A large-scale comparative genomics study reveals niche-driven and within-sample intra-species functional diversification in Lacticaseibacillus rhamnosus.

Lacticaseibacillus rhamnosus (L. rhamnosus) is widely recognized as a probiotic species, and it exists in a variety of environments including host gut and dairy products. This work aimed at conducting a large-scale comparative genomics analysis of 384 L. rhamnosus genomes (257 whole-sequence or metagenomic-assembled genomes from gut-associated isolates [122 and 135 retrieved from the UHGG and NCBI databases, respectively] and 127 genomes from dairy isolates [34 from the NCBI database; 93 isolated from a cheese sample and sequenced here]). Our results showed that L. rhamnosus had a large and open pan-genome (15,253 pan-genes identified from all 384 genomes; 15,028 pan-genes if the 93 cheese-originated isolates were excluded). The core-gene phylogenetic tree constructed from the 384 L. rhamnosus genomes comprised five phylogenetic branches, with a random distribution of dairy and gut-associated isolates/genomes across the tree. No significant difference was identified in the overall profile of metabolism-related genes between dairy and gut-associated genomes; however, notably, the gut-associated strains/isolates contained more genes coding for specific metabolic pathways and carbohydrate-active enzymes, e.g., lacto-N-biosidase (EC 3.2.1.140; GT20) and lacto-N-biose phosphorylase/galacto-N-biose phosphorylase (EC 2.4.1.211; GH112). Further, we found that there was obvious intra-species diversification of the 93 cheese-originated L. rhamnosus isolates, forming three clades (Clades A, B, and C) in the reconstructed core-gene phylogenetic tree. There were numerous single nucleotide variations (over 10,000) across the three clades. Moreover, significant differences were observed in the content of metabolism-related genes across clades (p < 0.05, Adonis test), characterized by the enrichment in glycoside hydrolases in Clade C and the possession of unique metabolic pathways in each clade. These results implicated genomics/functional diversification of L. rhamnosus in a single food matrix and niche-driven adaptive evolution of isolates from dairy and host gut-associated origins. Our study shed insights into the selection of candidate strains for food industry applications.