| 1. |
Sánchez B,
Champomier-Vergès MC,
Stuer-Lauridsen B,
Ruas-Madiedo P,
Anglade P,
Baraige F,
de los Reyes-Gavilán CG,
Johansen E,
Zagorec M,
Margolles A,
( 2007 ) Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach. PMID : 17827318 : DOI : 10.1128/AEM.00637-07 PMC : PMC2074956 Abstract >>
Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.
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2. |
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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3. |
Masco L,
Van Hoorde K,
De Brandt E,
Swings J,
Huys G,
( 2006 ) Antimicrobial susceptibility of Bifidobacterium strains from humans, animals and probiotic products. PMID : 16698847 : DOI : 10.1093/jac/dkl197 Abstract >>
The aim of this study was to assess the antimicrobial susceptibility of a taxonomically diverse set of Bifidobacterium strains to different classes of antimicrobial agents using a recently described medium. The susceptibility of 100 strains encompassing 11 bifidobacterial species originating from humans, animals and probiotic products to 12 antimicrobial agents was tested by agar overlay disc diffusion. Based on these results, one or two strains per species were selected for susceptibility testing to nine antibiotics by broth microdilution using the Lactic acid bacteria Susceptibility test Medium (LSM) supplemented with cysteine. The genotypic basis of atypical tetracycline resistance was further characterized using PCR, Southern blotting and partial sequencing. Based on the distribution of inhibition zone diameters and MIC values, all strains tested were susceptible to amoxicillin, chloramphenicol, erythromycin, quinupristin/dalfopristin, rifampicin and vancomycin. Our data also reinforce earlier observations indicating that bifidobacteria are intrinsically resistant to gentamicin, sulfamethoxazole and polymyxin B. Susceptibility to trimethoprim, trimethoprim/sulfamethoxazole, ciprofloxacin, clindamycin, tetracycline and minocycline was variable. The tet(W) gene was responsible for tetracycline resistance in 15 strains including 7 probiotic isolates belonging to the taxa Bifidobacterium animalis subsp. lactis and Bifidobacterium bifidum. This gene was present in a single copy on the chromosome and did not appear to be associated with the conjugative transposon TnB1230 previously found in tet(W)-containing Butyrivibrio fibrisolvens. The use of the LSM + cysteine medium allowed us to discriminate between intrinsic and atypical resistance properties of bifidobacteria and sets the scene for future definition of epidemiological cut-off values for all important Bifidobacterium species. The presence of an acquired tet(W) gene in several probiotic product isolates stresses the need for a minimal safety evaluation during the selection of Bifidobacterium strains for probiotic use.
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4. |
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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5. |
Ventura M,
Canchaya C,
Bernini V,
Del Casale A,
Dellaglio F,
Neviani E,
Fitzgerald GF,
van Sinderen D,
( 2005 ) Genetic characterization of the Bifidobacterium breve UCC 2003 hrcA locus. PMID : 16332909 : DOI : 10.1128/AEM.71.12.8998-9007.2005 PMC : PMC1317471 Abstract >>
The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and transcriptional regulators, including the DnaJ and the HrcA proteins. Genome analysis of Bifidobacterium breve UCC 2003 revealed a second copy of a dnaJ gene, named dnaJ2, which is flanked by the hrcA gene in a genetic constellation that appears to be unique to the actinobacteria. Phylogenetic analysis using 53 bacterial dnaJ sequences, including both dnaJ1 and dnaJ2 sequences, suggests that these genes have followed a different evolutionary development. Furthermore, the B. breve UCC 2003 dnaJ2 gene seems to be regulated in a manner that is different from that of the previously characterized dnaJ1 gene. The dnaJ2 gene, which was shown to be part of a 2.3-kb bicistronic operon with hrcA, was induced by osmotic shock but not significantly by heat stress. This induction pattern is unlike those of other characterized dnaJ genes and may be indicative of a unique stress adaptation strategy by this commensal microorganism.
