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Research article
Description and Genomic Characteristics of Weissella fermenti sp. nov., Isolated from Kimchi
1Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea
2Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju, 61755, Republic of Korea
3Department of Agro-Food Resources, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea
J. Microbiol. Biotechnol. 2023; 33(11): 1448-1456
Published November 28, 2023 https://doi.org/10.4014/jmb.2306.06010
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Since the genus
Many
Material and Methods
Isolation and Cultivation
Strain BK2T was isolated from fermented baechu kimchi prepared using baechu cabbage (
Phylogenetic Analysis Based on 16S rRNA Gene Sequences
To obtain nearly complete sequences of the 16S rRNA genes of strains BK2T and LMG 11983, PCR amplification was performed using the F1 and R13 primers. The resulting PCR amplicons were subjected to sequencing using the 340F, 518R (5'-ATT ACC GCG GCT GCT GG-3'), and 805F (5'-GAT TAG ATA CCC TGG TAG TC-3') primers, and the resulting nucleotide sequences were assembled as described previously [23]. The 16S rRNA gene sequence similarities between strains BK2T and LMG 11983 and other bacterial type strains were calculated using the EzBioCloud server. The 16S rRNA gene sequences of strains BK2T and LMG 11983 and closely related valid type strains were aligned using the secondary-structure aware Infernal aligner [25]. Phylogenetic trees were constructed using the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) algorithms with bootstrap values (1,000 replications) in MEGA11 [26]. The Kimura two-parameter model, nearest-neighbor-interchange heuristic search method, and complete deletion options were used for the NJ, ML, and MP tree constructions, respectively.
Genome Sequencing and Phylogenomic Analysis
The gDNA of strains BK2T and LMG 11983 was extracted using a Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturer's instructions. The gDNA of strain BK2T was sequenced using a hybrid combination of the Oxford Nanopore MinION platform (Nanopore, UK) in our laboratory and the Illumina HiSeq X platform (Illumina, USA) with 151 bp paired-end sequencing reads at Macrogen. On the other hand, the gDNA of strain LMG 11983 was sequenced using the Oxford Nanopore MinION platform. Sequencing reads derived from the Nanopore MinION sequencing were de novo-assembled using Flye ver. 2.9.1 [27]. The assembled long contigs of strain BK2T were polished with Illumina sequencing reads using Pilon ver. 1.24 [28], and multiple rounds of polishing were performed until no additional corrections were accomplished. The quality of the assembled genomes was assessed by evaluating their completeness and contamination rates using CheckM ver. 1.0.4 [29]. Finally, the genome sequences of strains BK2T and LMG 11983 were submitted to GenBank and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; www.ncbi.nlm.nih.gov/genome/annotation_prok/) [30].
The DNA G+C contents of the strains were determined based on their whole genome sequences. To perform genome-based phylogenomic analysis, 92 housekeeping core genes were extracted from the genomes of strains BK2T and LMG 11983, as well as from closely related
Phenotypic, Physiological, and Biochemical Analyses
Strains BK2T and LMG 11983 were tested for their growth on various culture media including MRS agar, Reasoner's 2A (R2A) agar (BD), tryptic soy agar (TSA; BD), nutrient agar (NA; BD), marine agar (MA; BD), and Luria-Bertani (LB) agar (BD) for 2 days at 30°C. To evaluate the growth temperature and pH ranges of strains BK2T and LMG 11983, they were incubated on MRS agar and in MRS broth at different temperatures (10–50°C at 5°C intervals) and pH values (4.0–10.0 at 1.0 pH unit intervals), respectively, for 2 days. MRS broths with pH 4.0–5.0, 6.0–8.0, and 9.0–10.0 were prepared using sodium citrate, Na2HPO4/NaH2PO4, and Tris-HCl buffers, respectively [35]. The pH values were adjusted if necessary after autoclaving at 121°C for 15 min. The MRS broths used for pH testing were incubated without agitation at 30°C. The growth of strains BK2T and LMG 11983 was also examined in MRS broth containing different concentrations of NaCl (0–5%, w/v, at 1.0% intervals), prepared in the laboratory according to the MRS formula.
The biochemical and physiological properties of strains BK2T and LMG 11983 were evaluated using cells grown on MRS agar for 2 days at 30°C. Gram staining was performed using PREVI Colour Gram (bioMérieux, France), following the manufacturer's instructions. The cell morphology of strains BK2T and LMG 11983 was examined using phase-contrast microscopy (Carl Zeiss, Germany) and transmission electron microscopy (JEM-1010; Jeol, Japan). Oxidase and catalase activities were determined by observing the oxidation of 1% (w/v) tetramethyl-
The phenotypic characteristics of strains BK2T and LMG 11983, along with four closely related reference strains, were examined under identical growth conditions. Hydrolysis of tyrosine, casein, esculin, gelatin, starch, Tween 20, and Tween 80 was assessed on MRS agar following previously described protocols [36, 37]. Additional biochemical features and enzymatic activities were determined using the API 20E and API ZYM systems (bioMérieux), respectively, according to the manufacturer's instructions. Acid production from various carbohydrates was evaluated using API 50 CH test strips (bioMérieux) with the API 50 CHL medium, following the manufacturer's guidelines. The configuration of lactic acids produced by strains BK2T and LMG 11983 and reference strains was determined using a DL-lactate test kit (Boehringer Mannheim/R-Biopharm, Germany).
