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Draft genome sequence of oligosaccharide-synthesizing Leuconostoc citreum SG255 isolated from radish kimchi in Korea
Korean J. Microbiol. 2021;57(1):69-71
Published online March 31, 2021
© 2021 The Microbiological Society of Korea.

Sungyoon Kim1, Sulhee Lee2, and Young-Seo Park1*

1Department of Food Science and Biotechnology, Gachon University, Seongnam 13120, Republic of Korea
2Research Group of Healthcare, Korea Food Research Institute, Wanju 55365, Republic of Korea
Correspondence to: E-mail: ypark@gachon.ac.kr;
Tel.: +82-31-750-5378;Fax: +82-31-750-5389
Received December 28, 2020; Revised March 14, 2021; Accepted March 15, 2021.
Abstract
Leuconostoc citreum SG255, which was isolated from radish kimchi, synthesized oligosaccharides using sucrose as a donor and maltose or lactose as an acceptor. In this study, the draft genome sequence of L. citreum SG255 was determined using Illumina Miseq platform. The draft genome consisted of 28 contigs and the genome size was 1,869,057 bp. The G + C content was 37.78% with 1,829 coding sequences, 3 rRNA genes, and 51 tRNA genes. Leuconostoc citreum SG255 has alternansucrase gene, which encodes for the enzyme synthesizing oligosaccharides.
Keywords : Leuconostoc citreum, alternansucrase, draft genome sequence, Illumina Miseq, lactic acid bacteria
Body

Leuconostoc citreum was first reported in 1989 as L. amelibiosum and L. citreum and was classified as same species, retaining the nomenclature L. citreum in 1992 (Takahashi et al., 1992). It is a Gram-positive, catalase-negative, non-motile, and facultative anaeorobe. It is known to synthesize exopolysaccharides, such as dextransucrase and alternansucrase, through glycosyltransferase. These enzymes hydrolyze sucrose and link one glucose molecule to another through different types of glycosidic linkages (van Hijum et al., 2006). Additionally, it is known that these enzymes can use other acceptor molecules. It was reported that alternansucrase from L. mesenteroides NRRL B-21297 catalyzes the synthesis of oligosaccharides using raffinose as an acceptor (Côté et al., 2009). Dextransucrases from L. mesenteroides B-512FM catalyze the synthesis of maltodextrins using maltose as an acceptor (Fu and Robyt, 1990). In preliminary experiments, L. citreum SG255 isolated from radish kimchi catalyzed the synthesis of oligosaccharides using sucrose as a donor and maltose or lactose as an acceptor (Park et al., 2019). Consequently, this strain was assumed to have glycosyltransferase.

The strain was incubated in MRS medium at 37°C for 16 h. Genomic DNA isolation was carried out using AccuPrep genomic DNA Extraction kit (Bioneer). Whole-genome sequencing of L. citreum SG255 was performed by Sanigen Co., Ltd. using the Illumina Miseq platform. Library construction was performed using Illumina TruSeq DNA library prep kit and sequencing was performed using Illumina Miseq. Illumina Miseq platform provided 482.8× coverage of the genome, which was assembled de novo into 28 contigs. Quality control was performed using FastQC v0.11.8 and trimming process was performed using Trimmomatic v0.38. Contamination was checked using BWA v0.7.17 for alignment followed by Samtools v1.9. De novo assembly was performed using Spades v3.13.0. Genome annotation was conducted through NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and coding sequences were detected using GenMarkS+. To calculate genomic distance with other L. citreum strains, orthologous average nucleotide identity (OrthoANI) was compared with 25 strains through OrthoANI tool. The formula, distance = 1 – (OrthoANI/100), was used to calculate the the orthoANI values (Lee et al., 2016). Each of the protein coding genes was classified according to its function through the assignment of a Clusters of Orthologous Group (COG) code and was sorted into COG categories (Tatusov et al., 1997). Carbohydrate active enzymes were analyzed through dbCAN2 meta server (Jhang et al., 2018).

The draft genome of L. citreum SG255 consisted of 28 contigs and 1,869,057 bp with G + C content of 38.8%. The number of protein coding sequences (CDSs) was 1,829 with 3 ribosomal RNA genes (5S, 16S, and 23S) and 51 transfer RNA genes (Table 1). Leuconostoc citreum SG255 was most similar to L. citreum NRRL B-742 at OrthoANI value 99.49%. Classification into COG categories revealed that 421 genes belonged to information storage and processing, 324 genes belonged to cellular processing, and 606 genes belonged to metabolism. Additionally, 28 genes belonged to code X (Mobilome: prophages, transposons). However, 444 genes were either not assigned or poorly identified. Compared with other strains with high OrthoANI value, the maximum difference was only 14 (code L) except that of not assigned, which was 74. When three search tools for carbohydrate active enzymes (CAZymes) annotation in dbCAN2 meta server, namely, HMMER, DIAMOND, and Hotpep, 36 carbohydrate active enzymes were commonly identified by all search tools. Among 36 enzymes, 5 enzymes belonged to glycoside hydrolase family (GH) 70.

