Isolation of lactic acid bacteria capable of reducing environmental alkyl and fatty acid hydroperoxides, and the effect of their oral administration on oxidative-stressed nematodes and rats.

Reinforcement of the hydroperoxide-eliminating activity in the small and large intestines should prevent associated diseases. We previously isolated a lactic acid bacterium, Pediococcus pentosaceus Be1 that facilitates a 2-electron reduction of hydrogen peroxide to water. In this study, we successfully isolated an alternative lactic acid bacterium, Lactobacillus plantarum P1-2, that can efficiently reduce environmental alkyl hydroperoxides and fatty acid hydroperoxides to their corresponding hydroxyl derivatives through a 2-electron reduction. Each strain exhibited a wide concentration range with regard to the environmental reducing activity for each hydroperoxide. Given this, the two lactic acid bacteria were orally administered to an oxygen-sensitive short-lived nematode mutant, and this resulted in a significant expansion of its lifespan. This observation suggests that P. pentosaceus Be1 and L. plantarum P1-2 inhibit internal oxidative stress. To determine the specific organs involved in this response, we performed a similar experiment in rats, involving induced lipid peroxidation by iron-overloading. We observed that only L. plantarum P1-2 inhibited colonic mucosa lipid peroxidation in rats with induced oxidative stress.

Characterisation of the bacterial community structures of sunki, a traditional unsalted pickle of fermented turnip leaves.

The bacterial community structure in 29 naturally fermented samples of sunki, an unsalted lactic-fermented pickle in Japan, was determined by 16S rRNA gene-targeted metagenomic analysis. The data revealed that genus Lactobacillus was dominant in all samples and various bacterial species, related to Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus intestinalis, and Lactobacillus gasseri, showed a range of dominance depending on the samples. Comparative analysis of the bacterial composition by principal coordinate analysis and hierarchical clustering classified the varied bacterial composition of the 29 samples into three types of bacterial community structure. These types comprised lactobacilli belonging to different phylogenetic groups: type A had a certain ratio of Lactobacillus fermentum (70.3-22.1%, average 41.2%) in combination with several species belonging to Lactobacillus delbrueckii-phylogenetic group, type B comprised remarkably high levels of species Lactobacillus delbrueckii (average 89.5%), and type C had combinations of species belonging to Lactobacillus plantarum- and Lactobacillus buchneri-phylogenetic groups. Interestingly, these types differed in the compositional profiles of water-soluble and volatile compounds, and statistically significant differences were observed in the levels of acetic acid, succinic acid, ethanol, ethyl acetate, glutamic acid, γ-aminobutyric acid, and isovaleraldehyde. Furthermore, metabolomic analysis revealed a correlation of Lactobacillus fermentum dominance with pH value and lactic and acetic acid levels, with high R values of 0.643, -0.642, and 0.528, respectively. The data reported in this study showed the characteristics of the bacterial composition in the unsalted sunki pickle and its potential relationship with the compositional profile.

NMR- and GC/MS-based metabolomic characterization of sunki, an unsalted fermented pickle of turnip leaves.

This study revealed the compositional characteristics of sunki, a traditional, unsalted, lactic acid-fermented pickle produced using turnip leaf in Kiso district, Japan. Comprehensive compositional analysis by two metabolomic approaches based on NMR and solid-phase microextraction-GC/MS methods was used to determine its chemical composition by annotating 54 water-soluble and 62 volatile compounds. Principal component analysis showed that samples had different compositions, depending on the agricultural processing factory and production year. This variation potentially resulted from the differences in the lactic acid bacterial community produced during the spontaneous fermentation of sunki and in the initial nutritional composition of the turnip leaf. Partial least squares regression revealed that the acetic acid level showed a strong positive correlation with pH (R = 0.810), in contrast to the negative correlations of lactic acid and ethanol levels (R =  -0.533 and -0.547). This indicated the crucial impact of acetic acid-related metabolism on acidification during sunki fermentation.

