Complete genome sequence of Bifidobacterium animalis subsp. lactis BLC1

Bifidobacterium animalis subsp. lactis BLC1 is a probiotic bacterium that is widely exploited by food industries as the active ingredient of various functional foods. Here we report the complete genome sequence of B. animalis subsp. lactis BLC1, which is expected to provide insights into the biology of this health-promoting microorganism and improve our understanding of its phylogenetic relatedness with other members of the B. animalis subsp. lactis taxon.     

Genome sequence of the probiotic strain Bifidobacterium animalis subsp. lactis CNCM I-2494

Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.     

Complete Genome Sequence of Probiotic Bifidobacterium animalis subsp. lactis Strain V9

Bifidobacterium animalis subsp. lactis strain V9 is a Chinese commercial bifidobacteria with several probiotic functions. It was isolated from a healthy Mongolian child in China. We present here the complete genome sequence of V9 and compare it to 3 other published genome sequences of B. animalis subsp. lactis strains. The result indicates the lack of polymorphism among strains of this subspecies from different continents.Bifidobacterium animalis subsp. lactis strain V9 was isolated from the feces of a healthy Mongolian child in China (5). It has shown a high level of tolerance to gastric acid and bile acids (5). This strain has been implemented in the industrial production of dairy starter cultures by Inner Mongolia Yili Industrial Group Company Limited, the largest dairy corporation in China.Whole-genome sequencing of B. animalis subsp. lactis V9 was performed with a combined strategy of 454 sequencing (8) and Solexa paired-end sequencing technology (2). Genomic libraries containing 7-kb inserts were constructed, and 325,824 paired-end reads and 67,177 single-end reads were generated using the GS FLX system, giving 36.0-fold coverage of the genome. A total of 96.0% of the reads were assembled into four large scaffolds, including 163 nonredundant contigs, using the 454 Newbler assembler (454 Life Sciences, Branford, CT). A total of 8,953,102 reads (2-kb library) were generated to reach a depth of 335-fold coverage with an Illumina Solexa Genome Analyzer IIx and mapped to the scaffolds using the Burrows-Wheeler Alignment (BWA) tool (7). The gaps between scaffolds were filled by sequencing PCR products using an ABI 3730 capillary sequencer. The analysis of the genome was performed as described previously (3, 4).The complete genome sequence of V9 contains a circular 1,944,050-bp chromosome, with a GC content of 60.5%. The genome size is slightly larger than the sequenced genome sizes of B. animalis subsp. lactis strains DSM 10140^T (1), Bl-04 (1), and AD011 (6) due to a unique insertion of 4,037 bp. The V9 genome contains 1,636 genes in total, including 1,572 coding genes, 4 rRNA operons, and 52 tRNAs.Comparison of the four B. animalis subsp. lactis genomes revealed nearly perfect synteny. AD011 is the most diverged strain, with more single nucleotide polymorphisms (SNPs) and indels than the other three strains. There are 197 SNPs in AD011, with 70 synonymous and 16 nonsynonymous SNPs, which means that there is only 1 SNP per 10 kb, indicating the high consistency within this subspecies. The other three strains are almost identical, with only 25 SNPs in V9, 13 SNPs in Bl-04, and 44 SNPs in DSM 10140^T. Strain V9 was isolated from the feces of a Mongolian child in Inner Mongolia, China, where traditional fermented milk has been consumed for thousands of years, and the other three strains were originally isolated from fecal samples (1, 6) or yogurt (1) in the United States of America, France, and Korea. The result indicated the lack of polymorphism among multiple lineages from different continents (1).Interestingly, compared to the other three sequenced B. animalis subsp. lactis strains, V9 has a large insertion, which encodes one putative transposase (BalV_1091) and two sugar metabolism-related proteins, an alpha-1,4-glucosidase (BalV_1092) and an ABC transporter solute-binding protein (BalV_1093). This insertion is a copy of the region at positions 1,860,164 to 1,864,073, which is commonly shared by all four B. animalis subsp. lactis strains.     

