| 1. |
Requena T,
Burton J,
Matsuki T,
Munro K,
Simon MA,
Tanaka R,
Watanabe K,
Tannock GW,
( 2002 ) Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene. PMID : 11976117 : DOI : 10.1128/aem.68.5.2420-2427.2002 PMC : PMC127544 Abstract >>
Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.
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2. |
Jian W,
Zhu L,
Dong X,
( 2001 ) New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences. PMID : 11594590 : DOI : 10.1099/00207713-51-5-1633 Abstract >>
The partial 60 kDa heat-shock protein (HSP60) genes of 36 Bifidobacterium strains representing 30 different Bifidobacterium species and subspecies and of the type strain of Gardnerella vaginalis were cloned and sequenced using a pair of universal degenerate HSP60 PCR primers. The HSP60 DNA sequence similarities were determined for the taxa at various ranks as follows: 99.4-100% within the same species, 96% at the subspecies level, and 73-96% (mean 85%) at the interspecies level (and 98% in the case of two groups of closely related species, Bifidobacterium animalis and Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis, whose 165 rRNA sequence similarities are all above 99%). The HSP60 DNA sequence similarities between different Bifidobacterium species and G. vaginalis, a closely related bacterium according to 16S rRNA analysis, ranged from 71 to 79% (mean 75%). Although the topology of the phylogenetic tree constructed using the HSP60 sequences determined was basically similar to that for 16S rRNA, it seemed to be more clear-cut for species delineation, and the clustering was better correlated with the DNA base composition (mol% G+C) than that of the 16S rRNA tree. In the HSP60 phylogenetic tree, all of the high-G+C (55-67 mol%) bifidobacteria were grouped into one cluster, whereas the low-G+C species Bifidobacterium inopinatum (45 mol %) formed a separate cluster with G. vaginalis (42 mol%) and Bifidobacterium denticolens (55 mol%); a Bifidobacterium species of intermediate G+C content formed another cluster between the two. This study demonstrates that the highly conserved and ubiquitous HSP60 gene is an accurate and convenient tool for phylogenetic analysis of the genus Bifidobacterium.
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3. |
Nagae M,
Tsuchiya A,
Katayama T,
Yamamoto K,
Wakatsuki S,
Kato R,
( 2007 ) Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-L-fucosidase from Bifidobacterium bifidum. PMID : 17459873 : DOI : 10.1074/jbc.M702246200 Abstract >>
1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.
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4. |
Aires J,
Doucet-Populaire F,
Butel MJ,
( 2007 ) Tetracycline resistance mediated by tet(W), tet(M), and tet(O) genes of Bifidobacterium isolates from humans. PMID : 17308188 : DOI : 10.1128/AEM.02459-06 PMC : PMC1855585 Abstract >>
MICs of tetracyclines were determined for 86 human Bifidobacterium isolates and three environmental strains. The tet(O) gene was found to be absent in these isolates. tet(W) and tet(M) were found in 26 and 7%, respectively, of the Bifidobacterium isolates, and one isolate contained both genes. Chromosomal DNA hybridization showed that there was one chromosomal copy of tet(W) and/or tet(M).
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5. |
Gueimonde M,
Noriega L,
Margolles A,
de los Reyes-Gavilán CG,
( 2007 ) Induction of alpha-L-arabinofuranosidase activity by monomeric carbohydrates in Bifidobacterium longum and ubiquity of encoding genes. PMID : 17031615 : DOI : 10.1007/s00203-006-0181-x Abstract >>
Bifidobacterium longum can be isolated from human faeces, some strains being considered probiotics. B. longum NIZO B667 produces an exo-acting alpha-L-arabinofuranosidase, AbfB, previously purified by us, that releases L-arabinose from arabinan and arabinoxylan. This activity was subjected to two-seven-fold induction by L-arabinose, D-xylose, L-arabitol and xylitol and to repression by glucose. Maximum activity was obtained at 48 h incubation except for D-xylose that was at 24 h. High concentrations (200 mM) of L-arabitol also caused repression of the arabinofuranosidase. A unique band of activity showing the same migration pattern as the purified AbfB was found in zymograms of cell free extracts, indicating that the activity was likely due to this sole enzyme. The assessment of the influence of inducers and repressors on the activity of AbfB and on the expression of the abfB gene by real time PCR indicated that regulation was transcriptional. DNA amplifications using a pair of degenerated primers flanking an internal fragment within alpha-L-arabinofuranosidase genes of the family 51 of glycoside hydrolases evidenced that these enzymes are widespread in Bifidobacterium. The aminoacidic sequences of bifidobacteria included a fragment of four to six residues in the position 136-141 that was absent in other microorganisms.