Detection and analysis of Lactobacillus paracasei penicillin-binding proteins revealed the presence of cholate-sensitive penicillin-binding protein 3 and an elongated cell shape in a cholate-sensitive strain.

Penicillin-binding proteins (PBPs) are responsible for peptidoglycan synthesis. By using biotinylated ampicillin, we detected PBPs of Lactobacillus paracasei strains. Ten PBPs were identified, 7 of which had apparent molecular sizes similar to those of Escherichia coli. In the presence of cholate, strain NRIC 0625 showed an elongated shape, and its putative PBP3 showed cholate-sensitive penicillin-binding activity. Furthermore, this strain was highly sensitive to cefalexin, which is known to inhibit cell division by inactivating PBP3. These results suggest that the septum synthetase PBP3 of lactic acid bacteria can be one of the targets of intestinal bile acid.

A rapid NMR-based method for discrimination of strain-specific cell wall teichoic acid structures reveals a third backbone type in Lactobacillus plantarum.

The lactic acid bacterium Lactobacillus plantarum is capable of producing strain-specific structures of cell wall teichoic acid (WTA), an anionic polysaccharide found in the Gram-positive bacterial cell wall. In this study, we established a rapid, NMR-based procedure to discriminate WTA structures in this species, and applied it to 94 strains of L. plantarum. Six previously reported glycerol- and ribitol-containing WTA subtypes were successfully identified from 78 strains, suggesting that these were the dominant structures. However, the level of structural variety differed markedly among bacterial sources, possibly reflecting differences in strain-level microbial diversity. WTAs from eight strains were not identified based on NMR spectra and were classified into three groups. Structural analysis of a partial degradation product of an unidentified WTA produced by strain TUA 1496L revealed that the WTA was 1-O-ß-d-glucosylglycerol. Two-dimensional NMR analysis of the polymer structure showed phosphodiester bonds between C-3 and C-6 of the glycerol and glucose residues, suggesting a polymer structure of 3,6΄-linked poly(1-O-ß-d-glucosyl-sn-glycerol phosphate). This is the third WTA backbone structure in L. plantarum, following 3,6΄-linked poly(1-O-α-d-glucosyl-sn-glycerol phosphate) and 1,5-linked poly(ribitol phosphate).

Isolation of lactic acid bacteria exhibiting high scavenging activity for environmental hydrogen peroxide from fermented foods and its two scavenging enzymes for hydrogen peroxide.

To obtain lactic acid bacteria that scavenge environmental hydrogen peroxide, we developed a specialized enrichment medium and successfully isolated Pediococcus pentosaceus Be1 strain from a fermented food. This strain showed vigorous environmental hydrogen peroxide scavenging activity over a wide range of hydrogen peroxide concentrations. High Mn-catalase and NADH peroxidase activities were found in the cell-free extract of the P. pentosaceus Be1 strain, and these two hydrogen peroxide scavenging enzymes were purified from the cell-free extract of the strain. Mn-catalase has been purified from several microorganisms by several researchers, and the NADH peroxidase was first purified from the original strain in this report. After cloning the genes of the Mn-catalase and the NADH peroxidase, the deduced amino acid sequences were compared with those of known related enzymes.

Identification of sirtuin and its target as the ribosomal protein S4 in Lactobacillus paracasei.

Sirtuin is a protein with an enzymatic activity of NAD(+)-dependent protein deacetylation. It was first identified in yeast and its homologous genes have been widely found in various organisms. In bacteria, sirtuin gene was first described as cobB, encoding a cobalamin processing enzyme; and later its potential involvement in regulating acetylation levels of metabolic enzymes, transcription factors, chemotactic proteins and others have been reported. In order to study its physiological relevance in probiotic lactic acid bacteria, we analyzed the whole genome of three L. paracasei strains. All strains tested had sirtuin homolog genes designated hereby as sirA, and one of them had an additional gene designated as sirB. Following confirmation of their coding sequences by individual gene cloning, corresponding recombinant proteins have been generated and purified. The enzymatic characterization revealed that the intrinsic NAD(+)-dependent deacetylation activity of LpSirA (protein encoded by sirA) is comparable to human SIRT1. Furthermore, by blocking sirtuin activity using nicotinamide in vivo, together with an in vitro deacetylation reaction using recombinant LpSirA, we identified one of the target proteins in the lactic acid bacteria as the 30S ribosomal protein S4 (rpsD product).

