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.

Table 1

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.

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.

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