Bourassa, J.-S. | Search bioRxiv

bioRxiv2026

High-throughput dual bioreporter screening reveals distributed regulation of biofilm matrix components in Staphylococcus aureus

Staphylococcus aureus biofilm formation is a key factor enabling persistent infections. However, the lack of efficient high-throughput tools previously limited systematic study of its regulatory mechanisms. Here, we used high-efficiency transduction to construct two luminescent bioreporter libraries, each probing a distinct biofilm regulatory pathway. Derived from the Nebraska Transposon Mutant Library, these libraries enabled rapid, quantitative screening of biofilm-associated gene expression in a high-throughput format. Our screens revealed a surprising lack of overlap in the regulation of the two biofilm components investigated: adhesin synthesis and extracellular DNA production. However, we identified mntR as a key gene involved in the expression of both biofilm components and confirmed the previously reported role of yjbH. Cross-lineage validation showed that these regulators retain conserved significance across multiple S. aureus backgrounds, although their phenotypic effects varied across strains. Collectively, this work provides a versatile, high-throughput framework to dissect the regulatory networks underlying complex phenotypes in S. aureus. ImportanceBiofilm formation is a major contributor to the persistence and treatment failure of Staphylococcus aureus infections, yet its regulatory network remains incompletely understood. We developed a high-throughput bioreporter platform that enables genome-wide screening of biofilm-associated gene expression across nearly 2,000 transposon mutants. Using this approach, we show that key biofilm processes, adhesion and extracellular DNA release, are controlled by largely distinct regulatory networks, and we identify mntR as a previously unrecognized regulator shared by both pathways. Beyond these biological insights, our work provides a versatile and readily adaptable strategy for dissecting complex regulatory systems in S. aureus and other bacterial species.

microbiology↗

bioRxiv2022

Second-generation transfer mediates efficient propagation of ICEBs1 in biofilms.

Horizontal gene transfer (HGT) by integrative and conjugative elements (ICEs) is an important mechanism in the spread of antibiotic resistance genes. However, little is known about the spatiotemporal dynamic of ICEs propagation in bacterial biofilms, which are multicellular structures ubiquitous in the natural and clinical environment. Using a fluorescently marked ICEBs1, we report here that its propagation in biofilms is favored in recipient cells expressing the biofilm matrix. Also, conjugation appears restricted to clusters of bacteria in a close neighborhood in which a high level of ICEBs1 transfer occurs. These conjugative clusters are heterogenously distributed in the biofilm, forming close to the air-biofilm interface. Importantly, we established that transconjugant cells are the main contributors to ICEBs1 propagation in biofilms. Our findings provide a novel spatiotemporal understanding of ICEs propagation in biofilms, which should have an important role in our understanding of horizontal gene transfer in relevant settings. ImportanceThe transfer of mobile genetic elements between bacteria is the main cause of the spread of antibiotic resistance genes. While biofilms are the predominant bacterial lifestyle both in the environment and in clinical settings, their impact on the propagation of mobile genetic elements is still poorly understood. In this study, we examined the spatiotemporal propagation of the well-characterized integrative and conjugative element (ICE) ICEBs1. Using the Gram-positive B. subtilis, we observed that the main actors of ICEBs1 propagation in biofilms are the newly formed transconjugants that allow rapid transfer of ICEBs1 to new recipients. Our study provides a better understanding of the spatiotemporal dynamic of conjugative transfer in biofilms.

microbiology↗