Isolation of endosulfan sulfate-degrading Rhodococcus koreensis strain S1-1 from endosulfan contaminated soil and identification of a novel metabolite, endosulfan diol monosulfate.

An aerobic endosulfan sulfate-degrading bacterium, Rhodococcus koreensis strain S1-1, was isolated from soil to which endosulfan had been applied annually for more than 10 years until 2008. The strain isolated in this work reduced the concentration of endosulfan sulfate (2) from 12.25 µM to 2.11 µM during 14 d at 30 °C. Using ultra performance liquid chromatography-electrospray ionization-mass spectroscopy (UPLC-ESI-MS), a new highly water-soluble metabolite possessing six chlorine atoms was found to be endosulfan diol monosulfate (6), derived from 2 by hydrolysis of the cyclic sulfate ester ring. The structure of 6 was elucidated by chemical synthesis of the candidate derivatives and by HR-MS and UPLC-MS analyses. Therefore, it was suggested that the strain S1-1 has a new metabolic pathway of 2. In addition, 6 was expected to be less toxic among the metabolites of 1 because of its higher water-solubility.

Characterization of flagellins isolated from a highly motile strain of Lactobacillus agilis.

BACKGROUND: Most lactic acid bacteria are non-motile but some of them are flagellated and exhibit motility. So far, motile lactobacilli have rarely been studied, and characteristics of their flagellins are poorly understood. In this study, a highly motile strain of Lactobacillus agilis was recruited for transcriptional analysis and characterization of its flagellins. RESULTS: Unlike another motile lactic acid bacteria of intestinal isolate, Lactobacillus ruminis, flagellar filaments of the L. agilis strain probably consist of two homologous but distinct flagellins. Glycosylation of the flagellar filaments and their resistance to heat, acid and SDS were also observed. The immunological activity of the flagellins was evaluated through the stimulation of Caco-2 cells. The results show that TLR5-stimulating activity of the protein is attenuated, likely due to an incomplete TLR5-recognition site. CONCLUSIONS: The flagella filaments of L. agilis BKN88 consist of two homologous glycosylated flagellins, which likely have an incomplete TLR5-recognition site. The characteristics of the flagellin are presumably a consequence of adaptation as a commensal microbe in the gastrointestinal tract.

Comparative genomics of Fructobacillus spp. and Leuconostoc spp. reveals niche-specific evolution of Fructobacillus spp.

BACKGROUND: Fructobacillus spp. in fructose-rich niches belong to the family Leuconostocaceae. They were originally classified as Leuconostoc spp., but were later grouped into a novel genus, Fructobacillus, based on their phylogenetic position, morphology and specific biochemical characteristics. The unique characters, so called fructophilic characteristics, had not been reported in the group of lactic acid bacteria, suggesting unique evolution at the genome level. Here we studied four draft genome sequences of Fructobacillus spp. and compared their metabolic properties against those of Leuconostoc spp. RESULTS: Fructobacillus species possess significantly less protein coding sequences in their small genomes. The number of genes was significantly smaller in carbohydrate transport and metabolism. Several other metabolic pathways, including TCA cycle, ubiquinone and other terpenoid-quinone biosynthesis and phosphotransferase systems, were characterized as discriminative pathways between the two genera. The adhE gene for bifunctional acetaldehyde/alcohol dehydrogenase, and genes for subunits of the pyruvate dehydrogenase complex were absent in Fructobacillus spp. The two genera also show different levels of GC contents, which are mainly due to the different GC contents at the third codon position. CONCLUSION: The present genome characteristics in Fructobacillus spp. suggest reductive evolution that took place to adapt to specific niches.

