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Complete genome sequence of Leuconostoc suionicum DSM 20241T provides insights into its functional and metabolic features
Standards in Genomic Sciencesvolume 12, Article number: 38 (2017)
The genome of Leuconostoc suionicum DSM 20241T (=ATCC 9135T = LMG 8159T = NCIMB 6992T) was completely sequenced and its fermentative metabolic pathways were reconstructed to investigate the fermentative properties and metabolites of strain DSM 20241T during fermentation. The genome of L. suionicum DSM 20241T consists of a circular chromosome (2026.8 Kb) and a circular plasmid (21.9 Kb) with 37.58% G + C content, encoding 997 proteins, 12 rRNAs, and 72 tRNAs. Analysis of the metabolic pathways of L. suionicum DSM 20241T revealed that strain DSM 20241T performs heterolactic acid fermentation and can metabolize diverse organic compounds including glucose, fructose, galactose, cellobiose, mannose, sucrose, trehalose, arbutin, salcin, xylose, arabinose and ribose.
The genus Leuconostoc comprises Gram-positive, facultatively anaerobic, intrinsically vancomycin-resistant, catalase-negative, spherical heterofermentative lactic acid bacteria which are involved in the fermentation of plant materials (such as kimchi), dairy products, meats, vegetable sausages and beverages [1,2,3,4,5,6,7]. Strain DSM 20241 T (=ATCC 9135 T =LMG 8159 T =NCIMB 6992 T) of the genus Leuconostoc was isolated in Sweden in 1972. It was originally classified as a subspecies of L. mesenteroides , but was recently reclassified as a novel species – L. suionicum –based on its whole genome sequence . Here, we present the taxonomic and genomic features of L. suionicum DSM 20241 T. In addition, we investigated the metabolic properties of L. suionicum DSM 20241 T and reconstructed the metabolic pathways of organic compounds to estimate the fermentative metabolites in L. suionicum DSM 20241 T.
Classification and features
L. suionicum DSM 20241 T belongs to the family Leuconostocaceae , order Lactobacillales , class Bacilli and phylum Firmicutes . Strain DSM 20241 T is a Gram-positive, facultatively anaerobic, non-motile, non-sporulating, catalase-negative coccus, with a diameter of 0.5–0.7 μm (Fig. 1). It can be grown in MRS broth at 10–40 °C, with an optimal growth temperature of 30 °C . Strain DSM 20241 T ferments a wide variety of carbon sources including d-glucose, arbutin, melibiose, sucrose, turanose, N-acetylglucosamine, cellobiose, galactose, gentiobiose, amygdalin, l-arabinose, esculin, ferric citrate, d-fructose, d-mannose, lactose, maltose, methyl α-d-glucopyranoside, salicin, trehalose, d-xylose, potassium 5-ketogluconate, mannitol and ribose to produce gas and acids (Table 1); however, it does not ferment glycerol, erythritol, d-arabinose, l-xylose, d-adonitol, methyl β-d-xylopyranoside, l-sorbose, methyl α-d-mannopyranoside, l-rhamnose, dulcitol, inositol, d-sorbitol, inulin, d-melezitose, starch, glycogen, xylitol, d-lyxose, d-tagatose, fucose, d-arabitol, l-arabitol, potassium gluconate, potassium 2-ketogluconate or raffinose [4, 8].
Phylogenetic analysis using the 16S rRNA gene sequences with validated type strains showed that L. suionicum DSM 20241 T is most closely related to the subspecies of the species L. mesenteroides : L. mesenteroides subsp. mesenteroides , L. mesenteroides subsp. jonggajibkimchii , L. mesenteroides subsp. cremoris , and L. mesenteroides subsp. dextranicum with very high 16S rRNA gene sequence similarities (>99.73%; Fig. 2).
