GH136-encoding gene (perB) is involved in gut colonization and persistence by Bifidobacterium bifidum PRL2010.

Bifidobacteria are commensal microorganisms that typically inhabit the mammalian gut, including that of humans. As they may be vertically transmitted, they commonly colonize the human intestine from the very first day following birth and may persist until adulthood and old age, although generally at a reduced relative abundance and prevalence compared to infancy. The ability of bifidobacteria to persist in the human intestinal environment has been attributed to genes involved in adhesion to epithelial cells and the encoding of complex carbohydrate-degrading enzymes. Recently, a putative mucin-degrading glycosyl hydrolase belonging to the GH136 family and encoded by the perB gene has been implicated in gut persistence of certain bifidobacterial strains. In the current study, to better characterize the function of this gene, a comparative genomic analysis was performed, revealing the presence of perB homologues in just eight bifidobacterial species known to colonize the human gut, including Bifidobacterium bifidum and Bifidobacterium longum subsp. longum strains, or in non-human primates. Mucin-mediated growth and adhesion to human intestinal cells, in addition to a rodent model colonization assay, were performed using B. bifidum PRL2010 as a perB prototype and its isogenic perB-insertion mutant. These results demonstrate that perB inactivation reduces the ability of B. bifidum PRL2010 to grow on and adhere to mucin, as well as to persist in the rodent gut niche. These results corroborate the notion that the perB gene is one of the genetic determinants involved in the persistence of B. bifidum PRL2010 in the human gut.

Characterization of a novel recombinant D-mannose isomerase from Bifidobacterium bifidum and its catalytic mechanism.

Due to the increasing demand for health-conscious and environmentally friendly products, D-mannose has gained significant attention as a natural, low-calorie sweetener. The use of D-mannose isomerases (D-MIases) for D-mannose production has emerged as a prominent area of research, offering superior advantages compared with conventional methods such as plant extraction and chemical synthesis. In this study, a gene encoding D-MIase was cloned from Bifidobacterium and expressed in E. coli BL21 (DE3). The heterologously expressed enzyme, Bifi-mannose, formed a trimer with a molecular weight of 146.3 kDa and a melting temperature (Tm) of 63.39 ± 1.3 °C. Bifi-mannose exhibited optimal catalytic activity at pH 7.5 and 55 °C, and retained more than 80% of its activity after a 3-hour incubation at 55 °C, demonstrating excellent thermal stability. The Km, Vmax, and kcat/Km values of Bifi-mannose for D-fructose isomerization were determined as 538.7 ± 62.5 mM, 11.7 ± 0.9 µmol·mg1·s1, and 1.02 ± 0.3 mM1·s1, respectively. Notably, under optimized conditions, catalytic yields of 29.4, 87.1, and 148.5 mg·mL1 were achieved when using 100, 300, and 500 mg·mL1 of D-fructose as substrates, resulting in a high conversion rate (29%). Furthermore, kinetic parameters and molecular docking studies revealed that His387 residue primarily participates in the opening of the pyranose ring, while His253 acts as a basic catalyst in the isomerization process.

Bifidobacterium bifidum BGN4 fractions ameliorate palmitic acid-induced hepatocyte ferroptosis by inhibiting SREBP1-CYP2E1 pathway.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is strongly associated with disturbances in the intestinal microbiota. Herein, the biological effects and mechanism of Bifidobacterium bifidum BGN4 fractions in regulating hepatocyte ferroptosis during MAFLD progression were investigated. To establish an in vitro model of MAFLD, LO2 cells were subjected to palmitic acid (PA). The mRNA and protein expressions were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. LO2 cell proliferation was examined using 5-diphenyltetrazolium bromide (MTT) and ethynyl-2'-deoxyuridine (EdU) assays, whereas its apoptosis was evaluated by flow cytometry. Furthermore, level of reactive oxygen species (ROS) was measured using 2', 7,-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Additionally, the levels of Fe2+, malondialdehyde (MDA), and glutathione (GSH), as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were detected using corresponding kits. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were performed to analyze the interaction between sterol-regulatory element binding protein 1 (SREBP1) and cytochrome P450-2E1 (CYP2E1) promoter. Our results revealed that Bifidobacterium bifidum BGN4 fractions effectively ameliorated PA-induced hepatocyte injury, oxidative stress, and ferroptosis. However, these beneficial effects of BGN4 fractions on PA-induced hepatocyte were dramatically reversed by SREBP1 overexpression, suggesting that BGN4 attenuated MAFLD by acting on SREBP1. Moreover, we observed that BGN4 fractions inhibited CYP2E1 transcription by suppressing SREBP1 nuclear translocation. In addition, CYP2E1 overexpression eliminated the inhibitory effect of BGN4 fractions on PA-induced hepatocyte oxidative stress and ferroptosis. These findings collectively indicated that BGN4 fractions reduced CYP2E1 expression by inhibiting SREBP1 nuclear translocation, thereby suppressing hepatocyte oxidative stress and ferroptosis during the development of MAFLD.

