Complete genome sequence of Latilactobacillus curvatus CACC879 and its functional probiotic properties

Lactic acid bacteria, such as Latilactobacillus, are useful microbes that produce healthy metabolites, including bacteriocins and organic acids (such as lactic acid), that can regulate the gut microbiome balance [1]. Lactic acid bacteria also confer health benefits via diverse mechanisms, such as acid and bile tolerance, epithelial cell adherence, intestinal barrier buildup, and immune system modulation [2]. Latilactobacillus curvatus is a potential probiotic strain that produces various bacteriocins and metabolites and exhibits immunomodulatory activity [3,4]. In this study, the genome of L. curvatus CACC879 was sequenced and fully assembled to elucidate the genetic factors associated with its probiotic characteristics.

L. curvatus CACC879 was isolated from swine feces in Korea, and the isolate was cultured in De Man, Rogosa, and Sharpe (MRS) medium for 18 h at 37°C. The genomic DNA of L. curvatus CACC879 was extracted and purified using the DNeasy UltraClean kit (Qiagen, Hilden, Germany) and sequenced using the PacBio Sequel (Pacific Biosciences, Menlo Park, CA, USA) sequencing platform. De novo assembly was performed using PacBio SMRT analysis software (version 2.3.0; Pacific Biosciences) [5]. The EggNOG 5.0 database (http://eggnog5.embl.de) was used to classify all genes into clusters of ortholog gene (COG) / non-supervised orthologous group (NOG) categories. Functional annotations of the predicted coding sequences (CDSs) were compared with the Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) [6]. The genome sequence of CACC879 was compared with other reference strains by Orthologous average nucleotide identity (OrthoANI; http://www.ezbiocloud.net/tools/orthoani) [7].

The whole genome of strain CACC879 consisted of one circular chromosome 1,398,247 bp in length (41.9% guanine-cytosine [GC]) along with three plasmids designated pCACC879-1 (591,981 bp, 42.2% GC), pCACC879-2 (14,542 bp, 45.2% GC), and pCACC879-3 (45,393 bp, 41.2% GC) (Table 1 and Fig. 1A). The genome of strain CACC879 contains 2,077 CDSs and 115 non-coding genes (25 rRNA and 90 tRNA genes) (Table 1). In addition, a total of 1,874 proteins (90.2%) were matched and classified into 19 COG functional categories (Fig. 1B). The most abundant COG categories were associated with replication, recombination, and repair (12.7%); translation, ribosomal structure, and biogenesis (7.8%); transcription (7.6%), carbohydrate transport, and metabolism (7.5%); and cell wall/membrane/envelope biogenesis (5.7%), excluding those with unknown function (29.7%). Compared with the genome sequence of reference strains, the genome of strain CACC879 was the most similar to that of the reference strains L. curvatus DSM 20019 (99.4%) and Wikim38 (99.0%) (Fig. 1C). The CACC879 strain showed common probiotic properties including the CRISPR-associated endonuclease (Cas9) for antiviral-related mechanisms and the biosynthesis of vitamin B groups (ribF and pduO), bacteriocin (lysM), and antioxidant (tpx), compared to the reference strains [8-10]. Additionally, we confirmed that strain CACC879 harbors genes associated with common probiotic properties, including acid tolerance (clpB and grpE), lactate synthesis (ldh and L-lactate dehydrogenase), and cell adhesion (sotA) (Table 2). Interestingly, the CACC879 genome contained the dltB and dltD genes associated with the modulation of the host immune response, but the reference strains did not. These findings will serve as a reference for further studies on L. curvatus and provide a scientific basis for functional probiotic development.

Fig. 1. Genome features of Latilactobacillus curvatus CACC879. (A) Circular genome mapping of L. curvatus CACC879. (B) Functional classification of clusters of orthologous groups (COG). (C) Orthologous average nucleotide identity (OrthoANI) values of L. curvatus CACC879 compared to other reference strains.

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