competence regulator ComX. In this species, it is directly repressed by global carbon and nitrogen regulators (CcpA and CodY) as well as the general stress system CovRS. Additionally, it was discovered that the Clp machinery degrading ComX plays a dominant role in the strict control of competence activation. In Gram-positive cocci, the hierarchical organization of these regulators for controlling competence development in L. lactis is unprecedented.

microbiology↗

bioRxiv2022

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence in Streptococcus salivarius

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a sharp time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR-interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococus salivarius. We show that initiation of competence increases upon cell-wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell-wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell-wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCESurvival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR- interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell-wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

microbiology↗