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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2023; 51(4): 526-530

https://doi.org/10.48022/mbl.2309.09003

Received: September 19, 2023; Revised: October 14, 2023; Accepted: October 20, 2023

Draft Genome Sequence of Latilactobacillus sakei subsp. sakei FBL10, a Putative Probiotic Strain Isolated from Saeujeot (salted fermented shrimp)

So-Yun Lee†, Dabin Kim†, Seung-Min Yang, Eiseul Kim, and Hae-Yeong Kim*

Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea

Correspondence to :
Hae-Yeong Kim,         hykim@khu.ac.kr

Abstract

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Here, we report the draft genome sequence of Latilactobacillus sakei subsp. sakei FBL10 isolated from Saeujeot (salted fermented shrimp). The draft genome consists of 2,285,672 bp with a G+C content of 41.1% and contains 2,282 coding genes. Genome analysis revealed that clusters associated with bacteriocin production were identified, in addition to several probiotic properties, such as stress resistance factors and aggregation. On the other hand, antibiotic resistance genes and virulence factors were not present. Pangenome analysis for 32 genomes showed 213 unique genes for FBL10 strain. These results demonstrate the beneficial properties of strain FBL10 as a putative probiotic.

Keywords: Latilactobacillus sakei, Probiotics, whole-genome sequencing, pangenome analysis

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Latilactobacillus sakei is a heterofermentative, rodshaped Gram-positive lactic acid bacteria. They have been isolated from various niches including vegetables, sourdoughs, meat, and fish. In addition, they play an important role in fermented foods, such as fermented cabbage (kimchi), fermented seafood (jeotgal), and fermented meat products. Indeed, L. sakei has been used as a starter culture in fermented sausage and is considered a potential biopreservative of meat products [1]. They also contribute to the safety of the final products due to the production of antimicrobial compounds including sakacin. L. sakei has high adaptability to some adverse environmental conditions, which often prevail during preservation, such as high salt concentration, low temperature, low pH, and oxidative stress [2]. Moreover, they exhibited some health benefits, such as anti-obesity effects and atopic dermatitis [3]. The complete genome sequence was first reported from L. sakei 23K which was isolated from French sausage [4]. Subsequently, the genomes of several strains have been sequenced, and genetic diversity and high variability have been revealed [5]. The genome size varied from 1,800 to 2,300 kb, with a variation of approximately 25%. Presently, 34 complete genomes have been sequenced. The purpose of this study was to analyze the draft genome sequence of L. sakei subsp. sakei FBL10 isolated from Saeujeot to gain deep insight into its probiotic properties.

Saeujeot samples were collected from a local market in Korea. For the isolation of L. sakei, 10 g of the sample was homogenized with 90 ml of phosphate-buffered saline. The diluted homogenate was spread on de Man-Rogora-Sharpe (MRS) agar (MBcell, Korea) and incubated at 30℃ for 48 h. The bacterial colonies were selected based on colony morphology and then identified by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. MALDI-TOF MS was performed using the Microflex LT mass spectrometer (Bruker Daltonics, Germany) with BioTyper database. Genomic DNA was isolated using DNeasy Blood and Tissue kit (Qiagen, Germany) according to the manufacturer’s instruction. The library was prepared using Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific, USA) and its quality was confirmed using an Agilent 2100 bioanalyzer with the high-sensitivity DNA kit. Draft genome sequencing was performed using an Ion Torrent Personal Genome Machine (Thermo Fisher Scientific) using a 318 semiconductor chip (400 bp sequencing reads). A total of 5,796,089 reads, with an average read length of 304 bp, were obtained. Referencebased assembly was carried out using SPAdes version 3.1.0 with L. sakei 23K (CR936503.1) as the reference genome. Assembly of the reads resulted in 59 contigs > 1,000 bp (1,499 to 615,703 bp; N50 length 138,163 bp). The draft genome size was 2,330,863 bp with a G+C content of 41.1% (Table 1). L. sakei subsp. sakei FBL10 has a genome size and G+C content similar to L. sakei wikim 22, L. sakei HUP1, and L. sakei WiKim0095. The assembled sequence was annotated by the National Center for Biotechnology Information (NCBI) prokaryotic genome annotation pipeline. The annotation showed that the genome of L. sakei subsp. sakei FBL10 had 2,282 coding genes.

Table 1 . Genome feature of L. sakei subsp. sakei FBL10.