Chemotaxonomic Analyses
For cellular fatty acid analysis, strains BK2T and LMG 11983, along with four reference strains, were cultivated in MRS broth at 30°C. Microbial cells were harvested during the exponential growth phase at an 0.8 optical density at 600 nm. The cellular fatty acids of the harvested cells were saponified, methylated, and extracted following the standard Sherlock Microbial Identification System (MIDI) protocol. Fatty acid methyl esters were analyzed by gas chromatography on the HP 6890 GC (Hewlett Packard, USA) and identified using the MIDI identification database RTSBA6 (ver. 6.0B). Polar lipids of strains BK2T and LMG 11983 were extracted from cells harvested during the exponential growth phase and analyzed by two-dimensional thin-layer chromatography following the protocol described by Minnikin
Results and Discussion
Phylogenetic Analysis Based on 16S rRNA Gene Sequences
Sequencing using the 340F, 518R, and 805F primers yielded nearly complete 16S rRNA gene sequences of strains BK2T (1,513 nucleotides) and LMG 11983 (1,504 nucleotides). The obtained sequences of strains BK2T and LMG 11983 were found to be very similar, showing a 99.9% sequence similarity, indicating their close phylogenetic relatedness. Comparative analysis of the 16S rRNA gene sequences revealed that strain BK2T exhibited the highest similarities to
Phylogenetic analysis using the NJ algorithm based on the 16S rRNA gene sequences demonstrated that strains BK2T and LMG 11983 formed a tight phyletic lineage with
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Fig. 1. A neighbor-joining tree (NJ) showing the phylogenetic relationships between strains BK2T and LMG 11983 and closely related species based on 16S rRNA gene sequences. Bootstrap values (based on 1,000 replications) exceeding 70% are indicated at branch points. The filled circles (●) indicate branches that were commonly recovered in NJ, maximum-likelihood, and maximum-parsimony algorithms.
Enterococcus plantarum CCM 7889T (MT758013) was used as the outgroup. The scale bar equals 0.01 changes per nucleotide position.
Genomic Features and Phylogenomic and Genome Relatedness
The
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Table 1 . General genomic features of strains BK2T and LMG 11983 and related
Weissella type strains and their genome relatedness.Characteristic 1 2 3 4 5 6 Genomic features* Genome status Draft Draft Draft Draft Draft Draft No. of contigs 5 2 41 25 41 8 Genome size (Mb) 2.50 2.51 2.19 2.32 2.29 1.54 G+C content (mol%) 44.7 44.7 44.7 45.1 40.4 40.8 No. of total genes 2,433 2,506 2,121 2,198 2,345 1,546 No. of protein-coding genes 2,299 2,275 2,050 2,090 2,243 1,475 Proteins assigned to COG (%) 1,426 (62.0) 1,411 (62.0) 1,431 (69.8) 1,491 (71.3) 1,384 (61.7) 1,149 (77.9) No. of rRNAs (5S, 16S, and 23S) 9, 8, 8 9, 8, 8 1, 1, 1 7, 2, 2 4, 1, 1 1, 0, 1 No. of total tRNA genes 83 84 49 77 74 46 No. of noncoding RNAs 3 3 3 3 3 3 No. of CRISPRs† 1 1 – – – – Lactate dehydrogenase (LDH)a L-LDH (EC 1.1.1.27) 1 1 1 2 – 1 D- LDH (EC 1.1.1.28) 1 1 1 1 1 1 D- LDH (EC 1.1.5.12) 1 1 1 – – 1 ANI value (%)† Taxa dDDH value (%)† 1 – 99.7 48.1 23.5 23.6 22.6 2 99.9 – 48.1 23.5 23.6 22.6 3 92.3 92.4 – 23.2 20.9 20.1 4 78.4 78.3 78.5 – 20.2 19.8 5 70.5 70.3 70.4 70.6 – 20.3 6 70.8 70.8 70.7 70.7 69.4 – Taxa: 1, strain BK2T (JAOZFC000000000); 2, strain LMG 11983 (JAOZFD000000000); 3,
W. confusa NBRC 106469T (BJZE00000000); 4,W. cibaria JCM 12495T (BJEF00000000); 5,W. muntiaci 8H-2T (SDGZ00000000); 6,W. viridescens DSM 20410T (JQBM00000000). *The information was obtained from GenBank and annotated by the NCBI prokaryotic genome annotation pipeline (PGAP; www.ncbi.nlm.nih.gov/genome/annotation_prok/).†CRISPR, clustered regularly interspaced short palindromic repeat; ANI, average nucleotide identity; dDDH, digital DNA-DNA hybridization.
Clustered regularly interspaced short palindromic repeats (CRISPRs) have been widely recognized as a host defense mechanism against bacteriophage predation [40]. Interestingly, the genomes of strains BK2T and LMG 11983 were found to harbor a single CRISPR region, unlike other
Strain BK2T was found to possess three distinct types of the lactate dehydrogenase gene (
The ANI and dDDH values between strain BK2T and strain LMG 11983 were determined to be 99.9% and 99.7%, respectively, which surpass the established thresholds (ANI ~95%; dDDH 70%) for prokaryotic species delineation [42]. This indicates that strains BK2T and LMG 11983 belong to the same species. Furthermore, the ANI and dDDH values between strain BK2T and closely related reference type strains, including
-
Fig. 2. A phylogenomic tree showing the phylogenetic relationships between strains BK2T and LMG 11983 and closely related species, based on the concatenated amino acid sequences of 92 housekeeping core genes. Bootstrap values (based on 1,000 replications) exceeding 70% are indicated at branch points.
Enterococcus plantarum CCM 7889T (MT758013) was used as the outgroup. The scale bars indicate 0.1 changes per amino acid.
