Cloning and characterization of a bile salt hydrolase (<Emphasis Type="Italic">bsh</Emphasis>) from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis>

A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P_bsh) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.     

Resistance to Simulated Gastrointestinal Conditions and Adhesion to Mucus as Probiotic Criteria for Bifidobacterium longum Strains

Eight Bifidobacterium longum strains, including reported probiotic strains (commercial and noncommercial), collection strains, and laboratory isolates, were investigated for their ability to adhere to mucin as well as their ability to tolerate acid and bile. Strains could be discriminated based on their sensitivity at pH values of 2.0 to 2.5 and bile concentrations of 0.5% to 2.0%. B. longum NCC 2705, a strain known for its probiotic properties, showed the highest resistance to gastrointestinal conditions, whereas the commercial probiotic strains B. longum BB 536 and SP 07/3 were the least resistant. In parallel, the human isolate B. longum BIF 53 showed the highest adhesion to mucin, whereas the commercial probiotic strains B. longum W 11, BB 536, and SP 07/3 were the least adhesive. The bacterial adhesion to mucin of strains B. longum NCC 2705 and BIF 53 could be reduced by lysozyme, indicating that cell-wall components are involved in the adhesion process. These results showed that there is no obvious link between adhesion and resistance to gastrointestinal conditions and the probiotic status of the studied strains. This calls for a definition of conditions for in vitro tests that better predict the in vivo functionality of probiotic strains.     

Cross-Feeding between Bifidobacterium longum BB536 and Acetate-Converting, Butyrate-Producing Colon Bacteria during Growth on Oligofructose

In vitro coculture fermentations of Bifidobacterium longum BB536 and two acetate-converting, butyrate-producing colon bacteria, Anaerostipes caccae DSM 14662 and Roseburia intestinalis DSM 14610, with oligofructose as the sole energy source, were performed to study interspecies interactions. Two clearly distinct types of cross-feeding were identified. A. caccae DSM 14662 was not able to degrade oligofructose but could grow on the fructose released by B. longum BB536 during oligofructose breakdown. R. intestinalis DSM 14610 could degrade oligofructose, but only after acetate was added to the medium. Detailed kinetic analyses of oligofructose breakdown by the last strain revealed simultaneous degradation of the different chain length fractions, in contrast with the preferential degradation of shorter fractions by B. longum BB536. In a coculture of both strains, initial oligofructose degradation and acetate production by B. longum BB536 took place, which in turn also allowed oligofructose breakdown by R. intestinalis DSM 14610. These and similar cross-feeding mechanisms could play a role in the colon ecosystem and contribute to the combined bifidogenic/butyrogenic effect observed after addition of inulin-type fructans to the diet.     

Oral administration of Bifidobacterium longum prevents gut-derived Pseudomonas aeruginosa sepsis in mice

Aims: The aim of the study was to evaluate the efficacy of probiotics on gut‐derived sepsis caused by Pseudomonas aeruginosa in immunocompromised mice. Methods and Results: After oral inoculation of P. aeruginosa, mice were treated with cyclophosphamide to induce leucopenia and translocation of the intestinal P. aeruginosa into blood, thereby producing gut‐derived sepsis. In this model, administration of 1 × 10⁹ CFU of Bifidobacterium longum strain BB536 for 10 days significantly (P < 0·01) increased the survival rate compared with groups of mice administered either with Bifidobacterium breve strain ATCC 15700 or excipients contained in the probiotic bacterial powder. Administration of B. longum significantly decreased viable counts of P. aeruginosa in the liver and blood compared with other groups. Culture of intestinal contents revealed a significantly lower viable count of P. aeruginosa in the jejunum of B. longum‐treated mice compared with other groups of mice. Furthermore, in vitro data demonstrated that B. longum possessed apparently higher adherent activity to Caco‐2 cell monolayers and significantly suppressed the adherence of P. aeruginosa to the monolayers of cells compared with other groups. Conclusion: Oral administration of B. longum protects mice against gut‐derived sepsis caused by P. aeruginosa, and the effect may be due to interference of P. aeruginosa adherence to intestinal epithelial cells. Significance and Impact of this Study: This study demonstrated that oral administration of B. longum BB536 is effective to protect against opportunistic infection with drug‐resistant bacteria such as P. aeruginosa. The results suggest that probiotics may play an important role even in the immunocompromised patients.     