FeatureValues
Genome size (bp)2,285,672
G+C content (%)41.1
Contig N50138,163
Contig L504
Number of contigs59
Number of coding genes2,282
Number of RNAs62
Number of subsystems298


The species prediction of L. sakei subsp. sakei FBL10 was performed by 16S rRNA gene analysis. The phylogenetic analysis showed that L. sakei subsp. sakei FBL10 was closest to the L. sakei subsp. sakei DSM 20017T (Fig. 1). The 16S rRNA gene similarity showed 99.7 and 99.5% for L. sakei subsp. sakei DSM 20017T and L. sakei subsp. carnosus CCUG 31331T, respectively. Also, the 16S rRNA gene similarity showed 97.7 to 99.2% identities between L. sakei subsp. sakei FBL10 and other Latilactobacillus species (L. fragifolii AMBP 162T, 99.2%; L. curvatus NBRC 15884T, 99.2%; L. graminis LMG 9825T, 98.7%; L. fuchuensis B5M10T, 97.7%). The taxonomic position of L. sakei subsp. sakei FBL10 was confirmed by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH). ANI and dDDH values were performed using the ANI calculator and Genome-to-Genome Distance Calculator, respectively. The proposed ANI cut-off for the species boundary is ≥ 95%, indicating that the strain FBL10 is the same species as L. sakei subsp. sakei ATCC 15521T (ANI value, 98.8%) and L. sakei subsp. carnosus DSM 15831T (ANI value, 97.2%). For the subspecies boundary, the dDDH cut-off value is > 79%. dDDH value showed that the strain FBL10 was L. sakei subsp. sakei ATCC 15521T (dDDH value, 91.5%) rather than L. sakei subsp. carnosus DSM 15831T (dDDH value, 75.3%).

Figure 1.Phylogenetic tree for L. sakei subsp. sakei FBL10 strain obtained from this study and other Latilactobacillus species based on 16S rRNA gene sequences using the maximum likelihood method. Bootstrap values for 1,000 replicates are displayed at the branching point. The scale bar indicates 0.02 nucleotide substitutes per position. L. sakei subsp. sakei FBL10 strain obtained from this study is indicated in red letters. The outgroup used Lactococcus lactis ATCC 19435T (M58837.1).

The probiotic-related genes in the L. sakei subsp. sakei FBL10 were identified by Rapid Annotations using Subsystem Technology version 2.0. The secondary metabolite biosynthesis gene cluster was detected using antiSMASH version 6.0. The genome of L. sakei subsp. sakei FBL10 contained putative genes involved in acid and bile tolerance, heat or oxidative stress tolerance, and adhesion to intestinal epithelial cells. Genes encoding F0F1 ATP synthase (Locus tag: MDR7924316.1 to MDR7924323.1) and chologlycine hydrolase (MDR7924149.1), which are associated with low pH and bile salt tolerance, were identified, thus posing the possibility of its survival in the gastrointestinal tract [6]. Genes encoding chaperone proteins, such as dnaK (MDR7924671.1), dnaJ (MDR7924672.1), groES (MDR7924603.1), and grpE (MDR7924670.1) were identified, which are beneficial in tolerating temperature stress [7]. Also, genes associated with the oxidative stress resistance were identified, including katE (MDR7924811.1), trxA (MDR7924119.1), and poxL (MDR7924701.1). The ability to adhere epithelial cells is critical for probiotic bacteria to persist and thrive [7]. L. sakei subsp. sakei FBL10 possesses genes encoding cell surface proteins, including tuf (MDR7923682.1), srtA (MDR7925201.1), and tpiA (MDR7923228.1). Cell surface proteins are known to promote bacterial adhesion to epithelial cells [8]. In addition, L. sakei subsp. sakei FBL10 has gene clusters associated with secondary metabolites production, such as type III polyketide synthase and AgrD-like cyclic lactone autoinducer (Fig. 2). These gene clusters are involved in bacteriocin production [9, 10]. To ensure the safety of L. sakei subsp. sakei FBL10, antibiotic resistance-associated genes and virulence factor-coding genes were searched for using ResFinder version 4.1 (https://cge.food.dtu.dk/services/ResFinder/ResFinder/) and VirulenceFinder version 2.0 (https://cge.food.dtu.dk/services/VirulenceFinder/). The result showed that the genome of L. sakei subsp. sakei FBL10 did not have genes associated with antibiotic resistance and virulence. Pathogenicity analysis by Pathogen-Finder version 1.1 (https://cge.food.dtu.dk/services/PathogenFinder/) showed that this strain was not a pathogen.

