The Variable Regions of Lactobacillus rhamnosus Genomes Reveal the Dynamic Evolution of Metabolic and Host-Adaptation Repertoires

Abstract

Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes (core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer (variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total (the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides (EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat (CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core (shared) proteins matched with the clade distribution obtained from the presence-absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains' core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification.

Keywords: comparative genomics; core and pan-genome; diversity; niche adaptation; probiotic.

Fig. 1.

Hierarchical map and clustering of the 40 L. rhamnosus genomes based on presence–absence of genes (green, presence and red, absence of genes). OGs of genes could be classified in four groups noted cluster A–D. Cluster D includes 10% of core genes to allow a shared OGs skewed classification of the clusters. At strain level there are eight recognizable clades of strains (1–8). The niche from which the strains were isolated is indicated by the color-coding of the strain’s ID-code: yellow: dairy; black: human feces; red: clinical (blood); light green: healthy intestine; blue: vagina; purple: goat feces; dark green: beer; gray: type strain (unknown).

Fig. 2.

Number of clade-specific genes and their functionality. Annotated genes are displayed; the number of clade-specific genes with unknown functionality is added on top of each bar.

Fig. 3.

LPxTG proteins with host interaction potential. LPxTG proteins not included in this figure: sortases, hydrolases, lyases, proteases.

Fig. 4.

Summary of mobile elements present in L. rhamnosus strains. Panel 1: Number of genes annotated as mobile elements in the pan-genome, including plasmids, phages, integrases, transposases (lighter colors), and number of genomic islands (dark colors). Panel 2: Type and distribution of mobile elements in each genome: plasmids, phages, cas genes and CRISPR spacers. Gray represents gene presence and white gene absence. For the spacers’ analysis, only spacers that present a hit in any of the databases are represented. In the columns Hits in rhamnosus and Hits in nt, the colors represent the type of hit: yellow: unknown; pink: plasmid; green: phage genes; white: not found. Strains are organized by genetic clades separated by vertical lines.

Fig. 5.

Lactobacillus rhamnosus genome-based phylogenetic relatedness, (Panel A) based on core proteome SAP distribution and (panel B) based on hierarchical clustering of variome gene distribution. The scale for the presence–absence tree represents the number of variable OGs the clustering is based on. Arrows connecting the same strains of both trees aims at highlighting the common groups of strains, which are also marked by the variome-based clade numbering (panel B).