Heterologous expression of cholesterol oxidase in<Emphasis Type="Italic"> Bifidobacterium longum</Emphasis> under the control of 16S rRNA gene promoter of bifidobacteria

We have constructed a constitutive high-level-expression vector for the genus Bifidobacterium and used it to express cholesterol oxidase from Streptomyces coelicola. The promoter region of the 16S rRNA gene was amplified by inverse PCR and used for the construction of pBES16PR. The optimal ribosome-binding site (RBS) for Bifidobacterium was incorporated in pBES16PR. In order to test the efficacy of this expression vector, we constructed pBES16PR-CHOL with the structural gene for cholesterol oxidase under the control of the 16S rRNA promoter, and used it to transform Bifidobacterium longum. The gene was successfully expressed and high level of cholesterol oxidase activity was obtained in B. longum. This is the first report of an expression vector for the genus Bifidobacterium using a 16S rRNA gene promoter and successful expression of cholesterol oxidase. Myeong Soo Park and Bin Kwon contributed equally.     

Oral Administration of an Immunostimulatory DNA Sequence from Bifidobacterium longum Improves Th1/Th2 Balance in a Murine Model

We have reported the antiallergic activities of the immunostimulatory oligodeoxynucleotide (ODN) BL07S, identified from genomic DNA of Bifidobacterium longum BB536 from in vitro and in vivo studies. The present study evaluated the efficiency of ODN BL07S in preventing allergic responses by oral administration. Oral administration of BL07S suppressed serum ovalbumin (OVA)-specific immunoglobulin (Ig) E levels and improved the OVA-specific IgG2a/IgG1 ratio. ODN BL07S increased Th1 cytokine and decreased Th2 cytokine production in splenocytes. These results suggest that immunostimulatory ODNs are potentially associated with the antiallergic effects of probiotics.     

Expression of Rice Glutamate Decarboxylase in <Emphasis Type="Italic">Bifidobacterium Longum</Emphasis> Enhances γ-Aminobutyric Acid Production

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for γ-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC⁻) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Conditions affecting cell surface properties of human intestinal bifidobacteria

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.     

Expression and Secretion of Bifidobacterium adolescentis Amylase by Bifidobacterium Longum

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli–Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium. Both authors contributed equally Received 17 June 2005; Revisions requested 13 July 2005 and 26 September 2005; Revisions received 12 September 2005 and 8 November 2005; Accepted 11 November 2005     

Isolation and molecular characterization of a cryptic plasmid from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

An isolate from the fecal samples of children was identified as Bifidobacterium longum. A plasmid isolated from it pBIFA24 was 4,892 bp with three open reading frames, ORFI, ORFII, and ORFIII. ORFI encoded a replication protein involved in a rolling-circle replication mechanism, and three sets of tandem repeat sequences featuring iteron structure were identified. Secondary structure prediction analysis of ORFII suggested it was a transmembrane protein. ORFIII showed high amino acid sequence identity with some mobilization proteins and contained an oriT sequence.     

Optimization of Bifidobacterium longum growth by use of calcium carbonate-alginate beads

High concentrations of ammonium and sodium ions inhibited Bifidobacterium longum growth more than a high calcium ion concentration. The optimal pH for B. longum growth was determined to be 5.0 due to the lower accumulation of ammonium ion. To reduce the accumulation of ammonium ion and obtain an enhanced growth of B. longum, the pH of the culture containing immobilized calcium carbonate beads was controlled to 5.0 with ammonia water. The concentrations of ammonium, sodium, and calcium ions in the culture were maintained at the desired level. The maximum cell mass increased to 16.8 g/l, 1.23 times higher than cultures without calcium carbonate beads. The number of viable cells in the culture increased to 5.0 × 10¹⁰, 1.67 times more than cultures without calcium carbonate beads.     