GH20 and GH84 ß-N-acetylglucosaminidases with different linkage specificities underpin mucin O-glycan breakdown capability of Bifidobacterium bifidum.

Intestinal mucous layers mediate symbiosis and dysbiosis of host-microbe interactions. These interactions are influenced by the mucin O-glycan degrading ability of several gut microbes. The identities and prevalence of many glycoside hydrolases (GHs) involved in microbial mucin O-glycan breakdown have been previously reported; however, the exact mechanisms and extent to which these GHs are dedicated to mucin O-glycan degradation pathways warrant further research. Here, using Bifidobacterium bifidum as a model mucinolytic bacterium, we revealed that two ß-N-acetylglucosaminidases belonging to the GH20 (BbhI) and GH84 (BbhIV) families play important roles in mucin O-glycan degradation. Using substrate specificity analysis of natural oligosaccharides and O-glycomic analysis of porcine gastric mucin (PGM) incubated with purified enzymes or B. bifidum carrying bbhI and/or bbhIV mutations, we showed that BbhI and BbhIV are highly specific for ß-(1→3)- and ß-(1→6)-GlcNAc linkages of mucin core structures, respectively. Interestingly, we found that efficient hydrolysis of the ß-(1→3)-linkage by BbhI of the mucin core 4 structure [GlcNAcß1-3(GlcNAcß1-6)GalNAcα-O-Thr] required prior removal of the ß-(1→6)-GlcNAc linkage by BbhIV. Consistent with this, inactivation of bbhIV markedly decreased the ability of B. bifidum to release GlcNAc from PGM. When combined with a bbhI mutation, we observed that the growth of the strain on PGM was reduced. Finally, phylogenetic analysis suggests that GH84 members may have gained diversified functions through microbe-microbe and host-microbe horizontal gene transfer events. Taken together, these data strongly suggest the involvement of GH84 family members in host glycan breakdown.

Bifidobacterium bifidum H3-R2 and Its Molecular Communication within the Context of Ulcerative Colitis.

Bifidobacteria are important mediators of immune system development within the gastrointestinal system and immunological homeostasis. The present study explored the anti-colitic activity of Bifidobacterium bifidum H3-R2 in a murine dextran sulfate sodium (DSS)-induced model of ulcerative colitis (UC). Moreover, this study offers novel insight regarding the molecular basis for the probiotic properties of B. bifidum H3-R2 by analyzing the underlying mechanisms whereby B. bifidum H3-R2-derived proteins affect the intestinal barrier. B. bifidum H3-R2 administration was sufficient to alleviate clinical manifestations consistent with DSS-induced colitis, restoring aberrant inflammatory cytokine production, enhancing tight junction protein expression, and positively impacting overall intestinal microecological homeostasis in these animals. Moreover, the bifidobacteria-derived GroEL and transaldolase (TAL) proteins were found to regulate tight junction protein expression via the NF-κB, myosin light chain kinase (MLCK), RhoA/Rho-associated protein kinase (ROCK), and mitogen-activated protein kinase (MAPK) signaling pathways, preventing the lipopolysaccharide (LPS)-mediated disruption of the intestinal epithelial cell barrier.