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6. |
Ventura M,
Zhang Z,
Cronin M,
Canchaya C,
Kenny JG,
Fitzgerald GF,
van Sinderen D,
( 2005 ) The ClgR protein regulates transcription of the clpP operon in Bifidobacterium breve UCC 2003. PMID : 16321946 : DOI : 10.1128/JB.187.24.8411-8426.2005 PMC : PMC1317013 Abstract >>
Five clp genes (clpC, clpB, clpP1, clpP2, and clpX), representing chaperone- and protease-encoding genes, were previously identified in Bifidobacterium breve UCC 2003. In the present study, we characterize the B. breve UCC 2003 clpP locus, which consists of two paralogous genes, designated clpP1 and clpP2, whose deduced protein products display significant similarity to characterized ClpP peptidases. Transcriptional analyses showed that the clpP1 and clpP2 genes are transcribed in response to moderate heat shock as a bicistronic unit with a single promoter. The role of a clgR homologue, known to control the regulation of clpP gene expression in Streptomyces lividans and Corynebacterium glutamicum, was investigated by gel mobility shift assays and DNase I footprint experiments. We show that ClgR, which in its purified form appears to exist as a dimer, requires a proteinaceous cofactor to assist in specific binding to a 30-bp region of the clpP promoter region. In pull-down experiments, a 56-kDa protein copurified with ClgR, providing evidence that the two proteins also interact in vivo and that the copurified protein represents the cofactor required for ClgR activity. The prediction of the ClgR three-dimensional structure provides further insights into the binding mode of this protein to the clpP1 promoter region and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.
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7. |
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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8. |
Janer C,
Rohr LM,
Peláez C,
Laloi M,
Cleusix V,
Requena T,
Meile L,
( 2004 ) Hydrolysis of oligofructoses by the recombinant beta-fructofuranosidase from Bifidobacterium lactis. PMID : 15214632 : Abstract >>
The ability of the beta-fructofuranosidase (EC 3.2.1.26) from Bifidobacterium lactis DSM 10140T to cleave a variety of fructooligosaccharides was characterised. We identified its gene on a cloned chromosomal DNA fragment by sequence similarity (69% identity) to the putative CscA protein encoded in the Bifidobacterium longum genome. The deduced amino acid sequence of 532 residues (59.4 kDa) appeared to be identical to the beta-fructofuranosidase from the same strain recently described by Ehrmann et al. (Curr. Microbiol. 2003, 46, 391-397). However, the characterisation of the heterologously expressed enzyme showed several discrepancies to the referred study. First, the B. lactis beta-fructofuranosidase gene was found to have 41% identity with CscA from E. coli in contrast to the 16% reported, therefore it was assigned to as CscA protein instead of BfrA. Second, we observed only low activity of the enzyme towards sucrose (6%) instead of the 100% previously reported. Instead, we measured highest activity (100%) of the enzyme with the oligofructose Raftilose as a substrate compared with the inulin of low degree of polymerisation Raftiline LS (29%) and the highly polymerised Raftiline HP (10%). Altogether, the enzyme showed high affinity to terminal beta(2-1) glycosyl linkages between fructose moieties. The Km values obtained for Raftilose, Raftiline LS and sucrose were 0.12, 7.08 and 8.37 mM, respectively, and V(max) values for the conversion to fructose were calculated to be 5, 21 and 17 micromol/min per mg of protein, respectively. Growth of B. lactis was supported by fructans of low degree of polymerisation (Raftilose and Raftiline LS), whereas we observed no growth with highly polymerised inulin (Raftiline HP).
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9. |
Turroni S,
Bendazzoli C,
Dipalo SC,
Candela M,
Vitali B,
Gotti R,
Brigidi P,
( 2010 ) Oxalate-degrading activity in Bifidobacterium animalis subsp. lactis: impact of acidic conditions on the transcriptional levels of the oxalyl coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes. PMID : 20601517 : DOI : 10.1128/AEM.00844-10 PMC : PMC2918965 Abstract >>
Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyperabsorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and can be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis and reducing the risk of kidney stone development. In this study, the oxalate-degrading activities of 14 bifidobacterial strains were measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-coenzyme A (CoA) decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of Bifidobacterium animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unraveling the presence of two other independently transcribed open reading frames, potentially responsible for the ability of B. animalis subsp. lactis to degrade oxalate. pH-controlled batch fermentations revealed that acidic conditions were a prerequisite for a significant oxalate degradation rate, which dramatically increased in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Oxalate-preadapted cells also showed a strong induction of the genes potentially involved in oxalate catabolism, as demonstrated by a transcriptional analysis using quantitative real-time reverse transcription-PCR. These findings provide new insights into the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease.
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10. |
Gueimonde M,
Flórez AB,
van Hoek AH,
Stuer-Lauridsen B,
Strøman P,
de los Reyes-Gavilán CG,
Margolles A,
( 2010 ) Genetic basis of tetracycline resistance in Bifidobacterium animalis subsp. lactis. PMID : 20348299 : DOI : 10.1128/AEM.03096-09 PMC : PMC2869156 Abstract >>
All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis.