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6. |
Vaugien L,
Prevots F,
Roques C,
( 2002 ) Bifidobacteria identification based on 16S rRNA and pyruvate kinase partial gene sequence analysis. PMID : 16887679 : DOI : 10.1016/S1075-9964(03)00025-8 Abstract >>
The lack of a simple and rapid identification system for Bifidobacterium species makes them difficult to use in industrial applications. To obtain valuable discriminating factor, we studied different strains, and human isolates by two molecular taxonomy methods. First method was based on chrono-differentiation. A metabolic gene (pyruvate kinase) was chosen to be used as a systematic discriminating factor. A comparison of about 40 pyruvate kinase protein sequences allowed us to synthesize two oligonucleotides that were able to amplify a fragment of this corresponding gene in our strains. Based on these partial pyruvate kinase gene sequences, several clusters could be identified. The second method used in this study was based on 16S rRNA sequences analysis. We compared sequences present in GenBank database, and this allowed to separate bifidobacteria species into different clusters. They were different from those obtained with partial pyruvate kinase gene sequences analysis. So, by combining both methods, we were able to identify our isolates, when only 10% of them could be strictly identified using the 16S rRNA method. Moreover, pyruvate kinase analysis allowed to differentiate very ambivalent groups such as B. animalis/B. lactis or B. infantis/B. longum, but created different clusters for B. infantis species group, questioning on the homogeneity of this species.
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7. |
Yamashita T,
Ashiuchi M,
Ohnishi K,
Kato S,
Nagata S,
Misono H,
( 2004 ) Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum. PMID : 15606767 : DOI : 10.1111/j.1432-1033.2004.04445.x Abstract >>
Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.
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8. |
Ventura M,
Zink R,
Fitzgerald GF,
van Sinderen D,
( 2005 ) Gene structure and transcriptional organization of the dnaK operon of Bifidobacterium breve UCC 2003 and application of the operon in bifidobacterial tracing. PMID : 15640225 : DOI : 10.1128/AEM.71.1.487-500.2005 PMC : PMC544267 Abstract >>
The incorporation and delivery of bifidobacterial strains as probiotic components in many food preparations expose these microorganisms to a multitude of environmental insults, including heat and osmotic stresses. We characterized the dnaK gene region of Bifidobacterium breve UCC 2003. Sequence analysis of the dnaK locus revealed four genes with the organization dnaK-grpE-dnaJ-ORF1, whose deduced protein products display significant similarity to corresponding chaperones found in other bacteria. Northern hybridization and real-time LightCycler PCR analysis revealed that the transcription of the dnaK operon was strongly induced by osmotic shock but was not induced significantly by heat stress. A 4.4-kb polycistronic mRNA, which represented the transcript of the complete dnaK gene region, was detected. Many other small transcripts, which were assumed to have resulted from intensive processing or degradation of this polycistronic mRNA, were identified. The transcription start site of the dnaK operon was determined by primer extension. Phylogenetic analysis of the available bifidobacterial grpE and dnaK genes suggested that the evolutionary development of these genes has been similar. The phylogeny derived from the various bifidobacterial grpE and dnaK sequences is consistent with that derived from 16S rRNA. The use of these genes in bifidobacterial species as an alternative or complement to the 16S rRNA gene marker provides sequence signatures that allow a high level of discrimination between closely related species of this genus.
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9. |
Kim GB,
Miyamoto CM,
Meighen EA,
Lee BH,
( 2004 ) Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains. PMID : 15345449 : DOI : 10.1128/AEM.70.9.5603-5612.2004 PMC : PMC520925 Abstract >>
Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.