Predominant genera of fecal microbiota in children with atopic dermatitis are not altered by intake of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07

The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.     

Comparison of the Complete Genome Sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes are dominant microbial phyla widely distributed in diverse ecosystems on the planet (10, 13, 20, 23, 33, 40, 51). Metagenomic analyses of the microbial landscape inhabiting various mammalian environments, notably the human gastrointestinal tract (GIT) and skin, have specifically identified Actinobacteria as an important and occasionally dominant phylum (18, 21, 33). Among the members of the large, diverse, and dynamic microbial community residing in the human GIT, Bifidobacterium is a dominant genus considered beneficial to humans and includes probiotic strains (live microorganisms which, when administered in adequate amounts, confer a health benefit on the host) (11). The population of bifidobacteria in the human intestine varies over time. Following vaginal delivery, the GIT of healthy newborns is typically colonized by bifidobacteria, especially in breast-fed infants, during the first few days of life (12). Interindividual variation, however, is remarkable in the human infant intestinal flora (41), and dominant genera are not always consistent across metagenomic analyses of the human gut flora (18, 30, 33, 41). Over time, the infant intestinal ecosystem becomes more complex as the diet becomes more diverse, with bifidobacteria typically remaining dominant until weaning (30).Bifidobacterium animalis subsp. lactis is a gram-positive lactic acid bacterium commonly found in the guts of healthy humans and has been identified in the infant gut biota, particularly in ileal, fecal, and mucosal samples (52, 56). Some strains of B. animalis subsp. lactis are able to survive in the GIT, to adhere to human epithelial cells in vitro, to modify fecal flora, to modulate the host immune response, or to prevent microbial gastroenteritis and colitis (4, 15, 20, 40, 52, 56). Additionally, B. animalis subsp. lactis has been reported to utilize nondigestible oligosaccharides, which may contribute to the organism''s ability to compete in the human gut. Carbohydrates resistant to enzymatic degradation and not absorbed in the upper intestinal tract are a primary source of energy for microbes residing in the large intestine. The benefits associated with probiotic strains of B. animalis subsp. lactis have resulted in their inclusion in the human diet via formulation into a large array of dietary supplements and foods, including dairy products such as yogurt. Deciphering the complete genome sequences of such microbes will provide additional insight into the genetic basis for survival and residence in the human gut, notably with regard to the ability to survive gastric passage and utilize available nutrients. Also, these genomes provide reference sequences for ongoing metagenomic analyses of the human environment, including the gut metagenome.Bifidobacterium animalis subsp. lactis is the most common bifidobacterium utilized as a probiotic in commercial dairy products in North America and Europe (22, 38). However, despite this commercial and probiotic significance, strain-level differentiation of B. animalis subsp. lactis strains has been hindered by the high genetic similarity of these organisms, as determined by pulsed-field gel electrophoresis and other nucleic acid-based techniques (6, 55, 56), and the lack of available genomic sequence information. The genome sequence of strain BB-12 (17) is not currently publicly available, and only a draft genome sequence in 28 contigs is available for strain HN019 (GenBank project 28807). The complete B. animalis subsp. lactis genome for strain AD011 (28) was only recently (2009) published. While this was an important first step, a single genome does not allow identification of unique targets for strain differentiation or comparative analyses within the subspecies.The objectives of this study were to determine the complete genome sequences of two B. animalis subsp. lactis strains, the type strain and a widely used commercial strain, to provide insights into the functionality of this species and into species identification and strain specialization.     

The extracellular proteome of Bifidobacterium animalis subsp. lactis BB-12 reveals proteins with putative roles in probiotic effects

Probiotics are live microorganisms that exert health-promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well-studied probiotic strain Bifidobacterium animalis subsp. lactis BB-12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2-DE coupled with MALDI-TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either outside the cell or on its surface. These proteins include solute-binding proteins for oligosaccharides, amino acids and manganese, cell wall-metabolizing proteins, and 18 proteins that have been described to interact with human host epithelial cells or extracellular matrix proteins. The potential functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues, or immunomodulation of the host immune system. The identification of proteins predicted to be involved in such interactions can pave the way towards well targeted studies of the protein-mediated contacts between bacteria and the host, with the goal to enhance the understanding of the mode of action of probiotic bacteria.     

