Phylogenetic and phenotypic features of the isolated LAB strain
A bacterial strain, designated WiKim0068, was isolated from a Korean fermented food, kimchi. In order to identify the phylogenetic similarity of strain WiKim0068, phylogenetic analysis based on the 16S rRNA gene sequences of strain WiKim0068 and the closely related strains indicated that L. raffinolactis NBRC 100932T was most closely related to strain WiKim0068 with 99.9% similarity (Fig. 1). This result indicated that strain WiKim0068 belong to the L. raffinolactis species. Positive reactions were observed for sugar assimilation/acid formation from galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, esculin, ferric citrate, salicin, cellobiose, maltose, melibiose, saccharose, trehalose, raffinose, and turanose. Enzyme detection with an API ZYM kit was positive for esterase, leucine arylamidase, and naphthol-AS-BI-phosphohydrolase.
General genomic features of strain WiKim0068
The PacBio RS II sequencing system generated 74,558 reads, with an average read length of 8,212 bp. The complete genome of strain WiKim0068 was composed of a circular 2.22 Mb chromosome and two circular plasmids, with a total size of 2.29 Mb. The chromosome contained 2,060 predicted protein-coding genes (CDSs), 13 rRNA genes (5S rRNA, 5; 16S rRNA, 4; 23S rRNA, 4), 55 tRNAs, and 3 other RNAs. The WiKim0068 genome was observed to contain 39.7 mol% G + C content (Table 1), which is within the reported range of 35.5–46.4% for Lactococcus species [10], and similar to the 39.25 mol% observed in two L. raffinolactis strains, 4877 (CALL00000000) and NBRC 100932T (BCVN00000000). For functional classification, WiKim0068 genome was analyzed using the clusters of orthologous genes (COG) database (http://www.ncbi.nlm.nih.gov/COG/), and 2,000 genes were annotated. The annotated genes were associated with the following categories: general function prediction only (R; 237 genes), carbohydrate transport and metabolism (G; 210 genes), function unknown (S; 190 genes), amino acid transport and metabolism (E; 180 genes), coenzyme transport and metabolism (H; 70 genes), defense mechanisms (V; 63 genes), and secondary metabolites biosynthesis, transport, and catabolism (Q; 15 genes; Supplementary Table S1). In addition, Rapid Annotation using Subsystem Technology analysis revealed categories containing genes related to stress response (2.62%), cofactors, vitamins, prosthetic groups, pigments (5.30%), and virulence, disease and defense (3.39%) (Supplementary Fig. S1). Stress response-related gene categories included: “osmotic stress” (5 genes), “oxidative stress” (17 genes), “cold shock” (1 gene), “heat shock” (15 genes), “detoxification” (9 genes), and “no subcategory” (1 gene). Category of cofactors, vitamins, prosthetic groups, pigments includes major “biotin” (15 genes), “NAD and NADP” (14 genes), “riboflavin, FMN, FAD” (8 genes), and “folate and pterines” (33 genes). Category of virulence, disease and defense includes major “bacteriocins, ribosomally synthesized antibacterial peptides” (8 genes), “resistance to antibiotics and toxic compounds” (29 genes), and “invasion and intracellular resistance” (15 genes). In particular, biotin, riboflavin, and folate are related to human health and digestion and cause various symptoms when deficient [11]. Bacteriocins are antimicrobial peptides produced from bacteria [12] and an alternative to antibiotic resistance bacteria. In addition, bacteriocins have been regarded an important feature in the selection of probiotic strains. These genes were associated with the presence of useful probiotic characteristics, which play important roles in the food and pharmaceutical industries [13–15].
Table 1
Comparative genomic features of Lactococcus raffinolactis WiKim0068, L. raffinolactis 4877, and L. raffinolactis NBRC 100932T.
| Strain |
WiKim0068 | 4877 | NBRC 100932T |
Assembly size (Mb) | 2.29 | 2.28 | 2.18 |
DNA G + C content (mol%) | 39.7 | 38.7 | 39.8 |
CDSs* | 2,187 | 2,362 | 2,123 |
Scaffolds | 3 | 127 | 114 |
Genes | 2,258 | 2,409 | 2,141 |
Proteins | 2,123 | 2,070 | 2,030 |
rRNAs | 13 | 12 | 2 |
tRNAs | 55 | 48 | 29 |
Finishing quality | Complete | Scaffold | Contig |
*CDSs, Coding Sequences |
Comparative genomic analysis
Analysis of the orthologous average nucleotide identity (orthoANI) values among Lactococcus genome sequences showed that strain WiKim0068 had 68.55-98.73% genome sequence similarities with other species and subspecies. The genome was most closely related to that of L. raffinolactis NBRC 100932T (98.73%), followed by L. raffinolactis 4877 (87.02%), L. piscium MKFS47 (76.57%), L. lactis subsp. lactis IL 1403 (69.41%), L. fujiensis JCM 16395 (68.12%), and L. garvieae ATCC 49156 (68.55%; Fig. 2). These results indicated that strain WiKim0068 belongs to the species L. raffinolactis by comparative whole genome sequence analysis [16]. Its similarity to its two closest relative species (L. raffinolactis NBRC 100932T and L. raffinolactis 4877), based on BLAST comparison, is shown in Fig. 3. In addition, we searched for CRISPRs using the CRISPRFinder platform, but no confirmed CRISPRs were found in the WiKim0068 genome.