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10. |
Ventura M,
Canchaya C,
Zink R,
Fitzgerald GF,
van Sinderen D,
( 2004 ) Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses. PMID : 15466567 : DOI : 10.1128/AEM.70.10.6197-6209.2004 PMC : PMC522111 Abstract >>
The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes, including the GroEL and GroES proteins. The groES and groEL genes are highly conserved among eubacteria and are typically arranged as an operon. Genome analysis of Bifidobacterium breve UCC 2003 revealed that the groES and groEL genes are located in different chromosomal regions. The heat inducibility of the groEL and groES genes of B. breve UCC 2003 was verified by slot blot analysis. Northern blot analyses showed that the cspA gene is cotranscribed with the groEL gene, while the groES gene is transcribed as a monocistronic unit. The transcription initiation sites of these two mRNAs were determined by primer extension. Sequence and transcriptional analyses of the region flanking the groEL and groES genes of various bifidobacteria revealed similar groEL-cspA and groES gene units, suggesting a novel genetic organization of these chaperones. Phylogenetic analysis of the available bifidobacterial groES and groEL genes suggested that these genes evolved differently. Discrepancies in the phylogenetic positioning of groES-based trees make this gene an unreliable molecular marker. On the other hand, the bifidobacterial groEL gene sequences can be used as an alternative to current methods for tracing Bifidobacterium species, particularly because they allow a high level of discrimination between closely related species of this genus.
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11. |
Katayama T,
Sakuma A,
Kimura T,
Makimura Y,
Hiratake J,
Sakata K,
Yamanoi T,
Kumagai H,
Yamamoto K,
( 2004 ) Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). PMID : 15262925 : DOI : 10.1128/JB.186.15.4885-4893.2004 PMC : PMC451662 Abstract >>
A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the alpha-(1-->2) linkage of 2'-fucosyllactose, and a gene encoding 1,2-alpha-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2'-fucosyllactose was determined to be inversion by using (1)H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).
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12. |
Ventura M,
Canchaya C,
van Sinderen D,
Fitzgerald GF,
Zink R,
( 2004 ) Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny. PMID : 15128574 : DOI : 10.1128/aem.70.5.3110-3121.2004 PMC : PMC404453 Abstract >>
The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.
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13. |
Ventura M,
Canchaya C,
Meylan V,
Klaenhammer TR,
Zink R,
( 2003 ) Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification. PMID : 14602655 : DOI : 10.1128/aem.69.11.6908-6922.2003 PMC : PMC262312 Abstract >>
We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.
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14. |
Asakuma S,
Hatakeyama E,
Urashima T,
Yoshida E,
Katayama T,
Yamamoto K,
Kumagai H,
Ashida H,
Hirose J,
Kitaoka M,
( 2011 ) Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria. PMID : 21832085 : DOI : 10.1074/jbc.M111.248138 PMC : PMC3186357 Abstract >>
The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.
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15. |
Kiyohara M,
Tanigawa K,
Chaiwangsri T,
Katayama T,
Ashida H,
Yamamoto K,
( 2011 ) An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates. PMID : 21036948 : DOI : 10.1093/glycob/cwq175 Abstract >>
Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different �\-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-�\-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-�\-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 �XC. Notably, 80% activity remained after 30 min incubation at 80 �XC, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the �\2,3-linkage rather than �\2,6- and �\2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-�\-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.
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16. |
Miwa M,
Horimoto T,
Kiyohara M,
Katayama T,
Kitaoka M,
Ashida H,
Yamamoto K,
( 2010 ) Cooperation of �]-galactosidase and �]-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure. PMID : 20581010 : DOI : 10.1093/glycob/cwq101 Abstract >>
Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Gal�]1-3GlcNAc�]1-3Gal�]1-4Glc; type 1 chain) and lacto-N-neotetraose (Gal�]1-4GlcNAc�]1-3Gal�]1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one �]-galactosidase (BbgIII) and two �]-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto-N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs.
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17. |
Cazzola M,
Tompkins TA,
Matera MG,
( 2010 ) Immunomodulatory impact of a synbiotic in T(h)1 and T(h)2 models of infection. PMID : 20929951 : DOI : 10.1177/1753465810379009 Abstract >>
The immunomodulatory activity of a synbiotic combination containing three bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium bifidum R0071) and short-chain fructooligosaccharide was examined in two distinct infectious rat models. In the T(h)1 model, Wistar rats were administered the synbiotic combination for 2 weeks prior to challenge with a single oral dose of enterotoxigenic Escherichia coli or vehicle. In the T(h)2 model, pretreated rats were challenged with a single subcutaneous dose of hook worm, Nippostrongylus brasiliensis. Blood samples were collected 3 hours or 4 days postchallenge and serum levels of pro- and anti-inflammatory cytokines were measured. Significant reductions in pro-inflammatory cytokines interleukin (IL)-1�\, IL-1�], IL-6, and tumour necrosis factor (TNF)-�\ were observed in both models suggesting a single, unifying mode of action on an upstream regulator. The N. brasiliensis study also compared the effect of the individual strains to synbiotic. For most of cytokines the combination appeared to average the effect of the individual strains with the exception of IL-4 and IL-10 where there was apparent synergy for the combination. Furthermore, the cytokine response varied by strain. It was concluded that this synbiotic combination of these three microbes could be beneficial in both T(h)1 and T(h)2 diseases.