Genome-wide siRNA screening reveals several host receptors for the binding of human gut commensal Bifidobacterium bifidum.

Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, the protective mechanisms of bifidobacteria on intestinal epithelium at molecular level are poorly understood. In this study, we developed a high-throughput in vitro screening assay to explore binding receptors of intestinal epithelial cells for Bifidobacterium bifidum. Short interfering RNAs (siRNA) were used to silence expression of each gene in the Caco-2 cell line one by one. The screen yielded four cell surface proteins, SERPINB3, LGICZ1, PKD1 and PAQR6, which were identified as potential receptors as the siRNA knock-down of their expression decreased adhesion of B. bifidum to the cell line repeatedly during the three rounds of siRNA screening. Furthermore, blocking of these host cell proteins by specific antibodies decreased the binding of B. bifidum significantly to Caco-2 and HT29 cell lines. All these molecules are located on the surface of epithelial cells and three out of four, SERPINB3, PKD1 and PAQR6, are involved in the regulation of cellular processes related to proliferation, differentiation and apoptosis as well as inflammation and immunity. Our results provide leads to the first steps in the mechanistic cascade of B. bifidum-host interactions leading to regulatory effects in the epithelium and may partly explain how this commensal bacterium is able to promote intestinal homeostasis.

A recombinant Bifidobacterium bifidum BGN4 strain expressing the streptococcal superoxide dismutase gene ameliorates inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a gastrointestinal disease characterized by diarrhea, rectal bleeding, abdominal pain, and weight loss. Recombinant probiotics producing specific proteins with IBD therapeutic potential are currently considered novel drug substitutes. In this study, a Bifidobacterium bifidum BGN4-SK strain was designed to produce the antioxidant enzymes streptococcal superoxide dismutase (SOD) and lactobacillus catalase (CAT), and a B. bifidum BGN4-pBESIL10 strain was proposed to generate an anti-inflammatory cytokine, human interleukin (IL)-10. In vitro and in vivo efficacy of these genetically modified Bifidobacterium strains were evaluated for colitis amelioration. RESULTS: In a lipopolysaccharide (LPS)-stimulated HT-29 cell model, tumor necrosis factor (TNF)-α and IL-8 production was significantly suppressed in the B. bifidum BGN4-SK treatment, followed by B. bifidum BGN4-pBESIL10 treatment, when compared to the LPS-treated control. Synergistic effects on TNF-α suppression were also observed. In a dextran sodium sulphate (DSS)-induced colitis mouse model, B. bifidum BGN4-SK treatment significantly enhanced levels of antioxidant enzymes SOD, glutathione peroxidase (GSH-Px) and CAT, compared to the DSS-only group. B. bifidum BGN4-SK significantly ameliorated the symptoms of DSS-induced colitis, increased the expression of tight junction genes (claudin and ZO-1), and decreased pro-inflammatory cytokines IL-6, IL-1ß and TNF-α. CONCLUSIONS: These findings suggest that B. bifidum BGN4-SK ameliorated DSS-induced colitis by generating antioxidant enzymes, maintaining the epithelial barrier, and decreasing the production of pro-inflammatory cytokines. Although B. bifidum BGN4-pBESIL10 exerted anti-inflammatory effects in vitro, the enhancement of IL-10 production and alleviation of colitis were very limited.

Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI promotes neuronal rejuvenation in aged mice.

An increasing number of studies have indicated that alterations in gut microbiota affect brain function, including cognition and memory ability, via the gut-brain axis. In this study, we aimed to determine the protective effect of Bifidobacterium bifidum BGN4 (B. bifidum BGN4) and Bifidobacterium longum BORI (B. longum BORI) on age-related brain damage in mice. We found that administration of B. bifidum BGN4 and B. longum BORI effectively elevates brain-derived neurotrophic factor expression which was mediated by increased histone 3 lysine 9 trimethylation. Furthermore, administration of probiotic supplementation reversed the DNA damage and apoptotic response in aged mice and also improved the age-related cognitive and memory deficits of these mice. Taken together, the present study highlights the anti-aging effects of B. bifidum BGN4 and B. longum BORI in the aged brain and their beneficial effects for age-related brain disorders.