Phage and pathogenesis-related genes
PHAST analysis was performed to identify prophage contamination in the WiKim0068 genome. The chromosome contained two intact, one incomplete, and one questionable prophage, while the first plasmid (pWiKim0068-1) contained only one incomplete prophage and the second plasmid (pWiKim0068-2) contained none (Supplementary Fig. S2). Intact prophage regions were located between positions 57,319–90,123 and 1,524,268–1,563,900 bp of the chromosome.
Carbon metabolic pathway
Predicted metabolic pathways in strain WiKim0068 were associated with diverse phosphotransferase (PTS) systems or permeases that transport various carbohydrates, including d-glucose, d-galactose, d-mannose, trehalose, sucrose, cellobiose, N-acetyl-glucosamine, fructose, maltose, mannitol, galactitol, and lactose. The presence of these transport genes indicated the possibility that strain WiKim0068 performs fermentation metabolism using various carbohydrates (Fig. 4). Based on the metabolic pathways in strain WiKim0068, it was confirmed that the strain had heterofermentative pathways. Hexoses (glucose, fructose, and mannose) in strain WiKim0068 can be converted to lactate, ethanol and carbon dioxide. d- and l-Lactate are produced from the reduction of pyruvate by d-lactate dehydrogenase (d-LDH) (EC 1.1.1.28) and l-lactate dehydrogenase (l-LDH) (EC 1.1.1.27), respectively. However, strain WiKim0068 harbors only l-LDH (locus tag: CMV25_RS07125). This is similar to previous studies in which l-LDH was identified in Lactococcus lactis, belonging to the same genus as strain WiKim0068 [17]. Since d-lactate produced by LAB may induce d-lactate acidosis in some individuals [18], it is important to develop LAB for the production of dairy products that produce only l-lactate.
Antibiotic resistance
Recently, interest in foods as mediators of antibiotic resistance has been increasing. LAB, which are widely used in probiotics or starter cultures, have the potential to serve as hosts for antibiotic resistance genes, and carry the risk of transferring genes from various LAB and bacterial pathogens [19]. Strain WiKim0068 showed susceptibility to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, penicillin, rifampin, tetracycline, and vancomycin (Table 2).
Table 2
Antibiotic susceptibility of Lactococcus raffinolactis WiKim0068.
Antibiotic | Antibiotic susceptibility test |
Amount | Inhibition zone diameter (mm) |
Ampicillin | 10 µg | 30 |
Chloramphenicol | 30 µg | 40 |
Ciprofloxacin | 5 µg | 28 |
Erythromycin | 15 µg | 48 |
Gentamicin | 10 µg | 18 |
Penicillin | 10 U | 64 |
Rifampin | 5 µg | 42 |
Tetracycline | 30 µg | 65 |
Vancomycin | 30 µg | 23 |
Amino acid and nicotinate and nicotinamide metabolism
The amino acid metabolism-related genes of strain WiKim0068 were annotated using the KEGG database. Among 163 genes involved in amino acid metabolism, strain WiKim0068 harbors the most genes involved in the amino acid metabolism of cysteine, methionine, alanine, aspartate, and glutamate (Fig. 5), suggesting that the strain possesses the biosynthesis and utilization ability of the various amino acids.
Vitamin B3, one of the eight B vitamins, is also known as nicotinate or niacin. This endogenous metabolite is an effective antioxidant, preventing oxidative damage [20]. In general, nicotinamide and nicotinate metabolites are frequently reported with Lactobacillus strains [21–23], while Lactococcus members were unknown about these metabolites. Interestingly, in silico analysis of WiKim0068 genome was predicted an almost complete complement metabolic pathway from nicotinamide and nicotinate metabolism genes (Fig. 6), and through this metabolic pathway, 0.932 mg L− 1 vitamin B3 was produced in cultured cells (Supplementary Fig. S3). These results indicated that nicotinate and nicotinamide metabolism occurs in strain WiKim0068.