Unraveling novel acid tolerance mechanisms in Lactococcus lactis through  physiological and multi-omics analyses
Lactococcus lactis (L. lactis) is a key lactic acid bacterium widely used as a starter culture in food fermentation. However, acid stress during industrial processes poses a major challenge, impairing cell viability, metabolic activity, and fermentation performance, ultimately compromising the quality and consistency of fermented products. To uncover novel mechanisms underlying acid tolerance, we screened 60 L. lactis isolates from traditional fermented dairy products and identified XGL9-3 as highly acid-tolerant and XZ5304 as acid-sensitive. Comparative physiological analyses revealed that isolate XGL9-3 maintained superior cell membrane integrity, increased fatty acid unsaturation, synthesized the protective C19-cyclopropane fatty acid, and sustained intracellular pH homeostasis through elevated H+-ATPase activity and ATP production. Whole-genome sequencing showed that XGL9-3 possessed an expanded repertoire of genes involved in carbohydrate metabolism, energy production, and cell envelope biogenesis. Transcriptomic profiling of XGL9-3 under acid stress identified 317 differentially expressed genes (202 upregulated, 115 downregulated), including key players in ABC transporters, purine metabolism, and cationic antimicrobial peptide resistance. Integrated multi-omics analysis of XGL9-3 uncovered a coordinated acid tolerance strategy involving membrane remodeling, enhanced proton efflux, metabolic reprogramming, and modulation of quorum sensing pathways. Notably, the most significantly upregulated gene was msmX, encoding an ABC transporter subunit. This study identifies previously undercharacterized mechanisms, particularly C19-cyclopropane fatty acid synthesis and quorum-sensing regulation, as central to acid resistance in L. lactis, moving beyond classical pathways focused on F0F1-ATPase or the glutamatedecarboxylase system. These findings provide novel insights into the molecular basis of acid adaptation inL. lactis and offer promising targets for engineering robust starter strains for acidic fermented food industry.