Phylogenomic Analysis of Oenococcus oeni Reveals Specific Domestication of Strains to Cider and Wines.

Oenococcus oeni is a lactic acid bacteria species encountered particularly in wine, where it achieves the malolactic fermentation. Molecular typing methods have previously revealed that the species is made of several genetic groups of strains, some being specific to certain types of wines, ciders or regions. Here, we describe 36 recently released O. oeni genomes and the phylogenomic analysis of these 36 plus 14 previously reported genomes. We also report three genome sequences of the sister species Oenococcus kitaharae that were used for phylogenomic reconstructions. Phylogenomic and population structure analyses performed revealed that the 50 O. oeni genomes delineate two major groups of 12 and 37 strains, respectively, named A and B, plus a putative group C, consisting of a single strain. A study on the orthologs and single nucleotide polymorphism contents of the genetic groups revealed that the domestication of some strains to products such as cider, wine, or champagne, is reflected at the genetic level. While group A strains proved to be predominant in wine and to form subgroups adapted to specific types of wine such as champagne, group B strains were found in wine and cider. The strain from putative group C was isolated from cider and genetically closer to group B strains. The results suggest that ancestral O. oeni strains were adapted to low-ethanol containing environments such as overripe fruits, and that they were domesticated to cider and wine, with group A strains being naturally selected in a process of further domestication to specific wines such as champagne.

Genome Sequences of Three Strains of Lactobacillus paracasei of Different Origins and with Different Cholate Sensitivities.

We report here the draft genome sequences of three strains of Lactobacillus paracasei (NRIC 0644, NRIC 1781, and NRIC 1917) isolated from different sources. The three genomes range from 2.95 to 3.15 Mb with a G+C content of 46% and contain approximately 2,700 protein coding sequences.

Alicyclobacillus dauci sp. nov., a slightly thermophilic, acidophilic bacterium isolated from a spoiled mixed vegetable and fruit juice product.

A novel, moderately thermophilic, acidophilic, Gram-variable, rod-shaped, endospore-forming bacterium was isolated from a spoiled mixed vegetable and fruit juice product that had the off-flavour of guaiacol. The bacterium, strain 4F(T), grew aerobically at 20-50 °C (optimum 40 °C) and pH 3.0-6.0 (optimum pH 4.0) and produced acid from glycerol, d-galactose and d-glucose. It contained menaquinone-7 (MK-7) as the major isoprenoid quinone and the DNA G+C content was 49.6 mol%. The predominant cellular fatty acids of strain 4F(T) were ω-alicyclic (ω-cyclohexane fatty acids), which are characteristic of the genus Alicyclobacillus. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belongs to the Alicyclobacillus cluster, and is related most closely to the type strains of Alicyclobacillus acidoterrestris (97.4 % similarity) and Alicyclobacillus fastidiosus (97.3 %). Strain 4F(T) produced guaiacol from vanillic acid. It can be distinguished from related species by its acid production type and guaiacol production. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness values, it can be concluded that the strain represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus dauci sp. nov. is proposed; the type strain is 4F(T) ( = DSM 28700(T) = NBRC 108949(T) = NRIC 0938(T)).

Detection and genomic characterization of motility in Lactobacillus curvatus: confirmation of motility in a species outside the Lactobacillus salivarius clade.

