Comparative genomics and methylome profiling of Pseudolactococcus laudensis reveal signatures of niche adaptation and strain-level variation in mobile genetic elements and phage defence.

Pseudolactococcus laudensis (formerly named Lactococcus laudensis) is an emerging lactic acid bacterium first isolated from raw milk in 2015 and subsequently detected in vegetables and dairy mesophilic starter cultures. Despite its recurrent isolation from diverse environments, the genetic basis of its niche adaptation, horizontal gene transfer and phage defence remains unexplored. Here, we perform the first comparative genomic and epigenomic analysis of P. laudensis using complete genomes of a plant-derived isolate (MCRI-603), a milk isolate (DSM 28961) and 20 strains from a Danish dairy mesophilic starter culture. Genomes were annotated and analysed using pangenomics, Clustering of Orthologous Genes and methylome profiling. Average nucleotide identity, pangenome and Clustering of Orthologous Genes analyses revealed niche-associated structure: dairy starter strains formed a tight cluster, while the plant isolate MCRI-603 and milk isolate DSM 28961 were more similar to each other than to the starter culture group. The pangenome comprised 4,946 genes, with 1,396 core genes. Dairy starter strains showed markedly elevated numbers of insertion sequences, pseudogenes, plasmids and genomic islands relative to MCRI-603, which was plasmid-free and carried very few insertion sequence elements or genomic islands. DSM 28961 displayed pseudogene count similar to the dairy starter strains but markedly fewer transposases. These patterns are consistent with a plant-associated origin of P. laudensis and progressive dairy specialization via mobile genetic element acquisition. The P. laudensis mobilome was found to carry key niche-related traits. Lactose utilization operons were plasmid-encoded, whereas exopolysaccharide-encoding loci, opp oligopeptide transport systems and several defence loci, including clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), were consistently encoded within chromosomal integrative elements. All strains harboured prophage-like elements, including putatively intact prophages in 13 of them, and ~67% of 238 predicted antiphage systems resided on mobile genetic elements, underscoring their central role in phage defence. Restriction-modification systems dominated the defensome, and three strains encoded CRISPR-Cas systems (including type III-A and type I-C), indicating a higher prevalence than has been reported for Lactococcus lactis and Lactococcus cremoris, where CRISPR-Cas has rarely been observed. Methylome analysis identified 43 distinct motifs, of which 25 were novel. The P. laudensis methylome was overwhelmingly dominated by N⁶-methyladenine, and most motifs were short, non-palindromic and largely associated with type III restriction-modification systems and some type I and II subtypes. Nearly all strains exhibited distinct methylation profiles, including those isolated from the same dairy starter culture, highlighting extensive epigenetic diversification in dairy environments. Altogether, the data reveals a highly dynamic genomic and epigenomic landscape in P. laudensis, greatly shaped by mobile genetic elements, and provides a foundation for future work in this species and other Pseudolactococci.