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18. |
Kim BJ,
Kim HY,
Yun YJ,
Kim BJ,
Kook YH,
( 2010 ) Differentiation of Bifidobacterium species using partial RNA polymerase {beta}-subunit (rpoB) gene sequences. PMID : 20061504 : DOI : 10.1099/ijs.0.020339-0 Abstract >>
Partial RNA polymerase �]-subunit gene (rpoB) sequences (315 bp) were determined and used to differentiate the type strains of 23 species of the genus Bifidobacterium. The sequences were compared with those of the partial hsp60 (604 bp) and 16S rRNA genes (1475 or 1495 bp). The rpoB gene sequences showed nucleotide sequence similarities ranging from 84.1 % to 99.0 %, while the similarities of the hsp60 sequences ranged from 78.5 % to 99.7 % and the 16S rRNA gene sequence similarities ranged from 89.4 % to 99.2 %. The phylogenetic trees constructed from the sequences of these three genes showed similar clustering patterns, with the exception of several species. The Bifidobacterium catenulatum-Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum subsp. pseudolongum-Bifidobacterium pseudolongum subsp. globosum and Bifidobacterium gallinarum-Bifidobacterium pullorum-Bifidobacterium saeculare groups were more clearly differentiated in the partial rpoB and hsp60 gene sequence trees than they were in the 16S rRNA gene tree. Based on sequence similarities and tree topologies, the newly determined rpoB gene sequences are suitable molecular markers for the differentiation of species of the genus Bifidobacterium and support various other molecular tools used to determine the relationships among species of this genus.
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19. |
Sato T,
Iino T,
( 2010 ) Genetic analyses of the antibiotic resistance of Bifidobacterium bifidum strain Yakult YIT 4007. PMID : 20051305 : DOI : 10.1016/j.ijfoodmicro.2009.12.014 Abstract >>
Bifidobacterium bifidum strain Yakult YIT 4007 (abbreviated as B. bifidum YIT 4007) is a commercial strain and resistant to erythromycin, neomycin, and streptomycin. Resistances to these antibiotics were endowed by sequential isolation of resistant mutants from its susceptible progenitor strain YIT 4001. Comparison of nucleotide sequences of various candidate genes of both strains led us to find that B. bifidum YIT 4007 had mutations on three copies of 23S ribosomal RNA genes, an 8 bp deletion of the rluD gene for pseudouridine synthase, and a mutation on the rpsL gene for ribosomal protein S12. The responsibility of these mutations to antibiotic resistances was supported by analyses of newly isolated mutants resistant to these antibiotics. The antibiotic resistances of B. bifidum YIT 4007 were evidently acquired by mutations of the structural genes on the chromosome and not associated with mobile genetic elements like insertion sequences, phages, and plasmids.
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20. |
Ashida H,
Miyake A,
Kiyohara M,
Wada J,
Yoshida E,
Kumagai H,
Katayama T,
Yamamoto K,
( 2009 ) Two distinct alpha-L-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates. PMID : 19520709 : DOI : 10.1093/glycob/cwp082 Abstract >>
Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.
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21. |
Shkoporov AN,
Efimov BA,
Khokhlova EV,
Steele JL,
Kafarskaia LI,
Smeianov VV,
( 2008 ) Characterization of plasmids from human infant Bifidobacterium strains: sequence analysis and construction of E. coli-Bifidobacterium shuttle vectors. PMID : 18652842 : DOI : 10.1016/j.plasmid.2008.06.005 Abstract >>
A survey of infant fecal Bifidobacterium isolates for plasmid DNA revealed that a significant portion of the strains, 17.6%, carry small plasmids. The majority of plasmid-harboring strains belonged to the Bifidobacterium longum/infantis group. Most of the plasmids could be assigned into two groups based on their sizes and the restriction profiles. Three plasmids, pB44 (3.6 kb) from B. longum, pB80 (4.9 kb) from Bifidobacterium bifidum, and pB21a (5.2kb) from Bifidobacterium breve were sequenced. While the former two plasmids were found to be highly similar to previously characterized rolling-circle replicating pKJ36 and pKJ56, respectively, the third plasmid, pB21a, does not share significant nucleotide homology with known plasmids. However, it might be placed into the pCIBb1-like group of bifidobacterial rolling-plasmids based on the homology of its Rep protein and the overall molecular organization. Two sets of Escherichia coli-Bifidobacterium shuttle vectors constructed based on pB44 and pB80 replicons were capable of transforming B. bifidum and B. breve strains with efficiency up to 3x10(4)cfu/microg DNA. Additionally, an attempt was made to employ a broad host range conjugation element, RP4, in developing of E. coli-Bifidobacterium gene transfer system.