General genomic features of L. citreum SG255

Features Chromosome
Genome size (bp) 1,869,057
Contigs 28
GC content (%) 38.8
rRNA genes 3
tRNA genes 51
Protein coding genes 1,829
Genes assigned to COGs 1,614


Among five GH70 enzymes, an enzyme which was encoded by a gene consisted of 6,174 bp, was supposed to be an alternansucrase [EC 2.4.1.140]. It was annotated as SH3-like domain-containing protein in NCBI (NCBI GenBank locus tag number: H2O16_RS08760). The SH3-like motif is a structural distinction of alternansucrase (Wangpaiboon et al., 2019), and synteny analysis with other L. citreum strains showed that the gene encoding this enzyme from L. citreum SG255 was identified as an alternansucrase gene (Oberto, 2013). The alternansucrase from L. citreum SG255 was expected to catalyze the synthesis of various types of oligosaccharides using different acceptor and receptor sugar molecules.

Leuconostoc citreum SG255 was deposited in Korean Culture Center of Microorganisms under the deposit number KCCM 43402.

Nucleotide sequence accession number

The draft genome sequence of Leuconostoc citreum SG255 has been deposited in NCBI GenBank under the accession number JACGMK010000001-JACGMK010000028.

적 요

깍두기에서 분리된 Leuconostoc citreum SG255는 sucrose를 공여체로 하고 maltose 혹은 lactose를 수용체로 하여 올리고당을 생성하였다. Leuconostoc citreum SG255의 유전체는 1,869,057 bp의 28개의 contig로 구성된 염색체로 조합되었으며 G + C의 비율은 37.78%로 나타났다. 1,829개의 코딩 유전자, 3개의 rRNA 유전자와 51개의 tRNA 유전자가 염색체 DNA에서 확인되었다. Leuconostoc citreum SG255는 올리고당을 합성할 수 있는 glycosyltransferase 중 alternansucrase를 가지고 있는 것으로 확인되었다.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1A2C1004950).

References
  1. Côté GL, Dunlap CA, and Vermillion KE. 2009. Glucosylation of raffinose via alternansucrase acceptor reactions. Carbohydr. Res. 344, 1951-1959.
    Pubmed CrossRef
  2. Fu DT and Robyt JF. 1990. Acceptor reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase. Arch. Biochem. Biophys. 283, 379-387.
    Pubmed CrossRef
  3. Jhang H, Yohe T, Huang L, Entwistle S, Wu P, Yang Z, Busk PK, Xu Y, and Yin Y. 2018. dbCAN2: a meta server for automated carbohydrate-active enzyme annotation. Nucleic Acids Res. 46, W95-W101.
    Pubmed KoreaMed CrossRef
  4. Lee I, Kim YO, Park SC, and Chun J. 2016. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66, 1100-1103.
    Pubmed CrossRef
  5. Oberto J. 2013. SyntTax: A web server linking synteny to prokaryotic taxonomy. BMC Bioinformatics 14, 4.
    Pubmed KoreaMed CrossRef
  6. Park J, Kwon A, Lee S, and Park Y. 2019. Screening of lactic acid bacteria for bioconversion to produce glycosyltransferase. Abstr. P12-149, p. 512. Abstr. 2019 Annu. Meet. Kor. Soc. Food Sci. Technol.
  7. Takahashi M, Okada S, Uchimura T, and Kozaki M. 1992. Leuconostoc amelibiosum Schillinger, Holzapfel, and Kandler 1989 is a later subjective synonym of Leuconostoc citreum Farrow, Facklam, and Collins 1989. Int. J. Syst. Bacteriol. 42, 649-651.
  8. Tatusov RL, Koonin EV, and Lipman DJ. 1997. A genomic perspective on protein families. Science 278, 631-637.
    Pubmed CrossRef
  9. van Hijum SAFT, Kralj S, Ozimek LK, Dijkhuizen L, and van Geel-Schutten IGH. 2006. Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria. Microbiol. Mol. Biol. Rev. 70, 157-176.
    Pubmed KoreaMed CrossRef
  10. Wangpaiboon K, Pitakchatwong C, Panpetch P, Charoenwongpaiboon T, Field R, and Pichyangkura R. 2019. Modified properties of alternan polymers arising from deletion of SH3-like motifs in Leuconostoc citreum ABK-1 alternansucrase. Carbohydr. Polym. 220, 103-109.
    Pubmed CrossRef


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