Lactobacillus is the largest genus within the lactic acid bacteria (LAB), with almost 180 species currently identified. Motility has been reported for at least 13 Lactobacillus species, all belonging to the Lactobacillus salivarius clade. Motility in lactobacilli is poorly characterized. It probably confers competitive advantages, such as superior nutrient acquisition and niche colonization, but it could also play an important role in innate immune system activation through flagellin­Toll-like receptor 5 (TLR5) interaction. We now report strong evidence of motility in a species outside the L. salivarius clade, Lactobacillus curvatus (strain NRIC0822). The motility of L. curvatus NRIC 0822 was revealed by phase-contrast microscopy and soft-agar motility assays. Strain NRIC 0822 was motile at temperatures between 15 °C and 37 °C, with a range of different carbohydrates, and under varying atmospheric conditions. We sequenced the L. curvatus NRIC 0822 genome, which revealed that the motility genes are organized in a single operon and that the products are very similar (>98.5% amino acid similarity over >11,000 amino acids) to those encoded by the motility operon of Lactobacillus acidipiscis KCTC 13900 (shown for the first time to be motile also). Moreover, the presence of a large number of mobile genetic elements within and flanking the motility operon of L. curvatus suggests recent horizontal transfer between members of two distinct Lactobacillus clades: L. acidipiscis in the L. salivarius clade and L. curvatus inthe L. sakei clade. This study provides novel phenotypic, genetic, and phylogenetic insights into flagellum-mediated motility in lactobacilli.

Lactobacillus furfuricola sp. nov., isolated from Nukadoko, rice bran paste for Japanese pickles.

Two strains of lactic acid bacteria, Nu27(T) and Nu29, were isolated from Nukadoko, rice bran paste for Japanese pickles. The isolates were Gram-stain-positive, rod-shaped, catalase-negative, non-motile and facultatively anaerobic lactic acid bacteria. The isolates showed identical 16S rRNA gene sequences. The closest relatives to strain Nu27(T) based on 16S rRNA gene sequence similarities were Lactobacillus versmoldensis KU-3(T) (98.9% 16S rRNA gene sequence similarity), Lactobacillus nodensis iz4b(T) (96.3%) and Lactobacillus tucceti CECT 5290(T) (97.2%). DNA-DNA relatedness values revealed genotype separation of the two isolates from the above three species. Based on the physiological, biochemical and genotypic characteristics provided, the isolates represent a novel species of the genus Lactobacillus, for which name is Lactobacillus furfuricola proposed. The type strain is Nu 27(T) ( = JCM 18764(T) = NRIC 0900(T) = DSM 27174(T)).

Fructophilic characteristics of Fructobacillus spp. may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene (adhE).

Fructophilic strains of Leuconostoc spp. have recently been reclassified to a new genus, i.e., Fructobacillus. Members of the genus are differentiated from Leuconostoc spp. by their preference for fructose on growth, requirement of an electron acceptor for glucose metabolism, and the inability to produce ethanol from the fermentation of glucose. In the present study, enzyme activities and genes involved in ethanol production were studied, since this is the key pathway for NAD(+)/NADH cycling in heterofermentative lactic acid bacteria. Fructobacillus spp. has a weak alcohol dehydrogenase activity and has no acetaldehyde dehydrogenase activity, whereas both enzymes are active in Leuconostoc mesenteroides. The bifunctional alcohol/acetaldehyde dehydrogenase gene, adhE, was described in Leuconostoc spp., but not in Fructobacillus spp. These results suggested that, due to the deficiency of the adhE gene, the normal pathway for ethanol production is absent in Fructobacillus spp. This leads to a shortage of NAD(+), and the requirement for an electron acceptor in glucose metabolism. Fructophilic characteristics, as observed for Fructobacillus spp., are thus due to the absence of the adhE gene, and a phenotype that most likely evolved as a result of regressive evolution.

An increased number of CD4+CD25+ cells induced by an oral administration of Lactobacillus plantarum NRIC0380 are involved in antiallergic activity.