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22. |
Kawasaki S,
Satoh T,
Todoroki M,
Niimura Y,
( 2009 ) b-type dihydroorotate dehydrogenase is purified as a H2O2-forming NADH oxidase from Bifidobacterium bifidum. PMID : 19060157 : DOI : 10.1128/AEM.02111-08 PMC : PMC2632149 Abstract >>
Our previous report showed the existence of microaerophilic Bifidobacterium species that can grow well under aerobic conditions rather than anoxic conditions in a liquid shaking culture. The difference in the aerobic growth properties between the O(2)-sensitive and microaerophilic species is due to the existence of a system to produce H(2)O(2) in the growth medium. In this study, we purified and characterized the NADH oxidase that is considered to be a key enzyme in the production of H(2)O(2). Bifidobacterium bifidum, an O(2)-sensitive bacterium and the type species of the genus Bifidobacterium, possessed one dominant active fraction of NADH oxidase and a minor active fraction of NAD(P)H oxidase activity detected in the first step of column chromatography for purification of the enzyme. The dominant active fraction was further purified and determined from its N-terminal sequence to be a homologue of b-type dihydroorotate dehydrogenase (DHOD), composed of PyrK (31 kDa) and PyrDb (34 kDa) subunits. The genes that encode PyrK and PryDb are tandemly located within an operon structure. The purified enzyme was found to be a heterotetramer showing the typical spectrum of a flavoprotein, and flavin mononucleotide and flavin adenine dinucleotide were identified as cofactors. The purified enzyme was characterized as the enzyme that catalyzes the DHOD reaction and also catalyzes a H(2)O(2)-forming NADH oxidase reaction in the presence of O(2). The kinetic parameters suggested that the enzyme could be involved in H(2)O(2) production in highly aerated environments.
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23. |
Ammor MS,
Flórez AB,
Alvarez-Martín P,
Margolles A,
Mayo B,
( 2008 ) Analysis of tetracycline resistance tet(W) genes and their flanking sequences in intestinal Bifidobacterium species. PMID : 18614524 : DOI : 10.1093/jac/dkn280 Abstract >>
The tet(W) gene provides tetracycline resistance to a wide range of anaerobic intestinal and ruminal bacteria, but little is known about the molecular organization of the tet(W) gene. The aim of this study was to gain new insights into the molecular organization of the tet(W) gene in bifidobacteria strains from humans. A segment of DNA encompassing the whole tet(W) gene and its immediate upstream and downstream sequences was analysed in 10 representative strains of four Bifidobacterium species, of which two have been shown to be tetracycline-susceptible. The non-conserved flanking regions of the tet(W) gene were further analysed in six strains. All 10 strains share a core DNA domain of 2154 bp [starting 250 bp upstream of the tet(W) gene start codon and ending 13 bp before the stop codon] with 98% to 100% DNA identity. Except for Bifidobacterium animalis E43, all other strains further share 408 bp upstream and 70 bp downstream of the tet(W) gene. An insertion-like element of 736 bp was found to interrupt the tet(W) coding sequence in Bifidobacterium longum M21, which may be the reason for its tetracycline susceptibility. However, genetic events explaining the susceptible phenotype of B. longum LMG 13197(T) were not observed. The tet(W) genes from all 10 strains shared 98% to 100% DNA and amino acid identity, though large variation was found in their flanking regions.
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24. |
Guglielmetti S,
Tamagnini I,
Mora D,
Minuzzo M,
Scarafoni A,
Arioli S,
Hellman J,
Karp M,
Parini C,
( 2008 ) Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells. PMID : 18539800 : DOI : 10.1128/AEM.00124-08 PMC : PMC2519326 Abstract >>
We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.