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11. |
Candela M,
Centanni M,
Fiori J,
Biagi E,
Turroni S,
Orrico C,
Bergmann S,
Hammerschmidt S,
Brigidi P,
( 2010 ) DnaK from Bifidobacterium animalis subsp. lactis is a surface-exposed human plasminogen receptor upregulated in response to bile salts. PMID : 20167618 : DOI : 10.1099/mic.0.038307-0 Abstract >>
Bifidobacterium animalis subsp. lactis lives in the gastrointestinal tract of most mammals, including humans. Recently, for the probiotic strain B. animalis subsp. lactis BI07, a dose-dependent plasminogen-binding activity was demonstrated and five putative plasminogen-binding proteins were identified. Here we investigated the role of surface DnaK as a B. animalis subsp. lactis BI07 plasminogen receptor. DnaK was visualized on the bacterial cell surface by transmission electron microscopy. The His-tagged recombinant DnaK protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. The capability to tolerate physiological concentrations of bile salts is a crucial feature for an intestinal symbiont micro-organism. By proteome analysis we demonstrated that the long-term exposure of B. animalis subsp. lactis BI07 to bile salts results in the upregulation of important surface plasminogen receptors such as DnaK and enolase. Moreover, adaptation of B. animalis subsp. lactis BI07 to physiological concentrations of bile salts significantly increased its capacity to interact with the host plasminogen system. By enhancing the bacterial capacity to interact with the host plasminogen, the gut bile environment may facilitate the colonization of the human host by B. animalis subsp. lactis BI07.
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12. |
Candela M,
Biagi E,
Centanni M,
Turroni S,
Vici M,
Musiani F,
Vitali B,
Bergmann S,
Hammerschmidt S,
Brigidi P,
( 2009 ) Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host. PMID : 19574304 : DOI : 10.1099/mic.0.028795-0 Abstract >>
The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.
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13. |
Ruas-Madiedo P,
Gueimonde M,
Arigoni F,
de los Reyes-Gavilán CG,
Margolles A,
( 2009 ) Bile affects the synthesis of exopolysaccharides by Bifidobacterium animalis. PMID : 19088310 : DOI : 10.1128/AEM.00908-08 PMC : PMC2643586 Abstract >>
By using cryo-scanning electron microscopy and quantification with lectin-conjugated probes, we have detected the production of exopolysaccharides (EPS) in Bifidobacterium animalis subsp. lactis in the presence of bile. In addition, the expression of gtf01207, which codifies a putative priming glycosyltransferase involved in EPS synthesis, was induced by bile.
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14. |
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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15. |
Lugli GA,
Mancino W,
Milani C,
Duranti S,
Mancabelli L,
Napoli S,
Mangifesta M,
Viappiani A,
Anzalone R,
Longhi G,
van Sinderen D,
Ventura M,
Turroni F,
( 2019 ) Dissecting the Evolutionary Development of the Species Bifidobacterium animalis through Comparative Genomics Analyses. PMID : 30709821 : DOI : 10.1128/AEM.02806-18 PMC : PMC6585482 Abstract >>
Bifidobacteria are members of the gut microbiota of animals, including mammals, birds, and social insects. In this study, we analyzed and determined the pangenome of Bifidobacterium animalis species, encompassing B. animalis subsp. animalis and the B. animalis subsp. lactis taxon, which is one of the most intensely exploited probiotic bifidobacterial species. In order to reveal differences within the B. animalis species, detailed comparative genomics and phylogenomics analyses were performed, indicating that these two subspecies recently arose through divergent evolutionary events. A subspecies-specific core genome was identified for both B. animalis subspecies, revealing the existence of subspecies-defining genes involved in carbohydrate metabolism. Notably, these in silico analyses coupled with carbohydrate profiling assays suggest genetic adaptations toward a distinct glycan milieu for each member of the B. animalis subspecies, resulting in a divergent evolutionary development of the two subspecies.IMPORTANCE The majority of characterized B. animalis strains have been isolated from human fecal samples. In order to explore genome variability within this species, we isolated 15 novel strains from the gastrointestinal tracts of different animals, including mammals and birds. The present study allowed us to reconstruct the pangenome of this taxon, including the genome contents of 56 B. animalis strains. Through careful assessment of subspecies-specific core genes of the B. animalis subsp. animalis/lactis taxon, we identified genes encoding enzymes involved in carbohydrate transport and metabolism, while unveiling specific gene acquisition and loss events that caused the evolutionary emergence of these two subspecies.
