| 1. |
Kim M,
Kwon T,
Lee HJ,
Kim KH,
Chung DK,
Ji GE,
Byeon ES,
Lee JH,
( 2003 ) Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme. PMID : 14514069 : Abstract >>
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-alpha-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.
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2. |
Margolles A,
de los Reyes-Gavilán CG,
( 2003 ) Purification and functional characterization of a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667. PMID : 12957891 : DOI : 10.1128/aem.69.9.5096-5103.2003 PMC : PMC194971 Abstract >>
The gene encoding a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70 degrees C), and it had an optimum pH of 6.0 and an optimum temperature of 45 degrees C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55 degrees C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu(2+), Hg(2+), and Zn(2+) markedly reduced enzymatic activity. The protein exhibited a high level of activity with p-nitrophenyl alpha-L-arabinofuranoside, with apparent K(m) and V(max) values of 0.295 mM and 417 U/mg, respectively. AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides.
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3. |
Ruas-Madiedo P,
Moreno JA,
Salazar N,
Delgado S,
Mayo B,
Margolles A,
de Los Reyes-Gavilán CG,
( 2007 ) Screening of exopolysaccharide-producing Lactobacillus and Bifidobacterium strains isolated from the human intestinal microbiota. PMID : 17483284 : DOI : 10.1128/AEM.02470-06 PMC : PMC1932768 Abstract >>
Using phenotypic approaches, we have detected that 17% of human intestinal Lactobacillus and Bifidobacterium strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains.
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4. |
Sánchez B,
Champomier-Vergès MC,
Collado Mdel C,
Anglade P,
Baraige F,
Sanz Y,
de los Reyes-Gavilán CG,
Margolles A,
Zagorec M,
( 2007 ) Low-pH adaptation and the acid tolerance response of Bifidobacterium longum biotype longum. PMID : 17720838 : DOI : 10.1128/AEM.00886-07 PMC : PMC2075061 Abstract >>
Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum biotype longum NCIMB 8809 and its acid-pH-resistant mutant B. longum biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pH-resistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in B. longum biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.
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5. |
Nishimoto M,
Kitaoka M,
( 2007 ) Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk. PMID : 17690443 : Abstract >>
A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500 mM of LNB was generated in 10-liter of reaction mixture initially containing 660 mM of sucrose and 600 mM of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose-hexose-1-phospate uridylyltransferase, UDP-glucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.
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6. |
Guglielmetti S,
Karp M,
Mora D,
Tamagnini I,
Parini C,
( 2007 ) Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system. PMID : 17151871 : DOI : 10.1007/s00253-006-0755-1 Abstract >>
In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids.
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7. |
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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8. |
Flórez AB,
Ammor MS,
Alvarez-Martín P,
Margolles A,
Mayo B,
( 2006 ) Molecular analysis of tet(W) gene-mediated tetracycline resistance in dominant intestinal Bifidobacterium species from healthy humans. PMID : 16936047 : DOI : 10.1128/AEM.00486-06 PMC : PMC1636146 Abstract >>
tet(W) was found responsible for tetracycline resistance (MICs, 4 to > or =32 microg ml(-1)) in dominant bifidobacterial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66 proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio fibrisolvens.
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9. |
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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10. |
Kazimierczak KA,
Flint HJ,
Scott KP,
( 2006 ) Comparative analysis of sequences flanking tet(W) resistance genes in multiple species of gut bacteria. PMID : 16870752 : DOI : 10.1128/AAC.01587-05 PMC : PMC1538676 Abstract >>
tet(W) is one of the most abundant tetracycline resistance genes found in bacteria from the mammalian gut and was first identified in the rumen anaerobe Butyrivibrio fibrisolvens 1.230, where it is highly mobile and its transfer is associated with the transposable chromosomal element TnB1230. In order to compare the genetic basis for tet(W) carriage in different bacteria, we studied sequences flanking tet(W) in representatives of seven bacterial genera originating in diverse gut environments. The sequences 657 bp upstream and 43 bp downstream of tet(W) were 96 to 100% similar in all strains examined. A common open reading frame (ORF) was identified downstream of tet(W) in five different bacteria, while another conserved ORF that flanked tet(W) in B. fibrisolvens 1.230 was also present upstream of tet(W) in a human colonic Roseburia isolate and in another rumen B. fibrisolvens isolate. In one species, Bifidobacterium longum (strain F8), a novel transposase was located within the conserved 657-bp region upstream of tet(W) and was flanked by imperfect direct repeats. Additional direct repeats 6 bp long were identified on each end of a chromosomal ORF interrupted by the insertion of the putative transposase and the tet(W) gene. This tet(W) gene was transferable at low frequencies between Bifidobacterium strains. A putative minielement carrying a copy of tet(W) was identified in B. fibrisolvens transconjugants that had acquired the tet(W) gene on TnB1230. Several different mechanisms, including mechanisms involving plasmids and conjugative transposons, appear to be involved in the horizontal transfer of tet(W) genes, but small core regions that may function as minielements are conserved.
