Genome-based assessment of safety characteristics of Lacticaseibacillus paracasei NY1301 and genomic differences in closely related strains marketed as probiotics.

The probiotic attributes of Lacticaseibacillus paracasei NY1301 were comprehensively characterized, and a comparison between the closely related LcA (Actimel) and LcY (Yakult) probiotic strains was conducted using genomic tools. All strains exhibited high genetic similarity and likely shared a common ancestor; differences were primarily expressed as minor chromosomal re-arrangements, substitutions, insertions, and deletions. Compared with LcY, NY1301 exhibited 125 single-nucleotide polymorphisms. NY1301 lacked virulence factors, antibiotic resistance genes, and mutations associated with antibiotic resistance and had a 46-kbp prophage. This prophage is spontaneously induced at low levels and remains in a non-lytic state under standard culture conditions. The observed causal adaptive mutations were likely related to niche adaptation within the respective laboratory or manufacturing processes that occurred during the maintenance of the strains. However, the phenotypic effects of these genomic differences remain unclear. To validate the safety of NY1301, we conducted an open-label trial with healthy participants who consumed excessive amounts of NY1301 (3.0 × 1011 cfu) daily for 28 days. The results of this trial and those of other in vivo studies, coupled with the long history of human consumption without established risks to humans, provide strong evidence confirming the safety of NY1301.

A Diet Including Red Bell Pepper Juice and Soy Protein Suppress Physiological Markers of Muscle Atrophy in Mice.

Although muscle atrophy can be caused by disuse and lifestyle-related syndromes, it may be possible to prevent this condition through dietary intervention. We hypothesized that a diet including red bell pepper juice (RBPJ) and soy protein isolate (SPI) would prevent muscle atrophy. Accordingly, an experimental diet containing RBPJ and/or SPI was administered for 18 d to normal C57BL/6J mice. The control group was administered a casein diet. Four days before the end of the test period, denervation-induced muscle atrophy and/or sham operation were performed. Anterior tibialis muscle samples were then obtained to assess muscle degradation and perform metabolome analysis. Under the denervation condition, the 20% SPI diet did not alter the mRNA expression levels of muscle atrophy marker genes compared with the 20% casein group. Although the diet comprising RBPJ and 20% casein did not prevent muscle atrophy compared with the control group, the diet containing RBPJ and 20% SPI did. Metabolome analysis revealed that a diet including RBPJ and SPI induced a greater than 1.5-fold change in the levels of 20 muscle atrophy-related metabolites. In particular, the level of S-adenosylmethionine, which concerned with energy metabolism and lifespan, showed a strong positive correlation with the muscle atrophy marker. These findings suggest that a diet including RBPJ and soy protein suppress gene expressions related with muscle atrophy. Further research in humans is needed to confirm whether a combination of RBPJ and SPI can indeed prevent muscle atrophy.

Relation between cell-bound exopolysaccharide production via plasmid-encoded genes and rugose colony morphology in the probiotic Lactobacillus brevis KB290.

The probiotic Lactobacillus brevis KB290 is a natural producer of cell-bound exopolysaccharide (EPS), and the plasmid-encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid-cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N-acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain.

Genome Sequence of Lactobacillus plantarum KB1253, a Gamma-Aminobutyric Acid (GABA) Producer Used in GABA-Enriched Tomato Juice Production.

Here, we present the draft genome sequence of Lactobacillus plantarum KB1253, isolated from a traditional Japanese pickle. Its genome comprises 3,097 genes and 3,305,456 nucleotides, with an average G+C content of 44.4%.

Plasmid-encoded glycosyltransferase operon is responsible for exopolysaccharide production, cell aggregation, and bile resistance in a probiotic strain, Lactobacillus brevis KB290.

