Mechanism insights into the regulation of the LuxS/AI-2 quorum sensing system on the formation of viable but nonculturable state in biofilm cells of beer-spoilage Lactiplantibacillus plantarum

Beer spoilage Lactiplantibacillus plantarum poses a significant challenge in brewing due to biofilm formation and entry into the viable but nonculturable (VBNC) state, evading detection and resisting sterilization. The LuxS/AI-2 quorum sensing (QS) system is implicated in stress adaptation, yet its regulatory role in biofilm-associated VBNC transition remains poorly understood OBJECTIVES: This study aims to elucidate how the luxS gene regulates VBNC-state formation in biofilm-embedded L. plantarum under hop-derived iso-α-acid stress and to identify molecular mechanisms underlying QS-mediated adaptation and resuscitation. Wild-type and genetically engineered L. plantarum strains (ΔluxS, luxS-overexpressing) were exposed to iso-α-acids to induce VBNC states. Morphological changes were analyzed via scanning electron microscopy. Temporal luxS expression and AI-2 levels were quantified using qRT-PCR and bioluminescence assays. Multi-omics (transcriptomics, proteomics, ChIP-seq) and phenotypic analyses assessed metabolic reprogramming, gene regulation, and resuscitation dynamics. Iso-α-acids (27 mg/L) induced complete VBNC transition in biofilms within 12 h, with cells exhibiting reduced volume and surface polymer deposition. luxS expression and AI-2 levels synchronized during induction, peaking at 4 h. Overexpression of luxS accelerated VBNC entry and enhanced resuscitation (>103 CFU/mL recovery), while ΔluxS impaired both processes. Multi-omics revealed luxS activation of carbohydrate metabolism (e.g., pgmB, rbsUDK operon) and stress-response genes (nrdD, pepc). ChIP-seq confirmed direct LuxS binding to promoter regions of these genes, orchestrating energy homeostasis and oxidative defense via pentose phosphate and ABC transporter pathways. The LuxS/AI-2 QS system governs biofilm-to-VBNC transition in L. plantarum by modulating metabolic resilience and stress adaptation. These findings provide a molecular framework for targeting QS pathways to mitigate biofilm persistence and spoilage risks in brewing industries.