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11. |
Sridhar VR,
Smeianov VV,
Steele JL,
( 2006 ) Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and alpha-galactosidase as selectable markers. PMID : 16834603 : DOI : 10.1111/j.1365-2672.2006.02898.x Abstract >>
We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumalpha-galactosidase gene (aglL) as selectable markers. The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC-), the resulting vector, pSUW611 (3.9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0.2% loss per generation). The second vector, pSUW711 (5.1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48-72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis.
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12. |
Price CE,
Reid SJ,
Driessen AJ,
Abratt VR,
( 2006 ) The Bifidobacterium longum NCIMB 702259T ctr gene codes for a novel cholate transporter. PMID : 16391136 : DOI : 10.1128/AEM.72.1.923-926.2006 PMC : PMC1352250 Abstract >>
Preexposure of Bifidobacterium longum NCIMB 702259T to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [14C]cholate.
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13. |
Yin X,
Chambers JR,
Barlow K,
Park AS,
Wheatcroft R,
( 2005 ) The gene encoding xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp) is conserved among Bifidobacterium species within a more variable region of the genome and both are useful for strain identification. PMID : 15899413 : DOI : 10.1016/j.femsle.2005.04.013 Abstract >>
The nucleotide sequence of the xfp-gene region in six known and two unknown species of Bifidobacterium was determined and compared with the published sequences of B. animalis subsp. lactis DSM10140 and B. longum biovar longum NCC2705. The xfp coding sequences were 73% identical and coded for 825 amino acids in all 10 sequences. Partial sequences of an adjacent gene, guaA, were 61% identical in six sequences for which data were available. The region between xfp and guaA was variable in both length and sequence. Oligonucleotide sequences from the conserved and variable xfp regions were used as PCR primers, in combinations of appropriate specificity, for the detection and identification of Bifidobacterium isolates.
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14. |
Tanaka K,
Samura K,
Kano Y,
( 2005 ) Structural and functional analysis of pTB6 from Bifidobacterium longum. PMID : 15725673 : DOI : 10.1271/bbb.69.422 Abstract >>
The complete nucleotide sequence for pTB6 (3,624 bp) from Bifidobacterium longum was determined. This plasmid is 95% homologous in nucleotide (nuc) sequence, and also 92% in RepB aa sequence, to rolling circle replication (RCR) plasmids pKJ36 and pB44, suggesting that pTB6 replicates by the rolling circle mechanism. The putative MembB, MobA, and protein encoding from orf (Orf) I detected were nonessential for plasmid replication. We constructed an immobile shuttle vector from pTB6 and pUC18, which transformed B. longum with a high efficiency of 2.5 x 10(6) transformants/microg DNA.
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15. |
Corneau N,
Emond E,
LaPointe G,
( 2004 ) Molecular characterization of three plasmids from Bifidobacterium longum. PMID : 15003705 : DOI : 10.1016/j.plasmid.2003.12.003 Abstract >>
The complete nucleotide sequences for pNAC1 (3538bp) from strain RW048 as well as for pNAC2 (3684bp) and pNAC3 (10,224bp) from strain RW041 of Bifidobacterium longum were determined. The largest ORF (repB) of pNAC1 encodes a putative protein similar to those involved in a rolling-circle (RC) replication mechanism, which was confirmed by demonstration of single-strand intermediates in the host cell. The putative RepB gene product of pNAC2 is most similar to the replication protein of pDOJH10L and pKJ36. A second gene (mob) is similar to mobilization proteins involved in conjugation. Plasmid pNAC3 is the largest bifidobacterial plasmid to be sequenced to date. Of the eight putative gene products coded by pNAC3, one is similar to replication proteins (RepB), and another (Orf2) to putative transfer proteins (Tra). Bifidobacterial plasmids were divided into five groups based on Rep amino acid sequence homology and the results suggest a new plasmid family for B. longum.