Genome sequencing information
Genome project history
L. suionicum DSM 20241 T was selected owing to its taxonomic significance for the species L. mesenteroides and was obtained from the German Collection of Microorganisms and Cell Cultures. The complete sequences of the chromosome and plasmid of strain DSM 20241 T were deposited in GenBank with the accession numbers CP015247–48. The project information and its association with MIGS version 2.0  are summarized in Table 2.
Growth conditions and genomic DNA preparation
L. suionicum DSM 20241 T was cultured in MRS broth (BD Biosciences, CA, USA) at 30 °C for 24 h until the early stationary phase. Genomic DNA was extracted according to a standard phenol-chloroform extraction and ethanol precipitation procedure . DNA quality (OD260/OD280 > 1.8) and concentration were measured using a NanoDrop ND-1000 spectrophotometer (Synergy Mx, Biotek, VT, USA).
Genome sequencing and assembly
The genome of strain DSM 20241 T was sequenced using PacBio RS SMRT technology based on a 10-kb SMRT-bell library at Macrogen (Seoul, Korea) as previously described ; 138,738 high-quality reads were generated, with an average length of 7656 bp. De novo assembly of sequencing reads derived from PacBio SMRT sequencing was performed using the hierarchical genome assembly process (HGAP; ver. 3.0) , which yielded a circular chromosome (2,026,850 bp) and a circular plasmid (21,983 bp) (Fig. 3).
Automated genome annotation of strain DSM 20241 T was performed using Prodigal as part of the Joint Genome Institute’s microbial genome annotation pipeline . In addition, predicted coding sequences were functionally annotated using the NCBI non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups, and InterPro. Structural RNA genes were identified by using HMMER 3.0rc1 (rRNAs)  and tRNAscan-SE 1.23 (tRNAs) . Other non-coding genes were searched using INFERNAL 1.0.2 . Additional annotation was performed within the Integrated Microbial Genomes—Expert Review platform .
The complete genome of L. suionicum strain DSM 20241 T consists of a circular chromosome (2,026,850 bp) and a circular plasmid (21,983 bp) with 37.6% and 37.0% G + C contents, respectively (Table 3). The genome contains 1997 protein coding genes and 93 RNA genes (72 tRNAs, 12 rRNAs and 9 other RNAs; Table 4). Additional genome statistics and the distribution of the genes into COG functional categories are presented in Tables 4 and 5, respectively.
Insights from the genome sequence
KEGG metabolic and regulatory pathways
The KEGG metabolic pathways of L. suionicum DSM 20241 T show that strain DSM 20241 T displays typical heterolactic acid fermentative capabilities, performing pentose phosphate metabolism, fructose and mannose metabolism, galactose metabolism, sucrose metabolism and pyruvate metabolism without the complete tricarboxylic acid cycle (Fig. 4a, see Additional file 1: Table S1) [17,18,19]. In addition, L. suionicum DSM 20241 T harbors genes related to riboflavin metabolism, fatty acid biosynthesis, purine and pyrimidine metabolism and amino acid biosynthesis (Fig. 4a). The regulatory pathways of strain DSM 20241 T indicate that it contains various phospho transferase systems, such as a sucrose-specific EII component (K02808, K02809 and K02810), a β-glucoside β-glucoside-specific EII component (K02755, K02756 and K02757), a cellobiose-specific EII component (K02759, K02760 and K02761), a mannose-specific EII component (K02793, K02794, K02795 and K02796) and an l-ascorbate-specific EII component (K02821, K02822 and K03475) (Fig. 4b), suggesting that strain DSM 20241 T possesses the ability to ferment various carbon sources.
