Riassunto analitico
Leuconostoc is a genus of heterofermentative lactic acid bacteria. Leuconostocs frequently occur in food, including meat and vegetables, where they can exert a bioprotective action against deterioration by other bacteria and can be directly involved in fermentation processes resulting in the positive evolution of organoleptic properties and/or in the spoilage of the product. To date 16 species of Leuconostoc are recognized based on phenotypic and morphological features, chemotaxonomic criteria such as DNA-DNA hybridization, G+C content, and the sequence of the gene encoding the 16S rRNA. Nowadays the sequencing of bacterial whole genomes and average nucleotide identity (ANI) is being increasingly utilized for the delineation of bacterial species.
In this study, all the available genomes of the genus Leuconostoc, i.e., 219 genomes belonging to 14 species and their subspecies, were subjected to ANI analysis. Of the genomes analyzed, 214 belonged to strains attributed to 14 of the 16 species and the remaining 5 to strains for which the species attribution was not provided. The 219 strains were distributed in 15 groups with pairwise ANI > 96%. The strains currently attributed to L. lactis were separated to G1 and G2. G1 could be referred to L. argentinum, while the strain previously defined as L. garlicum should merge with L. lactis into G2. The two subspecies L. gelidum subsp. gelidum and L. gelidum subsp. gasicomitatum should be recognized as two diverse species, corresponding to G8 and G9. The strains of L. inhae, including the type strain, are included in group G9 and should give the name to this group/species. The group G8 is referred to as L. gelidum, with the type strain. L. pseudomesenteoides should be split into two species, G11 and G12, the latter also incorporating the type strain of L. falkenbergense that should rename this group. Most of the strains of L. mesenteroides, including the subspecies L. mesenteroides subsp. cremoris, L. mesenteroides subsp. dextranicum, L. mesenteroides subsp. jonggajibkimkii, and L. mesenteroides subsp. mesenteroides correspond to G15 species, whereas some L. mesenteroides should form a diverse one (G13). Digital-DNA/DNA hybridization (dDDH) confirmed the proposed groups for species delineation. The ANI phylogenetic tree was compared with 16S-rRNA gene, rpoA, pheS, and core genome trees utilizing ‘generalized’ Robinson-Foulds metrics. All the metrics confirmed that clustering of strains was consistent for most groups, but single-gene molecular clocks such as 16s-rRNA gene, rpoA, and pheS, although widely utilized in phylogenetics, seem unable to capture the complete diversity among Leuconostoc species.
The metabolic pathways of all the Leuconostoc strains were reconstructed from their genome, thus predicting all the orthologous functions with KEGG annotation and reconstructing the metabolic modules, including the substrate transporters. The analysis of reconstructed metabolic pathways revelated that groups were greatly homogeneous inside them, and specific autotrophies and nutritional requirements (e.g. for vitamins and amino acids) could be predicted for the different groups. Principal coordinate analysis (PCoA) was carried out to reveal groups of strains according to shared predicted functions and to establish whether they coincided with phylogenomics groups identified by ANI. In the PCoA space, the strains of each ANI group were located very closely to each other and separate from other groups, due to a great number of shared functions resulting in little Jaccard’s distance, thus groups are expected to be phenotypically very homogeneous. These results seem promising in view of an overall taxonomic reorganization of Leuconostoc genus. To confirm the evidence provided in this phylogenomic comparative study, a targeted polyphasic taxonomy approach will be required, including a through comparison of physiological and biochemical properties.
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Abstract
Leuconostoc is a genus of heterofermentative lactic acid bacteria. Leuconostocs frequently occur in food, including meat and vegetables, where they can exert a bioprotective action against deterioration by other bacteria and can be directly involved in fermentation processes resulting in the positive evolution of organoleptic properties and/or in the spoilage of the product. To date 16 species of Leuconostoc are recognized based on phenotypic and morphological features, chemotaxonomic criteria such as DNA-DNA hybridization, G+C content, and the sequence of the gene encoding the 16S rRNA. Nowadays the sequencing of bacterial whole genomes and average nucleotide identity (ANI) is being increasingly utilized for the delineation of bacterial species.
In this study, all the available genomes of the genus Leuconostoc, i.e., 219 genomes belonging to 14 species and their subspecies, were subjected to ANI analysis. Of the genomes analyzed, 214 belonged to strains attributed to 14 of the 16 species and the remaining 5 to strains for which the species attribution was not provided. The 219 strains were distributed in 15 groups with pairwise ANI > 96%. The strains currently attributed to L. lactis were separated to G1 and G2. G1 could be referred to L. argentinum, while the strain previously defined as L. garlicum should merge with L. lactis into G2. The two subspecies L. gelidum subsp. gelidum and L. gelidum subsp. gasicomitatum should be recognized as two diverse species, corresponding to G8 and G9. The strains of L. inhae, including the type strain, are included in group G9 and should give the name to this group/species. The group G8 is referred to as L. gelidum, with the type strain. L. pseudomesenteoides should be split into two species, G11 and G12, the latter also incorporating the type strain of L. falkenbergense that should rename this group. Most of the strains of L. mesenteroides, including the subspecies L. mesenteroides subsp. cremoris, L. mesenteroides subsp. dextranicum, L. mesenteroides subsp. jonggajibkimkii, and L. mesenteroides subsp. mesenteroides correspond to G15 species, whereas some L. mesenteroides should form a diverse one (G13). Digital-DNA/DNA hybridization (dDDH) confirmed the proposed groups for species delineation. The ANI phylogenetic tree was compared with 16S-rRNA gene, rpoA, pheS, and core genome trees utilizing ‘generalized’ Robinson-Foulds metrics. All the metrics confirmed that clustering of strains was consistent for most groups, but single-gene molecular clocks such as 16s-rRNA gene, rpoA, and pheS, although widely utilized in phylogenetics, seem unable to capture the complete diversity among Leuconostoc species.
The metabolic pathways of all the Leuconostoc strains were reconstructed from their genome, thus predicting all the orthologous functions with KEGG annotation and reconstructing the metabolic modules, including the substrate transporters. The analysis of reconstructed metabolic pathways revelated that groups were greatly homogeneous inside them, and specific autotrophies and nutritional requirements (e.g. for vitamins and amino acids) could be predicted for the different groups. Principal coordinate analysis (PCoA) was carried out to reveal groups of strains according to shared predicted functions and to establish whether they coincided with phylogenomics groups identified by ANI. In the PCoA space, the strains of each ANI group were located very closely to each other and separate from other groups, due to a great number of shared functions resulting in little Jaccard’s distance, thus groups are expected to be phenotypically very homogeneous. These results seem promising in view of an overall taxonomic reorganization of Leuconostoc genus. To confirm the evidence provided in this phylogenomic comparative study, a targeted polyphasic taxonomy approach will be required, including a through comparison of physiological and biochemical properties.
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