Identification of plasmalogens in the cytoplasmic membrane of Bifidobacterium animalis subsp. lactis

Plasmalogens are ether-linked lipids that may influence oxidative stress resistance of eukaryotic cell membranes. Since bacterial membrane composition can influence environmental stress resistance, we explored the prevalence of plasmalogens in the cytoplasmic membrane of Bifidobacterium animalis subsp. lactis. Results showed plasmalogens are a major component of the B. animalis subsp. lactis membrane.     

Genome Sequence of the Probiotic Bacterium Bifidobacterium animalis subsp. lactis AD011

Bifidobacterium animalis subsp. lactis is a probiotic bacterium that naturally inhabits the guts of most mammals, including humans. Here we report the complete genome sequence of B. animalis subsp. lactis AD011 that was isolated from an infant fecal sample. Biological functions encoded in a single circular chromosome of 1,933,695 bp, smallest among the completely sequenced bifidobacterial genomes, are suggestive of their probiotic functions, such as utilization of bifidogenic factors and a variety of glycosidic enzymes and biosynthesis of polysaccharides.     

Transport of glucose by Bifidobacterium animalis subsp. lactis occurs via facilitated diffusion

Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of D-[U-(14)C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-beta-glucoside than methyl-alpha-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for K(t) and V(max) were 14.8 +/- 3.4 mM D-glucose and 0.13 +/- 0.03 micromol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake.     

Anti-inflammatory metabolite production in the gut from the consumption of probiotic yogurt containing Bifidobacterium animalis subsp. lactis LKM512

There is little evidence for a relationship between probiotic metabolites and host cytokine production. We investigated in the present study the possibility that anti-inflammatory metabolites can be produced in the gut by LKM512 yogurt consumption by using murine macrophage-like J774.1 cells and extracts prepared from the feces of elderly volunteers. These volunteers' acute inflammation had been inhibited by LKM512 yogurt consumption in a previous test. The tumor necrosis factor (TNF)-alpha production elicited in J774.1 cells stimulated by lipopolysaccharide (LPS) and in the fecal extracts obtained during the period of LKM512 yogurt consumption was significantly decreased (p<0.05) than the pre-consumption baseline level. These findings and previous data enable us to conclude that intestinal bacterial metabolites produced by LKM512 yogurt consumption contributed to suppressing the inflammatory cytokine produced by macrophages and that one of the anti-inflammatory metabolites in the fecal extracts was likely to have been a polyamine.     

Comparative Genomics of Bifidobacterium animalis subsp. lactis Reveals a Strict Monophyletic Bifidobacterial Taxon

Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group.     

Molecular characterization of bile salt hydrolase from Bifidobacterium animalis subsp. lactis Bi30

The present work describes the identification, purification, and characterization of bile salt hydrolase (BSH) from Bifidobacterium animalis subsp. lactis. The enzyme was purified to electrophoretic homogeneity by hydrophobic chromatography, ion-exchange chromatography and ultrafiltration. SDS-PAGE analysis of putative BSH and gel filtration revealed that the analyzed protein is presumably a tetramer composed of four monomers each of about 35 kDa. The purified enzyme was analyzed by liquid chromatography coupled to LTQ FT ICR mass spectrometry and unambiguously identified as a bile salt hydrolase from B. animalis. The isoelectric point of the studied protein was estimated to be around pH 4.9. The pH optimum of the purified BSH is between 4.7 to 6.5, and the temperature optimum is around 50 degrees C. The BSH of B. animalis could deconjugate all tested bile salts, with clear preference for glycine-conjugated bile salts over taurine-conjugated forms. Genetic analysis of the bsh showed high similarity to the previously sequenced bsh gene from B. animalis and confirmed the usefulness of bile salt hydrolase as a genetic marker for B. animalis identification.     