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16. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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17. |
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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18. |
Lee JH,
Hyun YJ,
Kim DH,
( 2011 ) Cloning and characterization of �\-L-arabinofuranosidase and bifunctional �\-L-arabinopyranosidase/�]-D-galactopyranosidase from Bifidobacterium longum H-1. PMID : 21851513 : DOI : 10.1111/j.1365-2672.2011.05128.x Abstract >>
This study focused on the cloning, expression and characterization of recombinant �\-l-arabinosidases from Bifidobacterium longum H-1. �\-l-Arabinofuranosidase (AfuB-H1) and bifunctional �\-l-arabinopyranosidase/�]-d-galactosidase (Apy-H1) from B. longum H-1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB-H1 (rAfuB-H1) was purified by single-step Ni(2+) -affinity column chromatography, whereas recombinant Apy-H1 (rApy-H1) was purified by serial Q-HP and Ni(2+) -affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB-H1 hydrolysed p-nitrophenyl-�\-l-arabinofuranoside (pNP-�\L-Af) and ginsenoside Rc, but did not hydrolyse p-nitrophenyl-�\-l-arabinopyranoside (pNP-�\L-Ap). On the other hand, rApy-H1 hydrolysed pNP-�\L-Ap, p-nitrophenyl-�]-d-galactopyranoside (pNP-�]D-Ga) and ginsenoside Rb2. Ginsenoside-metabolizing bifidobacterial rAfuB-H1 and rApy-H1 were successfully cloned, expressed, and characterized. rAfuB-H1 specifically recognized the �\-l-arabinofuranoside, whereas rApy-H1 had dual functions, that is, it could hydrolyse both �]-d-galactopyranoside and �\-l-arabinopyranoside. These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified �\-l-arabinosidases from Bifidobacterium breve K-110 and Clostridium cellulovorans.
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19. |
Bujacz A,
Jedrzejczak-Krzepkowska M,
Bielecki S,
Redzynia I,
Bujacz G,
( 2011 ) Crystal structures of the apo form of �]-fructofuranosidase from Bifidobacterium longum and its complex with fructose. PMID : 21418142 : DOI : 10.1111/j.1742-4658.2011.08098.x Abstract >>
We solved the 1.8 ? crystal structure of �]-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of �]-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed �]-propeller and a C-terminal �]-sandwich module. The active site is located in the centre of the �]-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 �]-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn �\-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 ? crystal structure of the �]-fructofuranosidase complex with �]-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.
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20. |
Fukiya S,
Sugiyama T,
Kano Y,
Yokota A,
( 2010 ) Characterization of an insertion sequence-like element, ISBlo15, identified in a size-increased cryptic plasmid pBK283 in Bifidobacterium longum BK28. PMID : 20547379 : DOI : 10.1016/j.jbiosc.2010.02.013 Abstract >>
The characteristics of mobile genetic elements in bifidobacteria are not well understood. We characterized an insertion sequence-like element of the IS200/IS605 family found in a size-increased cryptic plasmid in Bifidobacterium longum. During a plasmid profile analysis of B. longum BK strains, we encountered a 6.5-kbp cryptic plasmid pBK283 in B. longum BK28, the size of which has not been identified in bifidobacteria. Nucleotide sequence analysis indicated that an insertion sequence-like element was inserted into the 5.0-kbp pKJ50-like plasmid and resulted in a size increase of pBK283. The element, named ISBlo15, was 1593 bp in length and contained a single ORF encoding a putative transposase, which is similar to the transposase OrfB encoded by IS200/IS605 family elements. Several sequence characteristics, including conserved transposase motifs in OrfB and terminal palindromic sequences that differ from the typical terminal inverted repeats, strongly suggested that ISBlo15 is a member of the IS200/IS605 family. Sequences similar to ISBlo15 were widely distributed among the nine Bifidobacterium species tested, and those of highly homologous sequences were detected only in Bifidobacterium gallicum JCM8224(T).