Comparative Genomic Analysis of Bifidobacterium bifidum Strains Isolated from Different Niches.

The potential probiotic benefits of Bifidobacterium bifidum have received increasing attention recently. We used comparative genomic analysis to explore the differences in the genome and the physiological characteristics of B. bifidum isolated from the fecal samples of Chinese adults and infants. The relationships between genotypes and phenotypes were analyzed to assess the effects of isolation sources on the genetic variation of B. bifidum. The phylogenetic tree results indicated that the phylogeny of B. bifidum may be related to the geographical features of its isolation source. B. bifidum was found to have an open pan-genome and a conserved core genome. The genetic diversity of B. bifidum is mainly reflected in carbohydrate metabolism- and immune/competition-related factors, such as the glycoside hydrolase gene family, bacteriocin operons, antibiotic resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Additionally, the type III A CRISPR-Cas system was discovered in B. bifidum for the first time. B. bifidum strains exhibited niche-specific characteristics, and the results of this study provide an improved understanding of the genetics of this species.

Integration of Transcriptome and Metabolome Reveals the Genes and Metabolites Involved in Bifidobacterium bifidum Biofilm Formation.

Bifidobacterium bifidum strains, an important component of probiotic foods, can form biofilms on abiotic surfaces, leading to increased self-resistance. However, little is known about the molecular mechanism of B. bifidum biofilm formation. A time series transcriptome sequencing and untargeted metabolomics analysis of both B. bifidum biofilm and planktonic cells was performed to identify key genes and metabolites involved in biofilm formation. Two hundred thirty-five nonredundant differentially expressed genes (DEGs) (including vanY, pstS, degP, groS, infC, groL, yajC, tadB and sigA) and 219 nonredundant differentially expressed metabolites (including L-threonine, L-cystine, L-tyrosine, ascorbic acid, niacinamide, butyric acid and sphinganine) were identified. Thirteen pathways were identified during the integration of both transcriptomics and metabolomics data, including ABC transporters; quorum sensing; two-component system; oxidative phosphorylation; cysteine and methionine metabolism; glutathione metabolism; glycine, serine and threonine metabolism; and valine, leucine and isoleucine biosynthesis. The DEGs that relate to the integration pathways included asd, atpB, degP, folC, ilvE, metC, pheA, pstS, pyrE, serB, ulaE, yajC and zwf. The differentially accumulated metabolites included L-cystine, L-serine, L-threonine, L-tyrosine, methylmalonate, monodehydroascorbate, nicotinamide, orthophosphate, spermine and tocopherol. These results indicate that quorum sensing, two-component system and amino acid metabolism are essential during B. bifidum biofilm formation.

Comparative genomics of the gut commensal Bifidobacterium bifidum reveals adaptation to carbohydrate utilization.

Bifidobacterium bifidum is one of the most abundant members of the gut microbiota at the early stage of life. The established association of the bacterium with the human gut confers health benefits. Such successful persistence of B. bifidum necessitates metabolic adaptation to the host-derived carbohydrates, a process which is poorly understood. The current study focuses on revealing the genomic-based phylogeny (phylogenomics) of B. bifidum and utilizing comparative genomics to decipher the glycolytic abilities of bifidobacterial strains isolated from different human body niches (feces, human gut, vagina, and breast milk). When the phylogenomic analysis was performed on 95 B. bifidum strains, currently available on the RefSeq database, the bacterium was clearly distinguished from other members of the Bifidobacterium genus. Furthermore, a pairwise genomic comparison indicated that a large proportion of orthologous gene families were shared among the B. bifidum strains. These findings highlight the notion that the B. bifidum species is genetically similar and may perform similar functions in their host. When 15 B. bifidum genomes representing strains from different human body niches were annotated, the resulting functional profile showed the presence of enriched proteins involved in carbohydrate utilization. Moreover, mining the 15 B. bifidum genomes for the presence of Carbohydrate-Active Enzyme (CAZY) systems, the analysis found the existence of diverse protein families which include glycosyl hydrolases, glycosyl transferases, carbohydrate-binding modules, and carbohydrate esterases. Collectively, these CAZY systems enables B. bifidum to utilize host-derived glycans (e.g., mucin) and diet-derived carbohydrates (e.g., starch). In contrast, a correlation analysis revealed that B. bifidum strains isolated from the different body niches were indistinguishable in the context of presence-absence of CAZY systems. These findings emphasize the valuable use of comparative genomics in deciphering the glycolytic abilities of B. bifidum and consequently its adaptation to carbohydrate utilization in the human gut environment.

Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4.

BACKGROUND: Bifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines. RESULTS: The vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473 ± 300 ng/mL (n = 4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n = 6, p < 0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n = 6, p < 0.01) or TNFα-induced HT-29 cells (n = 6, p < 0.001). CONCLUSION: B. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Ecofriendly Biomolecule-Capped Bifidobacterium bifidum-Manufactured Silver Nanoparticles and Efflux Pump Genes Expression Alteration in Klebsiella pneumoniae.

Background: Klebsiella pneumoniae is currently considered as an immediate threat to human health due to its various multidrug efflux pumps. Microbially synthesized silver nanoparticles (AgNPs) are an attractive and eco-friendly approach to prevent antibiotic resistance in bacteria. In the present study, we compared the inhibitory effect of both commercial and green AgNPs by Bifidobacterium bifidum on OxqAB efflux pump genes in ciprofloxacin-resistant strains of K. pneumoniae. Materials and Methods: AgNPs were characterized by ultraviolet-visible spectrophotometer, Fourier transform infrared spectroscopy, X-ray diffraction, zeta potential, transmission electron microscopy, and scanning electron microscopy. Antibiogram was used to identify resistant isolates and the effect of the biosynthesized AgNPs against OxqAB efflux pump strains was assessed by the minimum inhibitory concentration (MIC) method. The expression levels of oxqAB genes were evaluated using real-time polymerase chain reaction (PCR) followed by exposure to subMICs of the AgNPs. Results: PCR results showed that 25 strains had OxqAB efflux pump and the MIC method indicated that AgNPs had an inhibitory effect on all resistant strains with OxqAB efflux pump. The efficacy of the synthetic nanoparticles was assessed by comparing the antiefflux pump activity with commercial AgNPs. In ciprofloxacin-resistant isolates, the oxqAB genes expression levels reduced in the subMIC of both AgNPs, whereas biosynthesized AgNPs had greater bactericidal effects compared with the commercial AgNPs. Conclusions: Efflux pumps could be an attractive target for our biosynthesized AgNPs. The oxqAB genes expression levels reduced in subMIC of both AgNPs, whereas biosynthesized AgNPs had greater bactericidal effects than the commercial AgNPs.

Human milk and mucosa-associated disaccharides impact on cultured infant fecal microbiota.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods.

Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances.

Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.

Selective Synthesis of Galactooligosaccharides Containing ß(1→3) Linkages with ß-Galactosidase from Bifidobacterium bifidum (Saphera).

The transglycosylation activity of a novel commercial ß-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-ß-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other ß-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-ß(1→3)-Gal-ß(1→4)-Glc (3'-O-ß-galactosyl-lactose), Gal-ß(1→6)-Glc (allolactose), Gal-ß(1→3)-Glc (3-galactosyl-glucose), Gal-ß(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-ß(1→3)-Gal-ß(1→3)-Gal-ß(1→4)-Glc. In general, B. bifidum ß-galactosidase showed a tendency to form ß(1→3) linkages followed by ß(1→6) and more scarcely ß(1→4).

Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered ß-hexosaminidase immobilized on solid support.

The D746E variant of Bifidobacterium bifidum ß-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective ß-1,3-glycosylation of lactose from a d-glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145-200 mM) at quantitative yield from the donor substrate. The wild-type ß-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d-glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80-100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.

Converting a ß-N-acetylhexosaminidase into two trans-ß-N-acetylhexosaminidases by domain-targeted mutagenesis.