Phenotypic, Physiological, and Biochemical Characteristics
Strains BK2T and LMG 11983 exhibited robust growth on MRS agar and demonstrated relatively favorable growth on LB agar and TSA, but displayed slow growth on R2A agar, NA, and MA. After 2 days of incubation at 30°C, both strains formed ivory-colored colonies on MRS agar, a characteristic that distinguishes them from closely related reference strains, which formed white colonies (Table 2). The cells of strains BK2T and LMG 11983 were observed to be Gram-stain positive, non-motile rods with dimensions of 0.7–0.8 μm in width and 2.0–2.2 μm in length (Fig. S2). These strains produced both the D- and L-enantiomers of lactic acid in an approximate ratio of 85:15, similar to the other reference strains, except
-
Table 2 . Differential phenotype characteristics of strains BK2T and LMG 11983 and closely related type strains of the genus
Weissella .Characteristic 1 2 3 4 5 6 Isolation source Kimchi Grass silage Sugar cane Chili bo Feces Cured meat Colony color Ivory Ivory White White White White Enzyme activity (API ZYM)* of: Alkaline phosphatase + + + + – – Leucine arylamidase + + + – + + α -Chymotrypsin+ + + – – – β -Glucuronidase,β -glucosidase, valine arylamidase+ + – – – – α -Glucosidase– – – – – + Enzyme activity (API 20E)* of: β -Galactosidase+ + – – – – Arginine dihydrolase – – + + + – Citrate utilization (API 20E) – – – – + + Fermentation (API 20E)* of: Amygdalin, sucrose + + + + – – D-Mannitol + + – – – – Acid production (API 50 CHL)* from: Arbutin, amygdalin, cellobiose, gentiobiose, salicin, D-galactose, sucrose + + + + – – Trehalose + + – – + + D-Mannitol + + – – – – 2-Keto-gluconate – – + – + + Maltose – – + + – – D-Arabinose – – – + + + Lactic acid configuration* d, l d, l d, l d, l d d, l Taxa: 1, strain BK2T (this study); 2, strain LMG 11983 (this study); 3,
W. confusa KACC 11841T [8]; 4,W. cibaria KACC 11862T [9]; 5,W. muntiaci NBRC 113537T [14]; 6,W. viridescens KACC 11850T [1].All strains are facultative anaerobic short rods and positive for the following characteristics: acetoin production, activity* of acid phosphatase, naphthol-AS-BI-phosphohydrolase, and tryptophan deaminase, hydrolysis of esculin, D-glucose fermentation*, and acid production* from D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, gluconate, esculin, and
N -acetyl-Dglucosamine. All strains are negative for the following characteristics: hydrolysis of casein, gelatin, starch, tyrosine, Tween 20, and Tween 80, production* of indole and H2S, activity* of catalase, oxidase, lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, esterase (C4), esterase lipase (C8), lipase (C14), crystine arylamidase, trypsin,α -galactosidase,α - mannosidase,α -fucosidase, andN -acetyl-β -glucosaminidase, fermentation* of inositol, L-rhamnose, D-melibiose, and Dsorbitol, and acid production* from glycerol, erythritol, L-arabinose, D-adonitol, D-lactose, L-rhamnose, D-melibiose, Dmelezitose, D-raffinose, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-xylose, L-sorbose, L-fucose, L-arabitol, dulcitol, inositol, sorbitol, inulin, starch, glycogen, xylitol, methylβ -D-xylopyranoside, methylα -D-mannoside, methylα -Dglucoside, and 5-keto-gluconate. Symbols: +, positive; –, negative.*These analyses were conducted under the same conditions in this study.
Several phenotypic properties were found to be shared between strains BK2T and LMG 11983 and their closely related
The ability of strain BK2T to thrive specifically in kimchi fermentation cannot be fully explained by genomic analysis or the examination of several metabolic and physiological characteristics alone. However, it is noteworthy that strain LMG 11983, belonging to the same species as strain BK2T, was isolated from grass silage, an environment similar to kimchi in terms of plant fermentation. Similarly,
Chemotaxonomic Characteristics
Strains BK2T and LMG 11983 exhibited appreciable amounts (>10% of total fatty acids) of C18:1
-
Table 3 . Cellular fatty acid compositions (%) of strains BK2T and LMG 11983 and closely related type strains of the genus
Weissella Fatty acid 1 2 3 4 5 6 Saturated: C14:0 0.9 1.0 1.1 1.4 7.7 – C16:0 23.4 22.5 20.8 21.3 24.1 17.9 C17:0 tr – – – – – C18:0 3.2 2.5 3.1 3.4 2.9 2.3 C20:0 – – – 0.8 3.5 – Unsaturated: C18:1 ω 9c 29.5 25.8 34.4 39.4 19.1 68.0 Branched: iso-C19:0 1.4 1.2 1.8 2.1 1.6 2.9 Hydroxy: C17:0 2-OH 1.5 1.5 1.5 2.1 1.7 2.3 Summed feature*: 3 3.9 5.4 6.1 3.9 5.9 1.3 7 29.7 32.9 16.6 20.7 29.6 – 8 6.1 7.4 14.8 5.0 3.6 4.5 Taxa: 1, strain BK2T; 2, strain LMG 11983; 3,
W. confusa KACC 11841T; 4,W. cibaria KACC 11862T; 5,W. muntiaci NBRC 113537T; 6,W. viridescens KACC 11850T. Fatty acids amounting to less than 0.5% in all strains are not shown. Major fatty acids (>10.0%) are denoted in bold; tr, trace amount (< 0.5%); –, not detected. All data were obtained in this study.*Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed features 3, 7, and 8 contain C16:1
ω 6c and/or C16:1ω 7c , C19:1ω 6c and/or C19:1ω 7c , and C18:1ω 6c , respectively.
In conclusion, based on the phylogenetic analysis, genome relatedness, and phenotypic and chemotaxonomic features, it is evident that strains BK2T and LMG 11983 represent a single novel species of the genus
Description of Weissella fermenti sp. nov.
