Pan-genomic and transcriptomic analyses of Leuconostoc mesenteroides provide insights into its genomic and metabolic features and roles in kimchi fermentation

Abstract

The genomic and metabolic features of Leuconostoc (Leu) mesenteroides were investigated through pan-genomic and transcriptomic analyses. Relatedness analysis of 17 Leu. mesenteroides strains available in GenBank based on 16S rRNA gene sequence, average nucleotide identity, in silico DNA-DNA hybridization, molecular phenotype, and core-genome indicated that Leu. mesenteroides has been separated into different phylogenetic lineages. Pan-genome of Leu. mesenteroides strains, consisting of 999 genes in core-genome, 1,432 genes in accessory-genome, and 754 genes in unique genome, and their COG and KEGG analyses showed that Leu. mesenteroides harbors strain-specifically diverse metabolisms, probably representing high evolutionary genome changes. The reconstruction of fermentative metabolic pathways for Leu. mesenteroides strains showed that Leu. mesenteroides produces various metabolites such as lactate, ethanol, acetate, CO₂, mannitol, diacetyl, acetoin, and 2,3-butanediol through an obligate heterolactic fermentation from various carbohydrates. Fermentative metabolic features of Leu. mesenteroides during kimchi fermentation were investigated through transcriptional analyses for the KEGG pathways and reconstructed metabolic pathways of Leu. mesenteroides using kimchi metatranscriptomic data. This was the first study to investigate the genomic and metabolic features of Leu. mesenteroides through pan-genomic and metatranscriptomic analyses, and may provide insights into its genomic and metabolic features and a better understanding of kimchi fermentations by Leu. mesenteroides.

Figure 1

A phylogenetic tree using the NJ algorithm based on 16S ribosomal RNA sequences showing the phylogenetic relationships among Leuconostoc mesenteroides strains and related taxa. Weissella viridescens NRIC 1536^T was used as an outgroup (not shown). The type strains are highlighted in bold. The scale bar equals 0.01 changes per nucleotide.

Figure 2

Pan- and core-genome plot (A) and flower plot diagram (B) of 17 Leu. mesenteroides strains. An ordered list of the 17 strains was randomly generated and 20 sets of the randomly ordered strains were subjected to pan- and core-genome analysis. The average number of core- and pan-genome sizes were plotted with standard deviations. The pan-genome represents the total genes of genomes in a subset sampled and the core-genome represents the genes shared by all genomes in the same subset. The flower plot diagram represents gene numbers in the core-genome (in the center) and unique-genome (in the petals) of Leu. mesenteroides pan-genome, and in the genome of each Leu. mesenteroides strain (in the parentheses). The type strains of Leu. mesenteroides subspecies are highlighted in bold.

Figure 3

A phylogenetic tree with bootstrap values (1,000 replicates) reconstructed using the concatenated amino acid sequences of Leu. mesenteroides core-genome (999 genes) showing the relationships among Leu. mesenteroides strains. Strain names as described in GenBank or validated names are used in the tree and the type strains are highlighted in bold. The bar indicates 0.001 substitutions per site.

Figure 4

Heat-map and hierarchical clustering of 17 Leu. mesenteroides strains based on the presence (red) or absence (blue) of genes. The type strains of Leu. mesenteroides subspecies are highlighted in bold.

Figure 5

Comparison of COG functional categories in the pan-genomes of Leu. mesenteroides strains and closely related bacterial taxa (Leuconostoc species except for Leu. mesenteroides, Fructobacillus species, and Weissella species) (A) and distribution of the COG functional categories in the core- and accessory/unique-genome of Leu. mesenteroides strains (B). The alphabetic codes represent COG functional categories as follows: C, energy production and conversion; D, cell division and chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme metabolism; I, lipid metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, DNA replication, recombination, and repair; M, cell envelope biogenesis, outer membrane; N, cell motility and secretion; O, post-translational modification, protein turnover, and chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms.

Figure 6

Metabolic (A) and regulatory (B) pathways of Leu. mesenteroides strains. The pathways were generated using the iPath v2 module based on KEGG Orthology numbers of genes identified from the genomes of 17 Leu. mesenteroides strains. Metabolic pathways identified from all 17 genomes, belonging to the core-genome, are depicted in blue and metabolic pathways identified from 15–16 genomes, belonging to the soft core-genome, are depicted in violet. Metabolic pathways identified from 1–14 genomes, belonging to accessory/unique-genome, are depicted in red. Line thickness is proportional to the numbers of Leu. mesenteroides strains harboring the metabolic pathways.

Figure 7

Proposed fermentative metabolic pathways of Leu. mesenteroides for carbohydrates and their transcriptional expressions during kimchi fermentation. Metabolic pathways that were present in all Leu. mesenteroides strains are depicted in blue (core-genome) and metabolic pathways that were present in 15–16 Leu. mesenteroides strains are depicted in violet (soft core-genome). Metabolic pathways that were present in 1–14 Leu. mesenteroides strains are depicted in red (unique- or accessory-genome). Line thickness in the pathways is proportional to the number of Leu. mesenteroides strains harboring the corresponding genes, which are indicated in parentheses before EC numbers. Carbohydrate transport systems with black arrows indicate unidentified transporting systems that may be present in Leu. mesenteroides genomes. The transcriptional expressions were visualized by heatmaps based on their RPKM values and the white boxes represent no transcriptional expression during kimchi fermentation. Kimchi samples for the metatranscriptomic analysis were obtained at 7, 13, 18, 25, and 29 days. UDP, uridine diphosphate.

Figure 8

Transcriptional expressions of the metabolic pathways of Leu. mesenteroides at 7 (A), 13 (B), 18 (C), 25 (D), and 29 (E) days during kimchi fermentation. The metabolic pathways were generated using the iPath v2 module based on KEGG Orthology numbers identified from the pan-genome of Leu. mesenteroides strains. The transcriptional expression levels of the metabolic pathways are depicted by line thickness and color change based on their RPKM values (based on a log₂ scale).