Phylogenomic Analysis of Lactobacillus curvatus Reveals Two Lineages Distinguished by Genes for Fermenting Plant-Derived Carbohydrates

Abstract

Lactobacillus curvatus is a lactic acid bacterium encountered in many different types of fermented food (meat, seafood, vegetables, and cereals). Although this species plays an important role in the preservation of these foods, few attempts have been made to assess its genomic diversity. This study uses comparative analyses of 13 published genomes (complete or draft) to better understand the evolutionary processes acting on the genome of this species. Phylogenomic analysis, based on a coalescent model of evolution, revealed that the 6,742 sites of single nucleotide polymorphism within the L. curvatus core genome delineate two major groups, with lineage 1 represented by the newly sequenced strain FLEC03, and lineage 2 represented by the type-strain DSM20019. The two lineages could also be distinguished by the content of their accessory genome, which sheds light on a long-term evolutionary process of lineage-dependent genetic acquisition and the possibility of population structure. Interestingly, one clade from lineage 2 shared more accessory genes with strains of lineage 1 than with other strains of lineage 2, indicating recent convergence in carbohydrate catabolism. Both lineages had a wide repertoire of accessory genes involved in the fermentation of plant-derived carbohydrates that are released from polymers of α/β-glucans, α/β-fructans, and N-acetylglucosan. Other gene clusters were distributed among strains according to the type of food from which the strains were isolated. These results give new insight into the ecological niches in which L. curvatus may naturally thrive (such as silage or compost heaps) in addition to fermented food.

Fig. 1.

—Phylogenomic clonal genealogy and population structure of Lactobacillus curvatus strains. On the left: Fifty-percent majority rule consensus tree inferred with CLONALFRAME software using the coalescent model (branch lengths are given in coalescent units). All branches are supported by a posterior probability of >95%. Strains are colored according to their lineage affiliation. On the right: Proportion of genetic material derived from each of K subpopulations as inferred by STRUCTURE (linkage model) and assuming K=2 or 3 populations. Ancestral subpopulations are colored in red (lineage1), blue (lineage 2), and green (an unlikely lineage 3), respectively. Clade 2B of lineage 2 is colored in dark cyan to highlight its divergence from Clade 2A and a strong degree of admixture between the two lineages.

Fig. 2.

—Progression of the core genome and pangenome of Lactobacillus curvatus. Each boxplot represents the pairwise evolution of the core genome (blue) and pangenome (yellow) of clusters of orthologous proteins calculated iteratively as genomes were added to the analysis, for a total of 2–13 genomes. Dashed lines represent the values obtained for the progression of the core genome (using a stringent or relaxed estimation; see text), the pangenome, and for another important step in the estimation of the accessory genome, the shell genome. The shell genome is a more realistic functional estimation of the pangenome that excludes mobile selfish DNA (mobile genome) and unique gene clusters found in only one strain (cloud genome) from the sum of accessory genes.

Fig. 3.

—Heatmap showing the clustering analysis of Lactobacillus curvatus strains based on the content of their accessory genomes. Unsupervised complete linkage clustering of L. curvatus strains based on the presence (orange) or absence (blue) of 901 orthologs that constitute the L. curvatus accessory genome (without mobile and cloud genomes). Names of strains are colored according to their phylogenomic clade and lineage as shown in figure 1. The five main groups of orthologs prevalent among the strains are indicated above the heatmap and inside the clustering tree (from groups A to E). Similarly, gene prevalence groups are colored based on their specificity to each of the phylogenomic clades. Eight gene clusters representative of each groups are boxed with dashed lines: CSC clusters (Cell Surface Complexes); pdu cluster (propanediol catabolic pathway); map1 and map2 clusters (lineage-specific maltose phosphorylase pathways); srl cluster (sorbitol phosphotransferase system); fli, mot, and che clusters (motility operons); ula cluster (ascorbate catabolic pathway).

Fig. 4.

—Overview of Lactobacillus curvatus accessory gene repertoire involved in fermentation of plant-derived carbohydrates. Carbohydrates are grouped (external ellipses) according to their type (glucan and fructans) or origin (plants, cereals, and insects). Each uptake and catabolic system is represented by a circle whose size depicts the degree of conservation among the L. curvatus strains. The clusters are numbered (small yellow circles) to facilitate their identification using supplementary file S1, Supplementary Material online. The inner circles illustrate the fate of these carbohydrates: into glucose 6P, fructose 6P, N-acetyl glucosamine 6P, or dihydroxy-acetone P (DHAP).

Fig. 5.

—Barplots showing the number of phage-related genes and transposase families in the Lactobacillus curvatus strains. The barplots indicate the numbers of phage genes (green) or transposase families (red) identified in each strain. The red shaded area depicts strains from Asian-type foods, in which a higher number of transposase families was found.