Genomic Analysis by Deep Sequencing of the Probiotic Lactobacillus brevis KB290 Harboring Nine Plasmids Reveals Genomic Stability

Abstract

We determined the complete genome sequence of Lactobacillus brevis KB290, a probiotic lactic acid bacterium isolated from a traditional Japanese fermented vegetable. The genome contained a 2,395,134-bp chromosome that housed 2,391 protein-coding genes and nine plasmids that together accounted for 191 protein-coding genes. KB290 contained no virulence factor genes, and several genes related to presumptive cell wall-associated polysaccharide biosynthesis and the stress response were present in L. brevis KB290 but not in the closely related L. brevis ATCC 367. Plasmid-curing experiments revealed that the presence of plasmid pKB290-1 was essential for the strain's gastrointestinal tract tolerance and tendency to aggregate. Using next-generation deep sequencing of current and 18-year-old stock strains to detect low frequency variants, we evaluated genome stability. Deep sequencing of four periodic KB290 culture stocks with more than 1,000-fold coverage revealed 3 mutation sites and 37 minority variation sites, indicating long-term stability and providing a useful method for assessing the stability of industrial bacteria at the nucleotide level.

Screening, Characterization and In Vitro Evaluation of Probiotic Properties Among Lactic Acid Bacteria Through Comparative Analysis

“…Among the studied probiotic microbiota, lactic acid bacteria (LAB) are the most predominant, widely distributed and common inhabitants of epithelial surface of humans [2]. Several genera of LAB are widely used in fermented foods that are of traditional and industrial importance.…”

Genomics of Lactic Acid Bacteria

“…lactis strains have been found to harbour several plasmids, which range in size from 2 to 80 kb (Davidson et al 1996). 17.9 kb Tolerance to oxidative stress Fukao et al (2013) For example, Lactoc. lactis SK11, a phage-resistant strain used in cheese making, contains four plasmids and was shown to have a substantial number of 'adaptation' genes (Siezen et al 2005).…”

“…The draft genome of L. plantarum strain 16 contains more than eight plasmids, whereas nine plasmids were found in the completed genome of L. brevis KB290 (Crowley et al 2013;Fukao et al 2013). In L. brevis KB290, all of the plasmids had putative replication systems, including repABC genes for essential plasmid replication and stability, which is likely to explain the co-existence of multiple plasmids.…”

“…Consistent with the presence of genes encoding EPS biosynthesis in the pKB290-1 plasmid, the plasmid appeared to be essential for the strains' GI tract tolerance and tendency to aggregate during plasmid-curing experiments. Genes encoding putative multidrug resistance transporters, enzymes associated with polyketide biosynthesis, cycling thioester proteins, and enzymes associated with the condensation of dipeptides with fatty acids and cysteine-dipeptide synthesis were found to be harboured by other plasmids and were found to be associated with the stress response (Fukao et al 2013). …”

Cell-bound exopolysaccharides of Lactobacillus brevis KB290 enhance cytotoxic activity of mouse splenocytes

“…Deep sequencing of the whole genome of KB290 revealed it contains nine plasmids (Fukao et al . ). Plasmid‐curing experiments also demonstrated that pKB290‐1 is essential for resistance of the bacterium to gastrointestinal juice and for its tendency to aggregate.…”