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21. |
Takahashi K,
Tagami U,
Shimba N,
Kashiwagi T,
Ishikawa K,
Suzuki E,
( 2010 ) Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium. PMID : 20674574 : DOI : 10.1016/j.febslet.2010.07.043 Abstract >>
The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5 kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.
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22. |
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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23. |
Moon GS,
Wegmann U,
Gunning AP,
Gasson MJ,
Narbad A,
( 2009 ) Isolation and characterization of a theta-type cryptic plasmid from Bifidobacterium longum FI10564. PMID : 19420998 : Abstract >>
A number of bifidobacterial species of human origin were screened for the presence of cryptic plasmids. One strain, Bifidobacterium longum FI10564, harbored plasmids of approximately 2.2 kb, 3.6 kb, and 4.9 kb in size. The smallest plasmid, pFI2576 (2,197 bp), was studied in detail and its complete nucleotide sequence was determined. Computer-assisted analysis of this novel plasmid (G+C content 62%) identified 9 putative open reading frames (orfs), 3 of which were shown to be probable genes. These putative genes are arranged in an operon-like structure, in which the overlapping orfs 1 and 2 encode putative Rep proteins and are highly homologous to the rep genes of the B. longum plasmid pMB1 (1,847 bp). The mechanism of replication of pFI2576 was investigated using Southern blot analysis of whole cell lysates, with and without S1 nuclease treatment, and atomic force microscopy (AFM). The results indicate that pFI2576 is likely to use the theta mode of replication.
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24. |
Audy J,
Labrie S,
Roy D,
Lapointe G,
( 2010 ) Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002. PMID : 19850611 : DOI : 10.1099/mic.0.033720-0 Abstract >>
The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080+/-120 mg EPS l(-1) (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512+/-63, 564+/-165 and 616+/-93 mg EPS l(-1), respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production (galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase (wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.
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25. |
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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26. |
Vitali B,
Turroni S,
Serina S,
Sosio M,
Vannini L,
Candela M,
Guerzoni ME,
Brigidi P,
( 2008 ) Molecular and phenotypic traits of in-vitro-selected mutants of Bifidobacterium resistant to rifaximin. PMID : 18462927 : DOI : 10.1016/j.ijantimicag.2008.02.002 Abstract >>
Nucleotide mutations inside a core region of the rpoB gene, encoding the beta subunit of RNA polymerase, were found in rifaximin-resistant mutants of Bifidobacterium. Five different missense mutations of codons 513, 516, 522 and 529 were identified. Further aspects of rifaximin resistance were investigated, using Bifidobacterium infantis BI07 as a model strain. Partial resistance of RNA polymerase of a BI07 mutant at a rifaximin concentration >10 microg/mL was observed by cell-free transcription assay. Mass spectrometry detection of rifaximin in the cellular pellet of the BI07 resistant mutant, as well as changes in biosynthesis of saturated and cyclopropane fatty acids during growth, suggested a reduction in membrane permeability for the antibiotic moiety.
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27. |
Suzuki R,
Wada J,
Katayama T,
Fushinobu S,
Wakagi T,
Shoun H,
Sugimoto H,
Tanaka A,
Kumagai H,
Ashida H,
Kitaoka M,
Yamamoto K,
( 2008 ) Structural and thermodynamic analyses of solute-binding Protein from Bifidobacterium longum specific for core 1 disaccharide and lacto-N-biose I. PMID : 18332142 : DOI : 10.1074/jbc.M709777200 Abstract >>
Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Galbeta1-3GlcNAc) and galacto-N-biose (GNB; Galbeta1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K(d) values of 0.087 and 0.010 microm, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.