We demonstrate here that exopolysaccharide (EPS) production, cell aggregation, and bile resistance in Lactobacillus brevis KB290 are conferred by three eps genes (gtf27, gtf28, and orf29) located on the 42.4-kb plasmid pKB290-1. The predicted products of gtf27 and gtf28 belong to the membrane-bound glycosyltransferase family whereas the orf29 gene product showed homology with the ABC transporter. On in silico analysis, these genes were found to be widely distributed among lactobacilli from publicly available genomes and metagenomes, and their function is not yet elucidated. RT-PCR analysis showed that the eps genes were organised in an operon and their expression was markedly lower in arabinose- and xylose-containing media than in a glucose-containing medium. The three eps genes were cloned and expressed in homologous and heterologous strains. Considerably less EPS was produced by the plasmid-cured KB1802 strain than by the parental KB290 strain, whereas a similar amount was produced by the KB1802 strain expressing the three eps genes. The KB1802 strain expressing gtf27 and gtf28 but not orf29 did not produce EPS. Cell aggregation and bile resistance were also decreased in KB1802 strains but were complemented by eps genes. Moreover, the three eps genes conferred these phenotypes to a Lactobacillus plantarum strain. In conclusion, the three eps genes in pKB290-1 were sufficient for EPS biosynthesis with glucose and N-acetylglucosamine, and were responsible for cell aggregation and bile resistance. We consider these phenotypes to be at least partly responsible for KB290-specific properties.

Cell-bound exopolysaccharides of Lactobacillus brevis KB290: protective role and monosaccharide composition.

We examined the survivability of Lactobacillus brevis KB290 and derivative strain KB392 in artificial digestive juices and bile salts. The strains have similar membrane fatty acids but different amounts of cell-bound exopolysaccharides (EPS). In artificial digestive juices, KB290 showed significantly higher survivability than KB392, and homogenization, which reduced the amount of EPS in KB290 but not in KB392, reduced the survivability only of KB290. In bile salts, KB290 showed significantly higher survivability than KB392, and cell-bound EPS extraction with EDTA reduced the survivability of only KB290. Transmission electron microscopy showed there to be a greater concentration of cell-bound EPS in KB290 than in either KB392 or EDTA-treated or homogenized KB290. We conclude that KB290's cell-bound EPS (which high performance liquid chromatography showed to be made up of glucose and N-acetylglucosamine) played an important role in bile salt tolerance.

Genomic analysis by deep sequencing of the probiotic Lactobacillus brevis KB290 harboring nine plasmids reveals genomic stability.

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.

Assessment of antibiotic resistance in probiotic strain Lactobacillus brevis KB290.

Our purpose was to investigate the safety of the probiotic strain Lactobacillus brevis KB290. The European Qualified Presumption of Safety (QPS) evaluation approach was applied to the strain. We determined the strain's antibiotic resistance, verified it at the genetic level, and determined whether it could be transferred to intestinal microflora. Of 14 antibiotics tested, 11 showed MICs within the limits of the QPS criteria. However, the L. brevis KB290 MICs of ciprofloxacin (a fluoroquinolone), tetracycline, and vancomycin were two, four, and eight times, respectively, the breakpoint MICs suggested by the European Scientific Committee on Animal Nutrition, and the MIC of tetracycline was eight times the breakpoint MIC suggested by the European Scientific Panel on Additives and Products or Substances Used in Animal Feed. Using analysis of gapped-genome sequences, we found no known transferable determinants for tetracycline or vancomycin resistance, and we found no mutations in the quinolone resistance-determining regions of the genes encoding GyrA or ParC for ciprofloxacin resistance associated with insertion sequences, integrons, or transposons. These data were confirmed by using PCR primers specific for the respective genes. We assessed the transferability of the resistance traits in conjugation experiments with enterococci and obtained no transconjugants, strongly suggesting that the resistance traits were not transferable. This study demonstrated that the antibiotic resistance observed in L. brevis KB290 was due not to dedicated mechanisms but to intrinsic resistance. According to the QPS criteria, these results provide safety assurance for the ongoing use of L. brevis KB290 as a probiotic.

Complete covalent structure of nisin Q, new natural nisin variant, containing post-translationally modified amino acids.

The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3-7, 8-11, 13-19, 23-26, and 25-28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A.

Identification of the lantibiotic nisin Q, a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan.

Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.