Plasmid-mediated modulation of Listeria monocytogenes biofilm formation via TCS/PTS signaling: Implications for food contamination control.

Abstract

Listeria monocytogenes thrives in diverse and often hostile environments by forming biofilms that act as protective physical barriers. While plasmids have been implicated in enhancing biofilm formation, the underlying regulatory mechanisms remain largely unexplored. In this study, representative wild-type L. monocytogenes strains and their plasmid-cured counterparts were selected from 33 food-derived isolates based on biofilm reduction rates. Their biofilm-forming ability was assessed under various food-relevant stress conditions, followed by comprehensive multi-omics analyses. Phenotypic differences in key regulatory pathways between wild-type and plasmid-cured strains were further validated to systematically elucidate the molecular mechanisms of plasmid-mediated biofilm regulation. The results identified three key plasmid-regulated pathways: (i) Flagellar assembly and exoprotein biosynthesis, which are regulated via two-component systems (TCS), are evidenced by the reduced initial aggregation capacity and extracellular protein content in plasmid-cured strains. (ii) Carbohydrate metabolism, particularly the modulation of fructose/mannose metabolism and d-glucose synthesis through the phosphotransferase system (PTS), was experimentally confirmed that this significantly reduces EPS content in plasmid-cured strains. (iii) Amino acid metabolism, specifically involving glycine, serine, and threonine pathways, was also affected; however, amino acid supplementation failed to restore biofilm formation to wild-type levels, suggesting a more complex regulatory interaction. Collectively, these findings provide the first systematic dissection of plasmid-mediated biofilm regulation in L. monocytogenes, linking mobile genetic elements to coordinated control of motility, metabolic reprogramming, and matrix production. This study deepens our understanding of L. monocytogenes biofilm physiology and offers a scientific foundation for developing targeted strategies to disrupt biofilm