Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms

The genomic DNA of SLC13 was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Germany), according to the manufacturer’s instructions. Total DNA was subjected to quality control by 2% agarose gel electrophoresis and quantified by a NanoDrop™ spectrophotometer. L. pentosus SLC13 was sequenced using the PacBio RS II platform (Pacific Biosciences, USA), and the reads were assembled using HGAP version 3.0. Sequences were further annotated at the RAST prokaryotic genome annotation server (http://​rast.​nmpdr.​org/​) [8].

ResFinder databases (http://​www.​genomicepidemiol​ogy.​org/​) were used to find the antibiotic resistance genes present in the plasmid pSLC13. The first step of the bacteriocin identification workflow was created by merging the BACTIBASE databases using BLASTN [9] and BAGEL (class I, II and III) [10]. RAST server was used for identifying eps gene cluster by comparative genomic analysis.

L. pentosus SLC13 was sequenced using the PacBio RS II platform, generating a library containing 58,744 single reads with an average length of 3936 bp. Reads were assembled and returned two contigs with the head segment was almost identical to the tail segment, indicating the circular nature of the contigs. As shown in Fig. 1 and Table 1, the complete genome sequence of SLC13 was composed of a circular 3,520,510-bp chromosome and one 62,498-bp plasmid named as pSLC13. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. The GC content and size of SLC13 chromosome was similar to other Lactobacillus strains, including L. plantarum GB-LP1 (3,040,388 bp, GC content: 44.9%) [11], L. rhamnosus CNCM I-3698 (2,966,480 bp, GC content: 46.69%) [12], L. casei BD-II (3,069,926 bp, GC content: 46.34%) [13], L. pentosus KCAI (3,418,159 bp, GC content: 46.4%) [14], but not L. jojnsonii FI9785 (1,755,993 bp, GC content: 34.49%) [15] (Table 1).

RAST annotation results showed that the genome contained 3172 coding sequences and 82 RNA genes (Table 1, Fig. 2). Moreover, all protein coding sequences were functionally annotated by RAST server in 350 subsystems (Fig. 2). Seventy-six protein-coding sequences were identified on the plasmids pSLC13. The sequence blast results revealed that pSLC13 showed high similarity to plasmid pLB1106-3 in L. brevis strain SRCM101106 (accession number: CP021675). In our previous study, we showed that SLC13 was resistant to penicillin G, cephalothin, cloxacillin, novobiocin, vancomycin, polymyxin B, rifampicin, tetercycline, kanamycin, gentamycin, neomycin, and streptomycin [7]. ResFinder databases were used to find the antibiotic resistance genes present in the plasmid pSLC13, and the results showed no antibiotic resistance genes were identified in pSLC13. Therefore, there is no further consideration for transmission of antibiotic resistant determinants by SLC13 as a candidate for probiotic development.

SLC 13 isolated from mustard pickles produced the largest amount of EPS (0.43 ± 0.04 g/l) among the 39 collected LAB isolates in our previous report [7]. To explore the gene cluster(s) that is (are) responsible for this phenotype, the comparative genomic analysis was performed by RAST server, and the genes that could be related to high-EPS biosynthesis were annotated in detail (Additional file 1: Figure S1). The genome of the SLC13 encoded 2 recognizable exopolysaccharide (eps) gene clusters. Comparison of the genome of L. pentosus SLC13 with that of strain L. plantarum WCFS1, indicated that the two EPS synthesis clusters assigned in SLC13 is highly conserved to the L. plantarum WCFS1 (Additional file 1: Figure S1). However, whether these two eps gene clusters responsible for high-EPS production in SLC13 is still unknown. Currently, Lee et al. reported that the abundance and sugar compositions of EPS diverse in three L. plantarum strains [16]. The diverse of sugar composition may affect its impact on probiotic-host interaction, for example, host cell adhesion [16]. Therefore, the composition of SLC13-EPS remains to be characterized in the future.

A plantaricin (pln) gene cluster for bacteriocin synthesis in SLC13 identified by RAST server was shown in Additional file 1: Figure S2a. Further investigation was then conducted with BAGEL3, a web-based bacteriocin mining tool. The result from BAGEL3 showed a putative class II bacteriocin, Pediocin PA-1 immunity protein, was identified on SLC13 chromosome, as shown in Additional file 1: Figure S2b (gene start position, 2,037,423 on the chromosome; length, 112 amino acids). Pediocin PA-1 was previous identified in probiotic Pediococcus acidilactici with antimicrobial activity against Listeria spp., Oenococcus oeni and other wine bacteria [17, 18]. However, the antimicrobial activity of pediocin in SLC13 remains to be verified.

RAST annotation results showed that four virulence factors were found in the SLC13 chromosome, including heat shock protein 33, Jag, YidD, and YidC. Heat shock protein 33 is an adhesin important for adhesion and colonization of the Streptococcus pyogenes on epithelial cells [19]. Jag, YidD, and YidC are part of the Mycobacterium virulence operon with unclear function. Currently, Thakur et al. reported that the translocase YidC controls respiratory metabolism in M. tuberculosis and is required for the intracellular survival of M. tuberculosis in human macrophages [20]. A type II toxin-antitoxin system, mRNA interferase toxin, which is ubiquitous in Lactobacillus strains with the ability to inhibit E. coli growth, was found in pSLC13 [21]. However, the function of virulence factors identified in SLC13 remains to be determined.

Together, the complete genome sequence of SLC13 allows us to better understand molecular basis of its antimicrobial activity, high EPS production and probiotic potentials. Future studies are needed to verify the probiotic properties and safety of this strain for its industry application.