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.     

In Vitro Kinetic Analysis of Oligofructose Consumption by Bacteroides and Bifidobacterium spp. Indicates Different Degradation Mechanisms

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F₂ and F₃) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F₄, GF₄, F₅, GF₅, and F₆) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A novel transglycosylation reaction from sucrose to l-ascorbic acid by a recombinant sucrose phosphorylase from Bifidobacterium longum was used to produce a stable l-ascorbic acid derivative. The major product was detected by HPLC, and confirmed to be 2-O-α-d-glucopyranosyl-l-ascorbic acid by LC-MS/MS analysis.     

Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups

The Actinobacteria constitute one of the main phyla of Bacteria. Presently, no morphological and very few molecular characteristics are known which can distinguish species of this highly diverse group. In this work, we have analyzed the genomes of four actinobacteria (viz. Mycobacterium leprae TN, Leifsonia xyli subsp. xyli str. CTCB07, Bifidobacterium longum NCC2705 and Thermobifida fusca YX) to search for proteins that are unique to Actinobacteria. Our analyses have identified 233 actinobacteria-specific proteins, homologues of which are generally not present in any other bacteria. These proteins can be grouped as follows: (i) 29 proteins uniquely present in most sequenced actinobacterial genomes; (ii) 6 proteins present in almost all actinobacteria except Bifidobacterium longum and another 37 proteins absent in B. longum and few other species; (iii) 11 proteins which are mainly present in Corynebacterium, Mycobacterium and Nocardia (CMN) subgroup as well as Streptomyces, T. fusca and Frankia sp., but they are not found in Bifidobacterium and Micrococcineae; (iv) 8 proteins that are specific for T. fusca and Streptomyces species, plus 2 proteins also present in the Frankia species; (v) 13 proteins that are specific for the Corynebacterineae or the CMN group; (vi) 14 proteins only found in Mycobacterium and Nocardia; (vii) 24 proteins unique to different Mycobacterium species; (viii) 8 proteins specific to the Micrococcineae; (ix) 85 proteins which are distributed sporadically in actinobacterial species. Additionally, many examples of lateral gene transfer from Actinobacteria to Magnetospirillum magnetotacticum have also been identified. The identified proteins provide novel molecular means for defining and circumscribing the Actinobacteria phylum and a number of subgroups within it. The distribution of these proteins also provides useful information regarding interrelationships among the actinobacterial subgroups. Most of these proteins are of unknown function and studies aimed at understanding their cellular functions should reveal common biochemical and physiological characteristics unique to either all actinobacteria or particular subgroups of them. The identified proteins also provide potential targets for development of drugs that are specific for actinobacteria.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Cloned Cytosine Deaminase Gene Expression of Bifidobacterium longum and Application to Enzyme/Pro-drug Therapy of Hypoxic Solid Tumors

Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors.     

Metabolism of chicory fructooligosaccharides by bifidobacteria

Two types of chicory fructooligosaccharides (Fibruline Instant and Fibrulose F97) were metabolised by Bifidobacterium longum, B. infantis and B. angulatum. Chromatographic analysis of the medium after 120 h revealed a consumption of all the fructose oligomers present in the commercial chicory fructooligosaccharide mixtures for all the strains. Maximum measurable degree of polymerisation of the substrates before fermentation was 41. The higher biomass production was reached with B. infantis (1.4 and 1.7 g dry wt l^–1) for its cultivation on medium complemented, respectively, with Fibruline Instant and Fibrulose F97 as substrate. These results give the opportunity to use chicory fructooligosaccharides as a prebiotic.