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28. |
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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29. |
Flórez AB,
Ammor MS,
Mayo B,
van Hoek AH,
Aarts HJ,
Huys G,
( 2008 ) Antimicrobial susceptibility profiles of 32 type strains of Lactobacillus, Bifidobacterium, Lactococcus and Streptococcus spp. PMID : 18061411 : DOI : 10.1016/j.ijantimicag.2007.09.003 Abstract >>
N/A
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30. |
Ma Y,
Xie TT,
Hu Q,
Qiu Z,
Song F,
( 2015 ) Sequencing analysis and characterization of the plasmid pBIF10 isolated from Bifidobacterium longum. PMID : 25587774 : DOI : 10.1139/cjm-2014-0581 Abstract >>
A resident plasmid, pBIF10, was isolated from Bifidobacterium longum B200304, and the full-length sequence of pBIF10 was analyzed. In this sequence, we identified at least 17 major open reading frames longer than 200 bp. A tetracycline resistance gene, tetQ, was identified and verified to confer antibiotic resistance to tetracycline. The plasmid replicon with replication protein B gene (repB) and a typical iteron was identified in pBIF10. An artificial clone vector was constructed with the replicon of pBIF10; the results showed that repB controlled plasmid replication in other bifidobacteria host cells at low transformation frequency. Taken together, the analysis and characterization of pBIF10 provided necessary information for the understanding of antibiotic resistance mediated by a plasmid in a Bifidobacterium strain. GC% and repB sequence analyses indicated that pBIF10 was a molecular hybrid of at least 2 other bacterial genera plasmids.
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31. |
Kanesaki Y,
Masutani H,
Sakanaka M,
Shiwa Y,
Fujisawa T,
Nakamura Y,
Yokota A,
Fukiya S,
Suzuki T,
Yoshikawa H,
( 2014 ) Complete Genome Sequence of Bifidobacterium longum 105-A, a Strain with High Transformation Efficiency. PMID : 25523770 : DOI : 10.1128/genomeA.01311-14 PMC : PMC4271160 Abstract >>
Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency.
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32. |
Nezametdinova VZ,
Zakharevich NV,
Alekseeva MG,
Averina OV,
Mavletova DA,
Danilenko VN,
( 2014 ) Identification and characterization of the serine/threonine protein kinases in Bifidobacterium. PMID : 24395073 : DOI : 10.1007/s00203-013-0949-8 Abstract >>
Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.
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33. |
Jung IH,
Lee JH,
Hyun YJ,
Kim DH,
( 2012 ) Metabolism of ginsenoside Rb1 by human intestinal microflora and cloning of its metabolizing �]-D-glucosidase from Bifidobacterium longum H-1. PMID : 22466563 : DOI : 10.1248/bpb.35.573 Abstract >>
To understand the role of intestinal microflora in expressing the pharmacological effect of ginsenoside Rb1, the metabolic activity of ginsenoside Rb1 by 148 fecal specimens was measured and its metabolizing �]-glucosidase was cloned. The average activities for p-nitrophenyl-�]-D-glucopyranoside and ginsenoside Rb1 were 0.097��0.059 �gmol/min/mg and 0.311��0.118 pmol/min/mg, respectively. These enzyme activities were not different between male and female, or between ages. A gene encoding �]-D-glucosidase (BglX) was cloned from Bifidobacterium longum H-1, which transformed ginsenoside Rb1 to compound K. The probe for cloning was synthesized from the genes encoding a �]-D-glucosidase of previously reported B. longum DJO10A. The sequences of the cloned gene revealed 2364 bp open reading frame (ORF) encoding a protein containing 787 amino acids (molecular weight of 95 kDa). The gene exhibited 99% homology (identities) to that of B. longum. The cloned gene was expressed under T7 promoter of the expression vector, pET-39b(+), in Escherichia coli BL21(DE3), and the expressed enzyme was purified by using HiTrap immobilized metal affinity chromatography (IMAC) HP. The enzyme potently biotransformed ginsenoside Rb1, loganin, arctiin and arbutin to ginsenoside Rd, loganetin, arctigenin and hydroquinone, respectively, but was not active in the case of hesperidin, and kakkalide. This is the first report on cloning and expression of �]-D-glucosidase from B. longum. Based on these findings, ginsenoside Rb1 may be metabolized to bioactive compound(s) by exo-�]-D-glucosidase(s) produced from the intestinal bacteria and its pharmacological effects may be dependent on intestinal bacterial exo-�]-D-glucosidase(s) activity.
