Multi-omics and functional validation profiling reveals the regulatory mechanism of biofilm-forming Tetragenococcus halophilus JY1 in response to salt tolerance

Biofilms are crucial for enhancing microbial productivity in industrial applications, yet the mechanisms underlying their stress resistance and formation in functional microorganisms remain inadequately explored. Tetragenococcus halophilus , a halophilic lactic acid bacterium, is widely utilized in traditional fermentation industries; however, research has focused on its planktonic state. To address this gap, this study investigated the molecular mechanisms of biofilm formation in T. halophilus JY1 under low-salt (0.2 M, LS) and high-salt (3 M, HS) stress conditions. Physiological analyses revealed that biofilms protected cellular integrity under LS and HS, as evidenced by marked elevations in ATPase activity and ATP concentration. Furthermore, salt stress altered the abundance and composition of JY1 biofilms. RNA-seq analysis identified upregulation of genes associated with cell surface composition, energy homeostasis, and ion transport. Metabolomic profiling further revealed elevated levels of glycerophospholipids and fatty acids in JY1 under LS and HS. Functional validation further demonstrated that overexpression of ohrR , yodB , isaA , icaA , ruvA , and ctsR significantly promoted biofilm formation in recombinant strains. In addition, exogenous monounsaturated fatty acids (C16:1, C14:1, and C20:1) markedly improved cell survival and biofilm formation under salt stress by modulating membrane lipid composition and enhancing osmotic adaptation. Overall, restructuring of biofilms emerged as a key adaptive strategy for JY1 in response to salinity, while prolonged salt exposure reduced physiological and metabolic activity. This study provides critical insights into the adaptive strategies of biofilm-forming microorganisms and opens new avenues for the application of biofilms in industrial processes.