Complete Genome Sequence of Leuconostoc citreum KM20

Abstract

Leuconostoc citreum is one of the most prevalent lactic acid bacteria during the manufacturing process of kimchi, the best-known Korean traditional dish. We have determined the complete genome sequence of L. citreum KM20. It consists of a 1.80-Mb chromosome and four circular plasmids and reveals genes likely involved in kimchi fermentation and its probiotic effects.

Kimchi is a popular fermented Korean food made from a variety of vegetables with assorted, often spicy seasonings such as hot pepper (Capsicum annuum var. annuum). The vegetables of choice for this traditional health food are most commonly a Korean cabbage called baechu or napa cabbage (Brassica rapa subsp. pekinensis) and/or an East Asian giant white radish called mu (Raphanus sativus var. niger). Analyses of the kimchi microflora, both culture based and metagenomic, indicated that Leuconostoc citreum is a dominant microbe during the early and mid-phases of kimchi fermentation (2, 4), and thus it can be useful as a starter culture (2). A strain of L. citreum, designated KM20, that can suppress the growth of pathogenic microorganisms such as Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium and is cytotoxic to HT-29 cells was isolated from baechu kimchi fermented at 10°C (data not shown).

The complete genome sequence of this strain was determined by the conventional whole-genome shotgun strategy. Genomic libraries harboring 1.2-kb, 2-kb, and 40-kb fragments were constructed, and a total of ∼56,000 chromatograms (16.3-fold coverage) were produced, either by an ABI 3730xl model genetic analyzer or RISA-384. Basecalling, fragment assembly, contig editing, and primer design were performed using Phred/Phrap/Consed software packages. Gap closure was carried out by primer walking on gap-spanning clones or PCR products. Physical gaps were closed by combinatorial PCR and custom primer walking on the amplified products. Misassemblies due to repetitive sequences were checked in terms of mate information and were corrected manually using Consed. The final error rate was estimated to be less than 1 bp per 900,000 bp (1.98 bp errors throughout the entire genome). Putative protein-coding genes, predicted by Yacop software (5), were assigned functions by a hierarchical information transfer from the best hits received from searches of public protein databases such as TIGRFAMs, UniProt, COG, KEGG, NCBI-NR, and Pfam, with the aid of AutoFACT (3).

The L. citreum KM20 genome is composed of one circular chromosome of 1,796,284 bp (39.0% G+C content) and four circular plasmids (pLCK1, 38,713 bp; pLCK2, 31,463 bp; pLCK3, 17,971 bp; and pLCK4, 12,183 bp) with slightly lower G+C contents. The entire genome contains 1,820 protein-coding genes, i.e., 1,702 genes on the chromosome and 49, 36, 29, and 13 genes on each plasmid, respectively, as well as four rRNA operons and 69 tRNA genes on the chromosome. No complete prophages were found, but several phage-related genes were identified, including one site-specific recombinase. The genome also contains five complete copies of the IS3 family of insertion sequences and five derivatives of the IS30 family elements. An average read depth of the contig sequences suggests that pLCK2, pLCK3, and pLCK4 are high-copy-number plasmids; the presence of mob-ori-rep for theta replication in each of them coincides with this observation.

Genome analysis of L. citreum KM20 revealed a complete gene set for heterolactic fermentation via the phosphoketolase pathway, with an incomplete tricarboxylic acid cycle and a limited biosynthetic capacity for various amino acids and cofactors. An extensive set of carbohydrate hydrolase and transporter genes implies its possible lifestyle associated with plant-derived materials. In addition, multiple genes for dextransucrase and alternansucrase implicate its potential for medical application and food microbiology. A plasmid-encoded putative cell wall-anchored protein with five mucus-binding domains suggests that L. citreum may colonize the surfaces of the gastrointestinal tract, which are reported to be the sites of major probiotic effects with many Lactobacillus species via immune modulation (1). This work provides scientific insights into the probiotic effects of L. citreum and may lead to new biotechnological applications of traditional fermented foods.

Nucleotide sequence accession numbers.

Genome information for the chromosome and four plasmids of L. citreum KM20 was deposited in GenBank under the accession numbers DQ489736 to DQ489740. The sequences and annotations are also available from the Genome Encyclopedia of Microbes (http://www.gem.re.kr/).