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34. |
Salonen N,
Turunen O,
Salonen K,
( 2012 ) Bifidobacterium longum L-arabinose isomerase--overexpression in Lactococcus lactis, purification, and characterization. PMID : 22763951 : DOI : 10.1007/s12010-012-9783-8 Abstract >>
Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum L-AI was characterized using D-galactose and L-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0-6.5. The enzyme showed the highest activity at 55 �XC during a 20-min incubation at pH 6.5. The K(m) value was 120 mM for L-arabinose and 590 mM for D-galactose. The V(max) was 42 U mg(-1) with L-arabinose and 7.7 U mg(-1) with D-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg(2+), Mn(2+)). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum L-AI as the catalyst at 35 �XC, equilibrium yields of 36 % D-tagatose and 11 % L-ribulose with 1.67 M D-galactose and L-arabinose, respectively, as the substrates were reached.
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35. |
Ha SJ,
Kim SR,
Choi JH,
Park MS,
Jin YS,
( 2011 ) Xylitol does not inhibit xylose fermentation by engineered Saccharomyces cerevisiae expressing xylA as severely as it inhibits xylose isomerase reaction in vitro. PMID : 21655987 : DOI : 10.1007/s00253-011-3345-9 Abstract >>
Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation. Although xylitol inhibited in vitro BsXI activity significantly (K(I) = 5.1 �� 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro.
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36. |
Jiang X,
Hall AB,
Arthur TD,
Plichta DR,
Covington CT,
Poyet M,
Crothers J,
Moses PL,
Tolonen AC,
Vlamakis H,
Alm EJ,
Xavier RJ,
( 2019 ) Invertible promoters mediate bacterial phase variation, antibiotic resistance, and host adaptation in the gut. PMID : 30630933 : DOI : 10.1126/science.aau5238 PMC : PMC6543533 Abstract >>
Phase variation, the reversible alternation between genetic states, enables infection by pathogens and colonization by commensals. However, the diversity of phase variation remains underexplored. We developed the PhaseFinder algorithm to quantify DNA inversion-mediated phase variation. A systematic search of 54,875 bacterial genomes identified 4686 intergenic invertible DNA regions (invertons), revealing an enrichment in host-associated bacteria. Invertons containing promoters often regulate extracellular products, underscoring the importance of surface diversity for gut colonization. We found invertons containing promoters regulating antibiotic resistance genes that shift to the ON orientation after antibiotic treatment in human metagenomic data and in vitro, thereby mitigating the cost of antibiotic resistance. We observed that the orientations of some invertons diverge after fecal microbiota transplant, potentially as a result of individual-specific selective forces.
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37. |
Oh SY,
Youn SY,
Park MS,
Baek NI,
Ji GE,
( 2018 ) Synthesis of Stachyobifiose Using Bifidobacterial �\-Galactosidase Purified from Recombinant Escherichia coli. PMID : 29363955 : DOI : 10.1021/acs.jafc.7b04703 Abstract >>
The prebiotic effects of GOS (galactooligosaccharides) are known to depend on the glycosidic linkages, degree of polymerization (DP), and the monosaccharide composition. In this study, a novel form of �\-GOS with a potentially improved prebiotic effect was synthesized using bifidobacterial �\-galactosidase (�\-Gal) purified from recombinant Escherichia coli. The carbohydrate produced was identified as �\-d-galactopyranosyl-(1��6)-O-�\-d-glucopyranosyl-(1��2)-[�\-d-galactopyranosyl-(1��6)-O-�]-d-fructofuranoside] and was termed stachyobifiose. Among 17 nonprobiotics, 16 nonprobiotics showed lower growth on stachyobifiose than �]-GOS. In contrast, among the 16 probiotics, 6 probiotics showed higher growth on stachyobifiose than �]-GOS. When compared with raffinose, stachyobifiose was used less by nonprobiotics than raffinose. Moreover, compared with stachyose, stachyobifiose was used less by Escherichia coli, Enterobacter cloacae, and Clostridium butyricum. The average amounts of total short-chain fatty acids (SCFA) produced were in the order of stachyobifiose > stachyose > raffinose > �]-GOS. Taken together, stachyobifiose is expected to contribute to beneficial changes of gut microbiota.