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25. |
Ruas-Madiedo P,
Gueimonde M,
Fernández-García M,
de los Reyes-Gavilán CG,
Margolles A,
( 2008 ) Mucin degradation by Bifidobacterium strains isolated from the human intestinal microbiota. PMID : 18223105 : DOI : 10.1128/AEM.02509-07 PMC : PMC2268317 Abstract >>
The presence of the genes engBF (endo-alpha-N-acetylgalactosaminidase) and afcA (1,2-alpha-L-fucosidase) was detected in several intestinal Bifidobacterium isolates. Two strains of Bifidobacterium bifidum contained both genes, and they were able to degrade high-molecular weight porcine mucin in vitro. The expression of both genes was highly induced in the presence of mucin.
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26. |
Nishimoto M,
Kitaoka M,
( 2007 ) Identification of the putative proton donor residue of lacto-N-biose phosphorylase (EC 2.4.1.211). PMID : 17587697 : DOI : 10.1271/bbb.70064 Abstract >>
Two lacto-N-biose phosphorylase (LNBP) isozyme genes were cloned from Bifidobacterium bifidum JCM1254. Alignment of the amino acid sequences of LNBP and its homologs identified 24 completely conserved acidic amino acid residues. All single mutants of Bifidobacterium longum LNBP at residues other than D313N retained considerable activity, suggesting that Asp313 is the putative proton donor residue in LNBP.
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27. |
Duranti S,
Milani C,
Lugli GA,
Turroni F,
Mancabelli L,
Sanchez B,
Ferrario C,
Viappiani A,
Mangifesta M,
Mancino W,
Gueimonde M,
Margolles A,
van Sinderen D,
Ventura M,
( 2015 ) Insights from genomes of representatives of the human gut commensal Bifidobacterium bifidum. PMID : 25523018 : DOI : 10.1111/1462-2920.12743 Abstract >>
Bifidobacteria are bacterial gut commensals of mammals, birds and social insects that are perceived to influence the metabolism/physiology of their host. In this context, members of the Bifidobacterium bifidum species are believed to significantly contribute to the overall microbiota of the human gut at infant stage. However, the molecular reasons for their adaptation to this environment are poorly understood. In this study, we analysed the pan-genome of B. bifidum species by decoding genomes of 15 B. bifidum strains, which highlighted the existence of a conserved gene uniquely present in this bifidobacterial taxon, underscoring a nutrient acquisition strategy that targets host-derived glycans, such as those present in mucin. Growth experiments and corresponding transcriptomic analyses confirmed the in silico data and supported these intriguing and unique host glycan-specific saccharolytic features. The ubiquity of the genetic features of B. bifidum for the breakdown of host glycans was confirmed by interrogating metagenomic datasets, thereby supporting the notion that metabolic access to host-derived glycans is a potent evolutionary force that has shaped B. bifidum genomes and consequently the ecology of the infant intestinal microbiota.
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28. |
De Bruyn F,
Beauprez J,
Maertens J,
Soetaert W,
De Mey M,
( 2013 ) Unraveling the Leloir pathway of Bifidobacterium bifidum: significance of the uridylyltransferases. PMID : 24014529 : DOI : 10.1128/AEM.02460-13 PMC : PMC3811521 Abstract >>
The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.
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29. |
( 1997 ) Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine. PMID : 9311137 : DOI : 10.1111/j.1574-6968.1997.tb12670.x Abstract >>
The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.
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30. |
Sato M,
Liebschner D,
Yamada Y,
Matsugaki N,
Arakawa T,
Wills SS,
Hattie M,
Stubbs KA,
Ito T,
Senda T,
Ashida H,
Fushinobu S,
( 2017 ) The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors. PMID : 28546425 : DOI : 10.1074/jbc.M117.777391 PMC : PMC5519364 Abstract >>
The �\-N-acetylgalactosaminidase from the probiotic bacterium Bifidobacterium bifidum (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAc�\1-Ser/Thr). NagBb is involved in assimilation of O-glycans on mucin glycoproteins by B. bifidum in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca2+, is involved in the recognition of the GalNAc N-acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-�\-N-acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.