Cells are Gram-stain-positive, facultatively anaerobic, and non-motile short rods (0.7–0.8 μm in width and 2.0–2.2 μm in length). Colonies on MRS agar are ivory-colored, circular, smooth, and convex. Growth occurs at 15–45°C (optimum, 30°C), pH 5.0–8.0 (optimum, 7.0), and 0–3.0% NaCl (optimum, 1.0% NaCl). Catalase- and oxidase-negative. Acetoin is produced, but not indole and H2S. Esculin is hydrolyzed, but not tyrosine, casein, gelatin, Tween 20, Tween 80, and starch. Positive for alkaline phosphatase, leucine arylamidase,
The type of strain is BK2T (=KACC 22833T=JCM 35750T), isolated from kimchi in South Korea. The GenBank accession numbers of the 16S rRNA gene and genome sequences of strain BK2T are OP376746 and JAOZFC000000000, respectively.
Supplemental Materials
Author Contributions
J.K.L., J.H.B., and C.O.J. designed the research; J.K.L. and J.H.B. performed the main experiments and analyses; D.M.H., S.H.L., and S.Y.K. performed data analyses; J.K.L. and J.H.B. wrote the manuscript; C.O.J. supervised the study and obtained funding; all authors read and approved the final version of the manuscript.
Acknowledgments
This work was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01710102), RDA; and the National Institute of Biological Resources (NIBR No. 2022-02-001), Republic of Korea.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Article
Research article
J. Microbiol. Biotechnol. 2023; 33(11): 1448-1456
Published online November 28, 2023 https://doi.org/10.4014/jmb.2306.06010
Copyright © The Korean Society for Microbiology and Biotechnology.
Description and Genomic Characteristics of Weissella fermenti sp. nov., Isolated from Kimchi
Jae Kyeong Lee1†, Ju Hye Baek1†, Dong Min Han1, Se Hee Lee2, So Young Kim3, and Che Ok Jeon1*
1Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea
2Microbiology and Functionality Research Group, World Institute of Kimchi, Gwangju, 61755, Republic of Korea
3Department of Agro-Food Resources, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea
Correspondence to:Che Ok Jeon, cojeon@cau.ac.kr
†These authors contributed equally to this work.
Abstract
A Gram-positive, non-motile, and non-spore-forming lactic acid bacterium, designated as BK2T, was isolated from kimchi, a Korean traditional fermented vegetable food, and the taxonomic characteristics of strain BK2T, along with strain LMG 11983, were analyzed. Both strains optimally grew at 30°C, pH 7.0, and 1.0% NaCl. Cells of both strains were heterofermentative and facultatively anaerobic rods, demonstrating negative reactions for catalase and oxidase. Major fatty acids (>10%) identified in both strains were C18:1 ω9c, C16:0, and summed feature 7 (comprising C19:1 ω6c and/or C19:1 ω7c). The genomic DNA G+C contents of both strains were 44.7 mol%. The 16S rRNA gene sequence similarity (99.9%), average nucleotide identity (ANI; 99.9%), and digital DNA-DNA hybridization (dDDH; 99.7%) value between strains BK2T and LMG 11983 indicated that they are different strains of the same species. Strain BK2T was most closely related to Weissella confusa JCM 1093T and Weissella cibaria LMG 17699T, with 100% and 99.4% 16S rRNA gene sequence similarities, respectively. However, based on the ANI and dDDH values (92.3% and 48.1% with W. confusa, and 78.4% and 23.5% with W. cibaria), it was evident that strain BK2T represents a distinct species separate from W. confusa and W. cibaria. Based on phylogenetic, phenotypic, and chemotaxonomic features, strains BK2T and LMG 11983 represent a novel species of the genus Weissella, for which the name Weissella fermenti sp. nov. is proposed. The type of strain is BK2T (=KACC 22833T=JCM 35750T).
Keywords: Weissella fermenti, new taxa, taxonomy, kimchi, genome
Introduction
Since the genus
Many
Material and Methods
Isolation and Cultivation
Strain BK2T was isolated from fermented baechu kimchi prepared using baechu cabbage (
Phylogenetic Analysis Based on 16S rRNA Gene Sequences
To obtain nearly complete sequences of the 16S rRNA genes of strains BK2T and LMG 11983, PCR amplification was performed using the F1 and R13 primers. The resulting PCR amplicons were subjected to sequencing using the 340F, 518R (5'-ATT ACC GCG GCT GCT GG-3'), and 805F (5'-GAT TAG ATA CCC TGG TAG TC-3') primers, and the resulting nucleotide sequences were assembled as described previously [23]. The 16S rRNA gene sequence similarities between strains BK2T and LMG 11983 and other bacterial type strains were calculated using the EzBioCloud server. The 16S rRNA gene sequences of strains BK2T and LMG 11983 and closely related valid type strains were aligned using the secondary-structure aware Infernal aligner [25]. Phylogenetic trees were constructed using the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) algorithms with bootstrap values (1,000 replications) in MEGA11 [26]. The Kimura two-parameter model, nearest-neighbor-interchange heuristic search method, and complete deletion options were used for the NJ, ML, and MP tree constructions, respectively.
Genome Sequencing and Phylogenomic Analysis
The gDNA of strains BK2T and LMG 11983 was extracted using a Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturer's instructions. The gDNA of strain BK2T was sequenced using a hybrid combination of the Oxford Nanopore MinION platform (Nanopore, UK) in our laboratory and the Illumina HiSeq X platform (Illumina, USA) with 151 bp paired-end sequencing reads at Macrogen. On the other hand, the gDNA of strain LMG 11983 was sequenced using the Oxford Nanopore MinION platform. Sequencing reads derived from the Nanopore MinION sequencing were de novo-assembled using Flye ver. 2.9.1 [27]. The assembled long contigs of strain BK2T were polished with Illumina sequencing reads using Pilon ver. 1.24 [28], and multiple rounds of polishing were performed until no additional corrections were accomplished. The quality of the assembled genomes was assessed by evaluating their completeness and contamination rates using CheckM ver. 1.0.4 [29]. Finally, the genome sequences of strains BK2T and LMG 11983 were submitted to GenBank and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; www.ncbi.nlm.nih.gov/genome/annotation_prok/) [30].