BACKGROUND: Our previous study showed that an oral administration of Lactobacillus plantarum NRIC0380 inhibited immunoglobulin E (IgE) production in a murine model, and that orally administered NRIC0380 induced CD4+CD25+ Foxp3+ T, i.e. regulatory T (Treg), cells in the spleen and Peyer's patch of mice. Although it has been reported that Treg cells might suppress the allergic symptoms, the involvement of the cells in the antiallergic activity of lactic acid bacteria has not been clearly demonstrated. We therefore examined in detail the antiallergic activity of Treg cells obtained from mice that had been fed NRIC0380. METHODS: Treg cells were obtained from mice that had been fed NRIC0380. The T cell-suppressive effect of the cells was analyzed by coculturing the cells with splenocytes of ß-lactoglobulin-immunized mice and ß-lactoglobulin. The effects of the Treg cells on the IgE production and cutaneous anaphylaxis reaction were then analyzed by transferring the cells into another mouse. RESULTS: The Treg cells obtained from the mice that had been fed NRIC0380 showed similar T cell-suppressive activity to those cells obtained from the control mice. The Treg cells obtained from the mice fed NRIC0380 significantly inhibited the IgE production and active cutaneous anaphylaxis reaction when transferred into another mouse that was subsequently immunized with the antigen. Furthermore, the Treg cells also significantly suppressed the passive cutaneous anaphylaxis reaction when cotransferred with the IgE antibody into another mouse. CONCLUSIONS: The induction of Treg cells by the oral administration of NRIC0380 would be involved in the antiallergic activity of NRIC0380.

Induction of ALDH activity in intestinal dendritic cells by Lactobacillus plantarum NRIC0380.

Lactic acid bacteria have been reported to have various immune-regulating activities. We also found in the previous study that the oral administration of heat-killed Lactobacillus plantarum NRIC0380 induced CD4(+)CD25(+)Foxp3(+) cells (Treg cells). We examine in this present study the influence of NRIC0380 on the function of intestinal dendritic cells (DCs) in vitro and in vivo. The aldehyde dehydrogenase (ALDH) activity was significantly induced in DCs obtained from the mesenteric lymph node (MLN) by culturing with NRIC0380. The oral administration of NRIC0380 also significantly increased ALDH-positive DCs in MLN. NRIC0380 significantly enhanced the production of TGF-ß from MLN cells in vitro. These effects were not apparent in cells from the Peyer's patch (PP) and spleen (SPL). NRIC0380 also significantly enhanced the expression of B7-H1 on DCs of all organs in vitro. The effects of NRIC0380 on DCs, especially those located in MLN, might be involved in its function to induce Treg cells.

Metabolic engineering of Lactobacillus plantarum for succinic acid production through activation of the reductive branch of the tricarboxylic acid cycle.

Biosynthesis of succinic acid is an alternative method from conventional chemical synthesis. For this application, several bacteria and fungi have been employed and genetically modified. Lactic acid bacteria (LAB) are gaining recognition as novel producers of useful compounds by metabolic engineering. Among LAB, Lactobacillus plantarum NCIMB 8826 is an interesting candidate for succinic acid production by metabolic engineering since it has an incomplete tricarboxylic acid (TCA) cycle and naturally produces small amounts of succinic acid. In this study, we constructed recombinant LAB and evaluated them as hosts of succinic acid production. We examined the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and malic enzyme for their potential to improve metabolic flux from glycolysis to the reductive TCA cycle in a lactate dehydrogenase-deficient strain of L. plantarum NCIMB 8826 (VL103). We investigated the effects of overexpression or coexpression of each enzyme on succinic acid production. Our results suggested that PC is the key enzyme for succinic acid production by L. plantarum VL103, whereas PEPCK is critical for increasing biomass. The highest yield of succinic acid was obtained through coexpression of PC and PEPCK in L. plantarum VL103. This recombinant strain produced a 22-fold higher amount of succinic acid than the wild-type and converted 25.3% of glucose to succinic acid.

Characterization of a Lactobacillus gasseri JCM 1131T lipoteichoic acid with a novel glycolipid anchor structure.

We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131(T). The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.