Figure 2.Genetic loci for type III polyketide synthase and AgrD-like cyclic lactone autoinducer. Arrows represent the direct of gene. The red, pink, blue, and gray arrows indicate core biosynthetic genes, additional biosynthetic genes, transport-related genes, and other genes, respectively.

For pangenome analysis between the L. sakei subsp. sakei FBL10 and other L. sakei strains, 31 publicly available genomes were downloaded from NCBI (Table 2). The pangenome analysis was confirmed using Bacterial Pan-genome Analysis version 1.3 (https://iicb.res.in/bpga/). The 32 genomes of L. sakei yield 3,672 orthologous genes. Among these, 1,217 genes comprise the core-genome. The accessory-genome and uniquegenome contained 1,482 and 973 genes, respectively. The core, accessory, and unique genes were assigned according to their features in the cluster of orthologous groups (COG) databases (https://www.ncbi.nlm.nih.gov/research/cog). In the core-genome, the group assigned as general function prediction only (R, 13.73%) was the most abundant. The accessory-genome was enriched with genes associated with proteins of carbohydrate transport & metabolism (G, 12.87%) and general function prediction only (R, 12.56%). The unique-genome was enriched with genes encoding proteins of cell wall/ membrane/envelope biogenesis (M, 17.54%). Among the 973 unique genes, L. sakei subsp. sakei FBL10 had 213 unique genes, including genes encoding Clp protease Clp P (MDR7924373.1), type II toxin-antitoxin system PemK/MazF family toxin (MDR7924721.1), molecular chaperone DnaK (MDR7924671.1), and hypothetical proteins. Our data provide a genetic basis for further research to elucidate the probiotic properties of L. sakei subsp. sakei FBL10 and promote its consideration as a potential probiotic strain.

Table 2 . Genomic information used for pangenome analysis.

Organism NameStrainLength (bp)Assembly accessionAssembly level
L. sakei subsp. sakei23K1,884,661GCA_000026065.1Complete Genome
L. sakeiFAM183112,056,563GCA_002224565.1Complete Genome
L. sakeiWiKim00632,075,520GCA_002250035.1Complete Genome
L. sakei subsp. sakeiLT-131,943,136GCA_002370355.1Complete Genome
L. sakeiLK-1451,994,264GCA_002370375.1Complete Genome
L. sakeiWiKim00722,032,521GCA_003288195.1Complete Genome
L. sakeiWiKim00742,037,873GCA_003288235.1Complete Genome
L. sakeiZFM2252,015,501GCA_003627235.1Complete Genome
L. sakeiZFM2202,015,521GCA_003627275.1Complete Genome
L. sakeiZFM2292,015,489GCA_003627315.1Complete Genome
L. sakeiLZ2172,015,495GCA_003627875.1Complete Genome
L. sakeiCBA36142,018,071GCA_009676365.1Complete Genome
L. sakeiCBA36352,062,275GCA_014081765.1Complete Genome
L. sakei subsp. sakeiob4.12,030,031GCA_018437525.1Complete Genome
L. sakeiTMW 1.462,079,718GCA_023734195.1Complete Genome
L. sakeiTMW 1.31,930,337GCA_023734215.1Complete Genome
L. sakei subsp. sakeiTMW 1.11891,942,056GCA_023734235.1Complete Genome
L. sakeiTMW 1.12391,975,742GCA_023734255.1Complete Genome
L. sakeiTMW 1.13961,935,698GCA_023734275.1Complete Genome
L. sakeiTMW 1.13982,066,572GCA_023734295.1Complete Genome
L. sakeiTMW 1.1141,976,338GCA_023734315.1Complete Genome
L. sakeiTMW 1.4171,980,463GCA_023734335.1Complete Genome
L. sakeiTMW 1.5781,976,312GCA_023734355.1Complete Genome
L. sakeiWiKim00952,119,284GCA_024022895.1Complete Genome
L. sakeiPMC1042,015,612GCA_029536225.1Complete Genome
L. sakeiHUP12,160,337GCA_029854175.1Complete Genome
L. sakeiFLEC011,957,804GCA_900234345.1Complete Genome
L. sakeiJ642,098,105GCA_900234355.1Complete Genome
L. sakeiMFPB16A14012,041,677GCA_900234375.1Complete Genome
L. sakeiJ542,011,339GCA_900234395.1Complete Genome
L. sakeiMFPB192,056,455GCA_900234405.1Complete Genome


Nucleotide Sequence Accession number. The draft genome of L. sakei subsp. sakei FBL10 has been deposited in GenBank under accession number of JAVKYY000000000.1.

Acknowledgments

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This research is funded by Graduate School Innovation office, Kyung Hee University (2023).

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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