The DNA G+C contents of the strains were determined based on their whole genome sequences. To perform genome-based phylogenomic analysis, 92 housekeeping core genes were extracted from the genomes of strains BK2T and LMG 11983, as well as from closely related
Phenotypic, Physiological, and Biochemical Analyses
Strains BK2T and LMG 11983 were tested for their growth on various culture media including MRS agar, Reasoner's 2A (R2A) agar (BD), tryptic soy agar (TSA; BD), nutrient agar (NA; BD), marine agar (MA; BD), and Luria-Bertani (LB) agar (BD) for 2 days at 30°C. To evaluate the growth temperature and pH ranges of strains BK2T and LMG 11983, they were incubated on MRS agar and in MRS broth at different temperatures (10–50°C at 5°C intervals) and pH values (4.0–10.0 at 1.0 pH unit intervals), respectively, for 2 days. MRS broths with pH 4.0–5.0, 6.0–8.0, and 9.0–10.0 were prepared using sodium citrate, Na2HPO4/NaH2PO4, and Tris-HCl buffers, respectively [35]. The pH values were adjusted if necessary after autoclaving at 121°C for 15 min. The MRS broths used for pH testing were incubated without agitation at 30°C. The growth of strains BK2T and LMG 11983 was also examined in MRS broth containing different concentrations of NaCl (0–5%, w/v, at 1.0% intervals), prepared in the laboratory according to the MRS formula.
The biochemical and physiological properties of strains BK2T and LMG 11983 were evaluated using cells grown on MRS agar for 2 days at 30°C. Gram staining was performed using PREVI Colour Gram (bioMérieux, France), following the manufacturer's instructions. The cell morphology of strains BK2T and LMG 11983 was examined using phase-contrast microscopy (Carl Zeiss, Germany) and transmission electron microscopy (JEM-1010; Jeol, Japan). Oxidase and catalase activities were determined by observing the oxidation of 1% (w/v) tetramethyl-
The phenotypic characteristics of strains BK2T and LMG 11983, along with four closely related reference strains, were examined under identical growth conditions. Hydrolysis of tyrosine, casein, esculin, gelatin, starch, Tween 20, and Tween 80 was assessed on MRS agar following previously described protocols [36, 37]. Additional biochemical features and enzymatic activities were determined using the API 20E and API ZYM systems (bioMérieux), respectively, according to the manufacturer's instructions. Acid production from various carbohydrates was evaluated using API 50 CH test strips (bioMérieux) with the API 50 CHL medium, following the manufacturer's guidelines. The configuration of lactic acids produced by strains BK2T and LMG 11983 and reference strains was determined using a DL-lactate test kit (Boehringer Mannheim/R-Biopharm, Germany).
Chemotaxonomic Analyses
For cellular fatty acid analysis, strains BK2T and LMG 11983, along with four reference strains, were cultivated in MRS broth at 30°C. Microbial cells were harvested during the exponential growth phase at an 0.8 optical density at 600 nm. The cellular fatty acids of the harvested cells were saponified, methylated, and extracted following the standard Sherlock Microbial Identification System (MIDI) protocol. Fatty acid methyl esters were analyzed by gas chromatography on the HP 6890 GC (Hewlett Packard, USA) and identified using the MIDI identification database RTSBA6 (ver. 6.0B). Polar lipids of strains BK2T and LMG 11983 were extracted from cells harvested during the exponential growth phase and analyzed by two-dimensional thin-layer chromatography following the protocol described by Minnikin
Results and Discussion
Phylogenetic Analysis Based on 16S rRNA Gene Sequences
Sequencing using the 340F, 518R, and 805F primers yielded nearly complete 16S rRNA gene sequences of strains BK2T (1,513 nucleotides) and LMG 11983 (1,504 nucleotides). The obtained sequences of strains BK2T and LMG 11983 were found to be very similar, showing a 99.9% sequence similarity, indicating their close phylogenetic relatedness. Comparative analysis of the 16S rRNA gene sequences revealed that strain BK2T exhibited the highest similarities to
Phylogenetic analysis using the NJ algorithm based on the 16S rRNA gene sequences demonstrated that strains BK2T and LMG 11983 formed a tight phyletic lineage with
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Figure 1. A neighbor-joining tree (NJ) showing the phylogenetic relationships between strains BK2T and LMG 11983 and closely related species based on 16S rRNA gene sequences. Bootstrap values (based on 1,000 replications) exceeding 70% are indicated at branch points. The filled circles (●) indicate branches that were commonly recovered in NJ, maximum-likelihood, and maximum-parsimony algorithms.
Enterococcus plantarum CCM 7889T (MT758013) was used as the outgroup. The scale bar equals 0.01 changes per nucleotide position.
