Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L. lactis plant isolates. Diagnostic sequencing of their genomes resulted in over 2.5 Mb of sequence for each strain. A high synteny was found with the genome of L. lactis IL-1403, which was used as a template for contig mapping and locating deletions and insertions in the plant L. lactis genomes. Numerous genes were identified that do not have homologs in the published genome sequences of dairy L. lactis strains. Adaptation to growth on substrates derived from plant cell walls is evident from the presence of gene sets for the degradation of complex plant polymers such as xylan, arabinan, glucans, and fructans but also for the uptake and conversion of typical plant cell wall degradation products such as ␣-galactosides, -glucosides, arabinose, xylose, galacturonate, glucuronate, and gluconate. Further niche-specific differences are found in genes for defense (nisin biosynthesis), stress response (nonribosomal peptide synthesis and various transporters), and exopolysaccharide biosynthesis, as well as the expected differences in various mobile elements such as prophages, plasmids, restrictionmodification systems, and insertion sequence elements. Many of these genes were identified for the first time in Lactococcus lactis. In most cases good correspondence was found with the phenotypic characteristics of these two strains.Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products. Because of its tremendous industrial importance, numerous studies have been dedicated to the elucidation of the physiology and molecular biology of many traits relevant for industrial applications, including the production of flavor compounds, vitamins and other nutraceuticals, and exopolysaccharides relevant for texture development (33,60,68). Moreover, due to the availability of a vast molecular toolbox for genetic engineering and recent genome sequencing efforts, L. lactis has gained a strong position as a model organism for low-GC gram-positive bacteria and lactic acid bacteria in particular (7,39,43). Much of the biochemical and genetic research has been conducted with a limited number of strains, mainly the plasmid-cured strains IL-1403 and MG1363, which originate from dairy fermentations.(Fermenting) plant material is a second important ecosystem occupied by L. lactis, where it typically occurs as an early colonizer that is later replaced by species that are more tolerant of low pH values (30, 31). Most plant-associated strains belong to Lactococcus lactis subsp. lactis, whereas Lactococcus lactis subsp. cremoris is typically found in dairy fermentations (30, 31). Fermenting ...