We have recently derived a ß-N-acetylhexosaminidase, BbhI, from Bifidobacterium bifidum JCM 1254, which could regioselectively synthesize GlcNAcß1-3Galß1-4Glc with a yield of 44.9%. Here, directed evolution of BbhI by domain-targeted mutagenesis was carried out. Firstly, the GH20 domain was selected for random mutagenesis using MEGAWHOP method and a small library of 1300 clones was created. A total of 734 colonies with reduced hydrolytic activity were isolated, and three mutants with elevated transglycosylation yields, GlcNAcß1-3Galß1-4Glc yields of 68.5%, 74.7%, and 81.1%, respectively, were obtained. Subsequently, nineteen independent mutants were constructed according to all the mutation sites in these three mutants. After transglycosylation analysis, Asp714 and Trp773 were identified as key residues for improvement in transglycosylation ability and were chosen for the second round of directed evolution by site-saturation mutagenesis. Two most efficient mutants D714T and W773R that acted as trans-ß-N-acetylhexosaminidase were finally achieved. D714T with the substitution at the putative nucleophile assistant residue Asp714 by threonine showed high yield of 84.7% with unobserved hydrolysis towards transglycosylation product. W773R with arginine substitution at Trp773 residue locating at the entrance of catalytic cavity led to the yield up to 81.8%. The kcat/Km values of D714T and W773R for hydrolysis of pNP-ß-GlcNAc displayed drastic decreases. NMR investigation of protein-substrate interaction revealed an invariable mode of the catalytic cavity of D714T, W773R, and WT BbhI. The collective motions of protein model showed the mutations Thr714 and Arg773 exerted little effect on the dynamics of the inside but a large effect on the dynamics of the outside of catalytic cavity.

Bifidobacterium bifidum ATCC 15696 and Bifidobacterium breve 24b Metabolic Interaction Based on 2'-O-Fucosyl-Lactose Studied in Steady-State Cultures in a Freter-Style Chemostat.

Infants fed breast milk harbor a gut microbiota in which bifidobacteria are generally predominant. The metabolic interactions of bifidobacterial species need investigation because they may offer insight into the colonization of the gut in early life. Bifidobacterium bifidum ATCC 15696 hydrolyzes 2'-O-fucosyl-lactose (2FL; a major fucosylated human milk oligosaccharide) but does not use fucose released into the culture medium. However, fucose is a growth substrate for Bifidobacterium breve 24b, and both strains utilize lactose for growth. The provision of fucose and lactose by B. bifidum (the donor) allowing the growth of B. breve (the beneficiary) conforms to the concept of syntrophy, but both strains will compete for lactose to multiply. To determine the metabolic impact of this syntrophic/competitive relationship on the donor, the transcriptomes of B. bifidum were determined and compared in steady-state monoculture and coculture using transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR). B. bifidum genes upregulated in coculture included those encoding alpha-l-fucosidase and carbohydrate transporters and those involved in energy production and conversion. B. bifidum abundance was the same in coculture as in monoculture, but B. breve dominated the coculture numerically. Cocultures during steady-state growth in 2FL medium produced mostly acetate with little lactate (acetate:lactate molar ratio, 8:1) compared to that in monobatch cultures containing lactose (2:1), which reflected the maintenance of steady-state cells in log-phase growth. Darwinian competition is an implicit feature of bacterial communities, but syntrophy is a phenomenon putatively based on cooperation. Our results suggest that the regulation of syntrophy, in addition to competition, may shape bacterial communities.IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the infant bowel) using in vitro experimentation with bacterial cultures maintained under controlled growth and environmental conditions. We studied the growth of bifidobacteria whose nutrition centered on the hydrolysis of a human milk oligosaccharide. The results revealed responses relating to metabolism occurring in a Bifidobacterium bifidum strain when it provided nutrients that allowed the growth of Bifidobacterium breve, and so discovered biochemical features of these bifidobacteria in relation to metabolic interaction in the shared environment. These kinds of experiments are essential in developing concepts of bifidobacterial ecology that relate to the development of the gut microbiota in early life.