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31. |
( 2013 ) Genetic diversity of bile salt hydrolases among human intestinal bifidobacteria. PMID : 23591474 : DOI : 10.1007/s00284-013-0362-1 PMC : PMC3722454 Abstract >>
This study analyzes the application of degenerative primers for the screening of bile salt hydrolase-encoding genes (bsh) in various intestinal bifidobacteria. In the first stage, the design and evaluation of the universal PCR primers for amplifying the partial coding sequence of bile salt hydrolase in bifidobacteria were performed. The amplified bsh gene fragments were sequenced and the obtained sequences were compared to the bsh genes present in GenBank. The determined results showed the utility of the designed PCR primers for the amplification of partial gene encoding bile salt hydrolase in different intestinal bifidobacteria. Moreover, sequence analysis revealed that bile salt hydrolase-encoding genes may be used as valuable molecular markers for phylogenetic studies and identification of even closely related members of the genus Bifidobacterium.
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32. |
Wang N,
Hang X,
Zhang M,
Liu X,
Yang H,
( 2017 ) Analysis of newly detected tetracycline resistance genes and their flanking sequences in human intestinal bifidobacteria. PMID : 28740169 : DOI : 10.1038/s41598-017-06595-0 PMC : PMC5524971 Abstract >>
Due to tetracycline abuse, the safe bifidobacteria in the human gastrointestinal intestinal tract (GIT) may serve as a reservoir of tetracycline resistance genes. In the present investigation of 92 bifidobacterial strains originating from the human GIT, tetracycline resistance in 29 strains was mediated by the tet(W), tet(O) or tet(S) gene, and this is the first report of tet(O)- and tet(S)-mediated tetracycline resistance in bifidobacteria. Antibiotic resistance genes harbored by bifidobacteria are transferred from other bacteria. However, the characteristics of the spread and integration of tetracycline resistance genes into the human intestinal bifidobacteria chromosome are poorly understood. Here, conserved sequences were identified in bifidobacterial strains positive for tet(W), tet(O), or tet(S), including the tet(W), tet(O), or tet(S) and their partial flanking sequences, which exhibited identity with the sequences in multiple human intestinal pathogens, and genes encoding 23 S rRNA, an ATP transporter, a Cpp protein, and a membrane-spanning protein were flanking by the 1920-bp tet(W), 1920-bp tet(O), 1800-bp tet(O) and 252-bp tet(S) in bifidobacteria, respectively. These findings suggest that tetracycline resistance genes harbored by human intestinal bifidobacteria might initially be transferred from pathogens and that each kind of tetracycline resistance gene might tend to insert in the vicinity of specific bifidobacteria genes.
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33. |
( 2013 ) Bifidobacterial succession and correlation networks in a large unselected cohort of mothers and their children. PMID : 23124244 : DOI : 10.1128/AEM.02359-12 PMC : PMC3553782 Abstract >>
Bifidobacteria are a major microbial component of infant gut microbiota, which is believed to promote health benefits for the host and stimulate maturation of the immune system. Despite their perceived importance, very little is known about the natural development of and possible correlations between bifidobacteria in human populations. To address this knowledge gap, we analyzed stool samples from a randomly selected healthy cohort of 87 infants and their mothers with >90% of vaginal delivery and nearly 100% breast-feeding at 4 months. Fecal material was sampled during pregnancy, at 3 and 10 days, at 4 months, and at 1 and 2 years after birth. Stool samples were predicted to be rich in the species Bifidobacterium adolescentis, B. bifidum, B. dentium, B. breve, and B. longum. Due to high variation, we did not identify a clear age-related structure at the individual level. Within the population as a whole, however, there were clear age-related successions. Negative correlations between the B. longum group and B. adolescentis were detected in adults and in 1- and 2-year-old children, whereas negative correlations between B. longum and B. breve were characteristic for newborns and 4-month-old infants. The highly structured age-related development of and correlation networks between bifidobacterial species during the first 2 years of life mirrors their different or competing nutritional requirements, which in turn may be associated with specific biological functions in the development of healthy gut.
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34. |
( 2013 ) Bifidobacterial �\-galactosidase with unique carbohydrate-binding module specifically acts on blood group B antigen. PMID : 23089618 : DOI : 10.1093/glycob/cws142 Abstract >>
Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O-linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different �]-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology. 22:361-368). The nonreducing termini of O-linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 �\-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed �\1,3-linked Gal in branched blood group B antigen [Gal�\1-3(Fuc�\1-2)Gal�]1-R], but not in a linear xenotransplantation antigen (Gal�\1-3Gal�]1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.
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