Carbon metabolic pathways
To investigate the fermentative metabolic properties of L. suionicum DSM 20241 T, metabolic pathways of various carbon sources were reconstructed based on predicted KEGG pathways and BLASTP analysis using reference protein sequences (Fig. 5). The predicted metabolic pathways identified motifs associated with the pentose phosphate pathway, fructose and mannose metabolism, galactose metabolism, sucrose metabolism, pyruvate metabolism, partial TCA cycle and incomplete glycolysis pathway in the genome of L. suionicum DSM20241 T, indicating that this strain performs typical heterolactic acid fermentation to produce lactate, ethanol and carbon dioxide (Fig. 5, Additional file 1: Table S1). It has been reported that mannitol, an important refreshing sweet agent in fermented vegetable foods such as sauerkraut, pickles and kimchi, is synthesized through fructose reduction by mannitol dehydrogenase (EC 22.214.171.124) through the consumption of NADH [20, 21]. The predicted metabolic pathways indicate that L. suionicum DSM 20241 T produces ethanol via the reduction of acetyl phosphate through the consumption of NADH; this strain may also produce acetate instead of ethanol due to the lack of NADH when the strain produces mannitol from fructose . L. suionicum DSM 20241 T harbors genes related to diverse PTSs or permeases that transport various glycosides or sugars including d-glucose, d-fructose, sucrose, d-mannose, trehalose, arbutin, salcin, cellobiose, d-xylose, arabinose, and d-ribose; this indicates that L. suionicum DSM 20241 T has versatile metabolic capabilities. d-lactate and l-lactate are produced from the reduction of pyruvate by d-lactate dehydrogenase (EC 126.96.36.199) and l-lactate dehydrogenase (EC 188.8.131.52), respectively. L. suionicum DSM 20241 T harbors four copies of d-lactate dehydrogenase (locus tags: Ga0151201_111849, Ga0151201_112070, Ga0151201_11385 and Ga0151201_111758) and one copy of l-lactate dehydrogenase (locus tag: Ga0151201_1175), suggesting that L. suionicum DSM 20241 T may produce more d-lactate than l-lactate; this is similar to other members of the genus Leuconostoc , which have been shown to produce more d-lactate than l-lactate under laboratory conditions [4, 22,23,24,25]. The predicted metabolic pathways show that L. suionicum DSM 20241 T produces diacetyl and acetoin, which are known as butter flavors in dairy products [26, 27]. Acetolactate synthase (EC 184.108.40.206) produces 2-acetolactate from pyruvate and converts it into deacetyl and CO2, which is emitted as a byproduct. Furthermore, 2-acetoin is produced from 2-acetolactate and diacetyl (acetolactate decarboxylase, EC 220.127.116.11; diacetyl reductase, EC 18.104.22.1684, respectively); but 2-acetoin is eventually converted to 2,3-butanediol, which lacks the butter flavoring property. In addition, the predicted metabolic pathways show that L. suionicum DSM 20241 T uses dextransucrase (EC 22.214.171.124) to produce dextran, a homopolysaccharide of glucose.
In this study, the complete genome of L. suionicum DSM 20241 T, consisting of a circular chromosome and a circular plasmid, was obtained by whole-genome sequencing using the PacBio SMRT sequencing system and de novo assembly using the HGAP method. In addition, the metabolic pathways of organic compounds in L. suionicum DSM 20241 T were reconstructed to estimate its fermentative properties and metabolites. The metabolic pathways show that strain DSM 20241 T performs typical heterolactic acid fermentations to produce lactate, ethanol and carbon dioxide and contains genes encoding various PTSs, permeases, and other enzymes to metabolize various organic compounds. In addition, strain DSM 20241 T synthesizes mannitol to produce acetate instead of ethanol through heterolactic acid fermentation, and produces butter flavoring compounds. The complete genome and reconstructed metabolic pathways of L. suionicum DSM 20241 T provide important insights into its functional and metabolic features during fermentation.
Clusters of orthologous groups
Kyoto Encyclopedia of Genes and Genomes
Single molecule real-time
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This work was supported the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01090604)” of Rural Development Administration and the World Institute of Kimchi funded by the Ministry of Science, ICT and Future Planning (KE1702–2), Republic of Korea.
The authors declare that they have no competing interests.
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List of genes of L. suionicum DMS 20241T in KEGG metabolic pathways. (PDF 411 kb)