Ecological diversification and isolate-specific acidification in Lactococcus lactis

Lactococcus lactis is a cornerstone of dairy fermentation, yet the links between its genomic diversity, ecological origin, and technological performance remain incompletely resolved. Here, we integrated whole-genome sequencing, functional annotation, phenotyping, and structural modeling across 147 globally sourced L. lactis isolates to test whether niche adaptation drives lineage-specific fermentation traits. Phylogenomic analysis revealed 5 clades strongly structured by isolation source, dairy versus plant/environmental, accompanied by clade-level differences in genome size, carbohydrate-active enzyme repertoires, clusters of orthologous genes' functional profiles, and bacteriocin biosynthetic potential. Dairy-associated clades showed lineage-dependent genomic architectures and functional specialization, rather than a uniform pattern of genome reduction. In particular, clades A and B possessed relatively larger genomes, whereas other clades displayed more compact genome sizes, suggesting that niche adaptation in L. lactis may involve both gene retention and gene loss depending on lineage history. Although plant-derived clades retained broader carbohydrate utilization capabilities, surprisingly, acidification kinetics showed no significant clade-level differences; instead, fermentation performance was predominantly isolate-specific. Genome-wide association analysis, corrected for phylogeny, identified kdgA, oppC, recF, gpr, and xseB as key loci associated with titratable acidity. AlphaFold2 modeling revealed that fast- and slow-acidifying variants differed in localized structural flexibility rather than global fold. These findings demonstrate that whereas L. lactis has diversified along ecological lines, its core technological trait, acidification, is governed by fine-scale genetic variation. This work provides a rational basis for precision starter selection and advances understanding of bacterial domestication in food ecosystems.