Genomic Features and Phylogenomic and Genome Relatedness
The
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Table 1 . General genomic features of strains BK2T and LMG 11983 and related
Weissella type strains and their genome relatedness..Characteristic 1 2 3 4 5 6 Genomic features* Genome status Draft Draft Draft Draft Draft Draft No. of contigs 5 2 41 25 41 8 Genome size (Mb) 2.50 2.51 2.19 2.32 2.29 1.54 G+C content (mol%) 44.7 44.7 44.7 45.1 40.4 40.8 No. of total genes 2,433 2,506 2,121 2,198 2,345 1,546 No. of protein-coding genes 2,299 2,275 2,050 2,090 2,243 1,475 Proteins assigned to COG (%) 1,426 (62.0) 1,411 (62.0) 1,431 (69.8) 1,491 (71.3) 1,384 (61.7) 1,149 (77.9) No. of rRNAs (5S, 16S, and 23S) 9, 8, 8 9, 8, 8 1, 1, 1 7, 2, 2 4, 1, 1 1, 0, 1 No. of total tRNA genes 83 84 49 77 74 46 No. of noncoding RNAs 3 3 3 3 3 3 No. of CRISPRs† 1 1 – – – – Lactate dehydrogenase (LDH)a L-LDH (EC 1.1.1.27) 1 1 1 2 – 1 D- LDH (EC 1.1.1.28) 1 1 1 1 1 1 D- LDH (EC 1.1.5.12) 1 1 1 – – 1 ANI value (%)† Taxa dDDH value (%)† 1 – 99.7 48.1 23.5 23.6 22.6 2 99.9 – 48.1 23.5 23.6 22.6 3 92.3 92.4 – 23.2 20.9 20.1 4 78.4 78.3 78.5 – 20.2 19.8 5 70.5 70.3 70.4 70.6 – 20.3 6 70.8 70.8 70.7 70.7 69.4 – Taxa: 1, strain BK2T (JAOZFC000000000); 2, strain LMG 11983 (JAOZFD000000000); 3,
W. confusa NBRC 106469T (BJZE00000000); 4,W. cibaria JCM 12495T (BJEF00000000); 5,W. muntiaci 8H-2T (SDGZ00000000); 6,W. viridescens DSM 20410T (JQBM00000000). *The information was obtained from GenBank and annotated by the NCBI prokaryotic genome annotation pipeline (PGAP; www.ncbi.nlm.nih.gov/genome/annotation_prok/)..†CRISPR, clustered regularly interspaced short palindromic repeat; ANI, average nucleotide identity; dDDH, digital DNA-DNA hybridization..
Clustered regularly interspaced short palindromic repeats (CRISPRs) have been widely recognized as a host defense mechanism against bacteriophage predation [40]. Interestingly, the genomes of strains BK2T and LMG 11983 were found to harbor a single CRISPR region, unlike other
Strain BK2T was found to possess three distinct types of the lactate dehydrogenase gene (
The ANI and dDDH values between strain BK2T and strain LMG 11983 were determined to be 99.9% and 99.7%, respectively, which surpass the established thresholds (ANI ~95%; dDDH 70%) for prokaryotic species delineation [42]. This indicates that strains BK2T and LMG 11983 belong to the same species. Furthermore, the ANI and dDDH values between strain BK2T and closely related reference type strains, including
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Figure 2. A phylogenomic tree showing the phylogenetic relationships between strains BK2T and LMG 11983 and closely related species, based on the concatenated amino acid sequences of 92 housekeeping core genes. Bootstrap values (based on 1,000 replications) exceeding 70% are indicated at branch points.
Enterococcus plantarum CCM 7889T (MT758013) was used as the outgroup. The scale bars indicate 0.1 changes per amino acid.
Phenotypic, Physiological, and Biochemical Characteristics
Strains BK2T and LMG 11983 exhibited robust growth on MRS agar and demonstrated relatively favorable growth on LB agar and TSA, but displayed slow growth on R2A agar, NA, and MA. After 2 days of incubation at 30°C, both strains formed ivory-colored colonies on MRS agar, a characteristic that distinguishes them from closely related reference strains, which formed white colonies (Table 2). The cells of strains BK2T and LMG 11983 were observed to be Gram-stain positive, non-motile rods with dimensions of 0.7–0.8 μm in width and 2.0–2.2 μm in length (Fig. S2). These strains produced both the D- and L-enantiomers of lactic acid in an approximate ratio of 85:15, similar to the other reference strains, except
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Table 2 . Differential phenotype characteristics of strains BK2T and LMG 11983 and closely related type strains of the genus
Weissella ..Characteristic 1 2 3 4 5 6 Isolation source Kimchi Grass silage Sugar cane Chili bo Feces Cured meat Colony color Ivory Ivory White White White White Enzyme activity (API ZYM)* of: Alkaline phosphatase + + + + – – Leucine arylamidase + + + – + + α -Chymotrypsin+ + + – – – β -Glucuronidase,β -glucosidase, valine arylamidase+ + – – – – α -Glucosidase– – – – – + Enzyme activity (API 20E)* of: β -Galactosidase+ + – – – – Arginine dihydrolase – – + + + – Citrate utilization (API 20E) – – – – + + Fermentation (API 20E)* of: Amygdalin, sucrose + + + + – – D-Mannitol + + – – – – Acid production (API 50 CHL)* from: Arbutin, amygdalin, cellobiose, gentiobiose, salicin, D-galactose, sucrose + + + + – – Trehalose + + – – + + D-Mannitol + + – – – – 2-Keto-gluconate – – + – + + Maltose – – + + – – D-Arabinose – – – + + + Lactic acid configuration* d, l d, l d, l d, l d d, l Taxa: 1, strain BK2T (this study); 2, strain LMG 11983 (this study); 3,
W. confusa KACC 11841T [8]; 4,W. cibaria KACC 11862T [9]; 5,W. muntiaci NBRC 113537T [14]; 6,W. viridescens KACC 11850T [1]..All strains are facultative anaerobic short rods and positive for the following characteristics: acetoin production, activity* of acid phosphatase, naphthol-AS-BI-phosphohydrolase, and tryptophan deaminase, hydrolysis of esculin, D-glucose fermentation*, and acid production* from D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, gluconate, esculin, and
N -acetyl-Dglucosamine. All strains are negative for the following characteristics: hydrolysis of casein, gelatin, starch, tyrosine, Tween 20, and Tween 80, production* of indole and H2S, activity* of catalase, oxidase, lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, esterase (C4), esterase lipase (C8), lipase (C14), crystine arylamidase, trypsin,α -galactosidase,α - mannosidase,α -fucosidase, andN -acetyl-β -glucosaminidase, fermentation* of inositol, L-rhamnose, D-melibiose, and Dsorbitol, and acid production* from glycerol, erythritol, L-arabinose, D-adonitol, D-lactose, L-rhamnose, D-melibiose, Dmelezitose, D-raffinose, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-xylose, L-sorbose, L-fucose, L-arabitol, dulcitol, inositol, sorbitol, inulin, starch, glycogen, xylitol, methylβ -D-xylopyranoside, methylα -D-mannoside, methylα -Dglucoside, and 5-keto-gluconate. Symbols: +, positive; –, negative..*These analyses were conducted under the same conditions in this study..