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38. |
Barnaba TJ,
Gangiredla J,
Mammel MK,
Lacher DW,
Elkins CA,
Lampel KA,
Whitehouse CA,
Tartera C,
( 2018 ) Draft Genome Sequences of Bifidobacterium Strains Isolated from Dietary Supplements and Cultured Food Products. PMID : 29954907 : DOI : 10.1128/genomeA.00610-18 PMC : PMC6025948 Abstract >>
Here, we present the genome sequences of 23 Bifidobacterium isolates from several commercially available dietary supplements and cultured food products. Strains of this genus are natural inhabitants of the mammalian mouth, gastrointestinal tract, and vagina. Some species are considered beneficial to human health.
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39. |
Freitas AC,
Hill JE,
( 2017 ) Quantification, isolation and characterization of Bifidobacterium from the vaginal microbiomes of reproductive aged women. PMID : 28552417 : DOI : 10.1016/j.anaerobe.2017.05.012 Abstract >>
The vaginal microbiome plays an important role in women's reproductive health. Imbalances in this microbiota, such as the poorly defined condition of bacterial vaginosis, are associated with increased susceptibility to sexually transmitted infections and negative reproductive outcomes. Currently, a "healthy" vaginal microbiota in reproductive aged women is understood to be dominated by Lactobacillus, although "atypical" microbiomes, such as Bifidobacterium-dominated profiles, have been described. Despite these observations, vaginal bifidobacteria remain relatively poorly characterized, and questions remain regarding their actual abundance in the microbiome. In this study, we used quantitative PCR to confirm the relative abundance of Bifidobacterium in the vaginal microbiomes of healthy reproductive aged women (n = 42), previously determined by deep sequencing. We also isolated and phenotypically characterized vaginal bifidobacteria (n = 40) in the context of features thought to promote reproductive health. Most isolates were identified as B. breve or B. longum based on cpn60 barcode sequencing. Fermentation patterns of vaginal bifidobacteria did not differ substantially from corresponding type strains of gut or oral origin. Lactic acid was produced by all vaginal isolates, with B. longum strains producing the highest levels, but only 32% of isolates produced hydrogen peroxide. Most vaginal bifidobacteria were also able to tolerate high levels of lactic acid (100 mM) and low pH (4.5 or 3.9), conditions typical of vaginal fluid of healthy women. Most isolates were resistant to metronidazole but susceptible to clindamycin, the two most common antibiotics used to treat vaginal dysbiosis. These findings demonstrate that Bifidobacterium is the dominant member of some vaginal microbiomes and suggest that bifidobacteria have the potential to be as protective as lactobacilli according to the current understanding of a healthy vaginal microbiome.
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40. |
( 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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41. |
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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42. |
( 2013 ) Sequence analysis of plasmid pSP02 from Bifidobacterium longum M62 and construction of pSP02-derived cloning vectors. PMID : 23228478 : DOI : 10.1016/j.plasmid.2012.11.006 Abstract >>
Replicons from bifidobacteria species are required for the construction of general- and special-purpose vectors that would allow the undertaking of molecular studies of these bacteria. In this work, pSP02, a cryptic plasmid from Bifidobacterium longum M62, was cloned, sequenced and characterized. pSP02 was found to consist of 4896bp with four ORFs coding for proteins over 50 amino acids long. Among the deduced protein sequences only a replicase (RepA) and a mobilization-like protein (MobA) showed known functional domains. Similar to previously described bifidobacterial plasmids, the organization of the putative ori region of pSP02 resembles that of the theta-replicating plasmids of Gram-positives. In spite of this, hybridization experiments detected single stranded (ss)-DNA as an intermediate product in the pSP02 replication, demonstrating it follows the rolling-circle (RC) replication mode. The ori region of pSP02 was used to construct a series of first generation cloning vectors able to replicate in many bifidobacterial species. Real time quantitative PCR established the copy number of pSP02 and its derived vectors to be around 12 copies per chromosome equivalent. pSP02-derivatives showed full segregational and structural stability even in the absence of antibiotic selection.
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43. |
( 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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