摄入含嗜酸乳杆菌NCFM和乳双歧杆菌Bi-07的益生菌补充剂增强免疫功能的动物实验研究

目的测定与验证摄入含特定乳酸菌株组合（嗜酸乳杆菌NCFM和乳双歧杆菌Bi-07）一定剂量的益生菌补充剂对动物（小鼠）免疫功能的影响。方法SPF昆明种小白鼠经口连续分别给予0．25、0．50、1．50g／kgBW的益生菌补充剂4周,进行迟发型变态反应试验、溶血素滴度测定、NK细胞活性测定等。结果在小白鼠迟发型变态反应试验中,中、高剂量组足跖增厚值均高于对照组,差异有显著性（P〈0．05）;对受试动物血清溶血素抗体滴度水平的影响的实验中,中、高剂量组的抗体积数高于对照组,差异均有显著性（P〈0．05）。NK细胞活性测定中,高剂量组脾NK细胞活性增强,差异有非常显著性（P〈0．01）。结论含该两种特定乳酸菌株的益生菌补充剂被小自鼠摄入一定剂量后,起到了增强小白鼠细胞免疫、体液免疫功能及NK细胞活性的作用。     

Chemical structure of the cell wall-associated polysaccharide of Bifidobacterium animalis subsp. lactis LKM512

We have demonstrated that Bifidobacterium animalis subsp. lactis LKM512 had some probiotic properties in vivo and in vitro. To further understand their mechanisms, the chemical structure of the extracellular polysaccharide that constructs the cell envelope was determined. The strain was anaerobically cultured in MRS broth at 37 °C for 20 h, then the bacterial cells were harvested by centrifugation and washed. The cell wall-associated polysaccharide (CPS) was prepared from the cell wall component digested by lysozyme. The results of anion exchange and gel filtration chromatography showed that the polysaccharide was negatively charged and had a high molecular mass. The CPS was found to compose of galactopyranosyl, galactofuranosyl, glucopyranosyl and rhamnopyranosyl residues in the molar ratio of 1:1:1:3 by using methylation analysis with GC-MS and HPLC profiling. From the results of the structural characterization by 1 dimensional and 2 dimensional NMR spectroscopy, the polysaccharide was established to be a hexasaccharide repeating unit with the following structure:      

Relevance of Bifidobacterium animalis subsp. lactis plasminogen binding activity in the human gastrointestinal microenvironment

Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species Bifidobacterium animalis subsp. lactis and the human host. However, up to now, only in vitro studies have been reported. Here, we demonstrate that the probiotic strain B. animalis subsp. lactis BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the B. lactis-plasmin(ogen) interaction in the human gastrointestinal tract.     

Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

Insights into physiological traits of Bifidobacterium animalis subsp. lactis BB-12 through membrane proteome analysis

Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two enrichment strategies to improve the identification of membrane proteins from BB-12 cultures grown on glucose and on xylo-oligosaccharides, the latter being an emerging prebiotic substrate recently reported to be fermented by BB-12. Our approach encompassed consecutive steps of detergent- and carbonate-treatment in order to generate inside-out membrane vesicles and to interfere with binding of membrane-associated proteins to the membrane, respectively. Proteins in the enriched membrane fraction and membrane-associated fraction were digested by lysyl endopeptidase and trypsin followed by peptide sequencing by LC-ESI-Q-TOF MS/MS. Ninety of a total of 248 identified unique proteins were predicted to possess transmembrane segments (TMSs), and 56 of these have more than one TMS. Seventy-nine of the identified proteins are annotated to be involved in transport of amino acids, oligosaccharides, inorganic ions, nucleotides, phosphate or exopolysaccharides, or to belong to the F1F0-ATP-synthetase complex and the protein translocation machinery, respectively.