Several phenotypic properties were found to be shared between strains BK2T and LMG 11983 and their closely related
The ability of strain BK2T to thrive specifically in kimchi fermentation cannot be fully explained by genomic analysis or the examination of several metabolic and physiological characteristics alone. However, it is noteworthy that strain LMG 11983, belonging to the same species as strain BK2T, was isolated from grass silage, an environment similar to kimchi in terms of plant fermentation. Similarly,
Chemotaxonomic Characteristics
Strains BK2T and LMG 11983 exhibited appreciable amounts (>10% of total fatty acids) of C18:1
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Table 3 . Cellular fatty acid compositions (%) of strains BK2T and LMG 11983 and closely related type strains of the genus
Weissella .Fatty acid 1 2 3 4 5 6 Saturated: C14:0 0.9 1.0 1.1 1.4 7.7 – C16:0 23.4 22.5 20.8 21.3 24.1 17.9 C17:0 tr – – – – – C18:0 3.2 2.5 3.1 3.4 2.9 2.3 C20:0 – – – 0.8 3.5 – Unsaturated: C18:1 ω 9c 29.5 25.8 34.4 39.4 19.1 68.0 Branched: iso-C19:0 1.4 1.2 1.8 2.1 1.6 2.9 Hydroxy: C17:0 2-OH 1.5 1.5 1.5 2.1 1.7 2.3 Summed feature*: 3 3.9 5.4 6.1 3.9 5.9 1.3 7 29.7 32.9 16.6 20.7 29.6 – 8 6.1 7.4 14.8 5.0 3.6 4.5 Taxa: 1, strain BK2T; 2, strain LMG 11983; 3,
W. confusa KACC 11841T; 4,W. cibaria KACC 11862T; 5,W. muntiaci NBRC 113537T; 6,W. viridescens KACC 11850T. Fatty acids amounting to less than 0.5% in all strains are not shown. Major fatty acids (>10.0%) are denoted in bold; tr, trace amount (< 0.5%); –, not detected. All data were obtained in this study..*Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed features 3, 7, and 8 contain C16:1
ω 6c and/or C16:1ω 7c , C19:1ω 6c and/or C19:1ω 7c , and C18:1ω 6c , respectively..
In conclusion, based on the phylogenetic analysis, genome relatedness, and phenotypic and chemotaxonomic features, it is evident that strains BK2T and LMG 11983 represent a single novel species of the genus
Description of Weissella fermenti sp. nov.
Cells are Gram-stain-positive, facultatively anaerobic, and non-motile short rods (0.7–0.8 μm in width and 2.0–2.2 μm in length). Colonies on MRS agar are ivory-colored, circular, smooth, and convex. Growth occurs at 15–45°C (optimum, 30°C), pH 5.0–8.0 (optimum, 7.0), and 0–3.0% NaCl (optimum, 1.0% NaCl). Catalase- and oxidase-negative. Acetoin is produced, but not indole and H2S. Esculin is hydrolyzed, but not tyrosine, casein, gelatin, Tween 20, Tween 80, and starch. Positive for alkaline phosphatase, leucine arylamidase,
The type of strain is BK2T (=KACC 22833T=JCM 35750T), isolated from kimchi in South Korea. The GenBank accession numbers of the 16S rRNA gene and genome sequences of strain BK2T are OP376746 and JAOZFC000000000, respectively.
Supplemental Materials
Author Contributions
J.K.L., J.H.B., and C.O.J. designed the research; J.K.L. and J.H.B. performed the main experiments and analyses; D.M.H., S.H.L., and S.Y.K. performed data analyses; J.K.L. and J.H.B. wrote the manuscript; C.O.J. supervised the study and obtained funding; all authors read and approved the final version of the manuscript.
Acknowledgments
This work was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01710102), RDA; and the National Institute of Biological Resources (NIBR No. 2022-02-001), Republic of Korea.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

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Table 1 . General genomic features of strains BK2T and LMG 11983 and related
Weissella type strains and their genome relatedness..Characteristic 1 2 3 4 5 6 Genomic features* Genome status Draft Draft Draft Draft Draft Draft No. of contigs 5 2 41 25 41 8 Genome size (Mb) 2.50 2.51 2.19 2.32 2.29 1.54 G+C content (mol%) 44.7 44.7 44.7 45.1 40.4 40.8 No. of total genes 2,433 2,506 2,121 2,198 2,345 1,546 No. of protein-coding genes 2,299 2,275 2,050 2,090 2,243 1,475 Proteins assigned to COG (%) 1,426 (62.0) 1,411 (62.0) 1,431 (69.8) 1,491 (71.3) 1,384 (61.7) 1,149 (77.9) No. of rRNAs (5S, 16S, and 23S) 9, 8, 8 9, 8, 8 1, 1, 1 7, 2, 2 4, 1, 1 1, 0, 1 No. of total tRNA genes 83 84 49 77 74 46 No. of noncoding RNAs 3 3 3 3 3 3 No. of CRISPRs† 1 1 – – – – Lactate dehydrogenase (LDH)a L-LDH (EC 1.1.1.27) 1 1 1 2 – 1 D- LDH (EC 1.1.1.28) 1 1 1 1 1 1 D- LDH (EC 1.1.5.12) 1 1 1 – – 1 ANI value (%)† Taxa dDDH value (%)† 1 – 99.7 48.1 23.5 23.6 22.6 2 99.9 – 48.1 23.5 23.6 22.6 3 92.3 92.4 – 23.2 20.9 20.1 4 78.4 78.3 78.5 – 20.2 19.8 5 70.5 70.3 70.4 70.6 – 20.3 6 70.8 70.8 70.7 70.7 69.4 – Taxa: 1, strain BK2T (JAOZFC000000000); 2, strain LMG 11983 (JAOZFD000000000); 3,
W. confusa NBRC 106469T (BJZE00000000); 4,W. cibaria JCM 12495T (BJEF00000000); 5,W. muntiaci 8H-2T (SDGZ00000000); 6,W. viridescens DSM 20410T (JQBM00000000). *The information was obtained from GenBank and annotated by the NCBI prokaryotic genome annotation pipeline (PGAP; www.ncbi.nlm.nih.gov/genome/annotation_prok/)..†CRISPR, clustered regularly interspaced short palindromic repeat; ANI, average nucleotide identity; dDDH, digital DNA-DNA hybridization..
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Table 2 . Differential phenotype characteristics of strains BK2T and LMG 11983 and closely related type strains of the genus
Weissella ..Characteristic 1 2 3 4 5 6 Isolation source Kimchi Grass silage Sugar cane Chili bo Feces Cured meat Colony color Ivory Ivory White White White White Enzyme activity (API ZYM)* of: Alkaline phosphatase + + + + – – Leucine arylamidase + + + – + + α -Chymotrypsin+ + + – – – β -Glucuronidase,β -glucosidase, valine arylamidase+ + – – – – α -Glucosidase– – – – – + Enzyme activity (API 20E)* of: β -Galactosidase+ + – – – – Arginine dihydrolase – – + + + – Citrate utilization (API 20E) – – – – + + Fermentation (API 20E)* of: Amygdalin, sucrose + + + + – – D-Mannitol + + – – – – Acid production (API 50 CHL)* from: Arbutin, amygdalin, cellobiose, gentiobiose, salicin, D-galactose, sucrose + + + + – – Trehalose + + – – + + D-Mannitol + + – – – – 2-Keto-gluconate – – + – + + Maltose – – + + – – D-Arabinose – – – + + + Lactic acid configuration* d, l d, l d, l d, l d d, l Taxa: 1, strain BK2T (this study); 2, strain LMG 11983 (this study); 3,
W. confusa KACC 11841T [8]; 4,W. cibaria KACC 11862T [9]; 5,W. muntiaci NBRC 113537T [14]; 6,W. viridescens KACC 11850T [1]..All strains are facultative anaerobic short rods and positive for the following characteristics: acetoin production, activity* of acid phosphatase, naphthol-AS-BI-phosphohydrolase, and tryptophan deaminase, hydrolysis of esculin, D-glucose fermentation*, and acid production* from D-ribose, D-xylose, D-glucose, D-fructose, D-mannose, gluconate, esculin, and
N -acetyl-Dglucosamine. All strains are negative for the following characteristics: hydrolysis of casein, gelatin, starch, tyrosine, Tween 20, and Tween 80, production* of indole and H2S, activity* of catalase, oxidase, lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, esterase (C4), esterase lipase (C8), lipase (C14), crystine arylamidase, trypsin,α -galactosidase,α - mannosidase,α -fucosidase, andN -acetyl-β -glucosaminidase, fermentation* of inositol, L-rhamnose, D-melibiose, and Dsorbitol, and acid production* from glycerol, erythritol, L-arabinose, D-adonitol, D-lactose, L-rhamnose, D-melibiose, Dmelezitose, D-raffinose, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-xylose, L-sorbose, L-fucose, L-arabitol, dulcitol, inositol, sorbitol, inulin, starch, glycogen, xylitol, methylβ -D-xylopyranoside, methylα -D-mannoside, methylα -Dglucoside, and 5-keto-gluconate. Symbols: +, positive; –, negative..*These analyses were conducted under the same conditions in this study..
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Table 3 . Cellular fatty acid compositions (%) of strains BK2T and LMG 11983 and closely related type strains of the genus
Weissella .Fatty acid 1 2 3 4 5 6 Saturated: C14:0 0.9 1.0 1.1 1.4 7.7 – C16:0 23.4 22.5 20.8 21.3 24.1 17.9 C17:0 tr – – – – – C18:0 3.2 2.5 3.1 3.4 2.9 2.3 C20:0 – – – 0.8 3.5 – Unsaturated: C18:1 ω 9c 29.5 25.8 34.4 39.4 19.1 68.0 Branched: iso-C19:0 1.4 1.2 1.8 2.1 1.6 2.9 Hydroxy: C17:0 2-OH 1.5 1.5 1.5 2.1 1.7 2.3 Summed feature*: 3 3.9 5.4 6.1 3.9 5.9 1.3 7 29.7 32.9 16.6 20.7 29.6 – 8 6.1 7.4 14.8 5.0 3.6 4.5 Taxa: 1, strain BK2T; 2, strain LMG 11983; 3,
W. confusa KACC 11841T; 4,W. cibaria KACC 11862T; 5,W. muntiaci NBRC 113537T; 6,W. viridescens KACC 11850T. Fatty acids amounting to less than 0.5% in all strains are not shown. Major fatty acids (>10.0%) are denoted in bold; tr, trace amount (< 0.5%); –, not detected. All data were obtained in this study..*Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed features 3, 7, and 8 contain C16:1
ω 6c and/or C16:1ω 7c , C19:1ω 6c and/or C19:1ω 7c , and C18:1ω 6c , respectively..
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