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16. |
Ejby M,
Guskov A,
Pichler MJ,
Zanten GC,
Schoof E,
Saburi W,
Slotboom DJ,
Abou Hachem M,
( 2019 ) Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on �]-mannan possess distinct manno-oligosaccharide-binding profiles. PMID : 30947380 : DOI : 10.1111/mmi.14257 Abstract >>
Human gut bifidobacteria rely on ATP-binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide-specific solute-binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associated to the �]-manno-oligosaccharide (MnOS) ABC transporter in Bifidobacterium animalis subsp. lactis. Despite similar overall specificity and preference to mannotriose (Kd ?80 nM), affinity of BlMnBP1 is up to 2570-fold higher for disaccharides than BlMnBP2. Structural analysis revealed a substitution of an asparagine that recognizes the mannosyl at position 2 in BlMnBP1, by a glycine in BlMnBP2, which affects substrate affinity. Both substitution types occur in bifidobacterial SBPs, but BlMnBP1-like variants prevail in human gut isolates. B. animalis subsp. lactis ATCC27673 showed growth on gluco and galactomannans and was able to outcompete a mannan-degrading Bacteroides ovatus strain in co-cultures, attesting the efficiency of this ABC uptake system. By contrast, a strain that lacks this transporter failed to grow on mannan. This study highlights SBP diversification as a possible strategy to modulate oligosaccharide uptake preferences of bifidobacterial ABC-transporters during adaptation to specific ecological niches. Efficient metabolism of galactomannan by distinct bifidobacteria, merits evaluating this plant glycan as a potential prebiotic.
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17. |
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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18. |
Viborg AH,
Katayama T,
Arakawa T,
Abou Hachem M,
Lo Leggio L,
Kitaoka M,
Svensson B,
Fushinobu S,
( 2017 ) Discovery of �\-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42. PMID : 29061847 : DOI : 10.1074/jbc.M117.792598 PMC : PMC5743082 Abstract >>
Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have �]-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for �\-l-arabinopyranoside and describe the first representative of this subfamily. We found that this enzyme (BlArap42B) from a probiotic Bifidobacterium species cannot hydrolyze �]-galactosides. However, BlArap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to �\-l-arabinopyranosyl-(1,6)-�]-d-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus Paeonia, is not hydrolyzed by classical GH42 �]-galactosidases. X-ray crystallography revealed a unique Trp345-X12-Trp358 sequence motif at the BlArap42B active site, as compared with a Phe-X12-His motif in classical GH42 �]-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Arap lacking this carbon. Automated docking of paeonolide revealed that it can fit into the BlArap42B active site. The Glcp moiety of paeonolide stacks onto the aromatic ring of the Trp252 at subsite +1 and C4-OH is hydrogen bonded with Asp249 Moreover, the aglycone stacks against Phe421 from the neighboring monomer in the BlArap42B trimer, forming a proposed subsite +2. These results further support the notion that evolution of metabolic specialization can be tracked at the structural level in key enzymes facilitating degradation of specific glycans in an ecological niche.
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19. |
( 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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20. |
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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21. |
( 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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( 2012 ) Identification of the beta-glucosidase gene from Bifidobacterium animalis subsp. lactis and its expression in B. bifidum BGN4. PMID : 23221535 : Abstract >>
beta-Glucosidase is necessary for the bioconversion of glycosidic phytochemicals in food. Two Bifidobacterium strains (Bifidobacterium animalis subsp. lactis SH5 and B. animalis subsp. lactis RD68) with relatively high beta- glucosidase activities were selected among 46 lactic acid bacteria. A beta-glucosidase gene (bbg572) from B. lactis was shotgun cloned, fully sequenced, and analyzed for its transcription start site, structural gene, and deduced transcriptional terminator. The structural gene of bbg572 was 1,383 bp. Based on amino sequence similarities, bbg572 was assigned to family 1 of the glycosyl hydrolases. To overexpress bbg572 in Bifidobacterium, several bifidobacteria expression vectors were constructed by combining several promoters and a terminator sequence from different bifidobacteria. The maximum activity of recombinant Bbg572 was achieved when it was expressed under its own promoter and terminator. Its enzyme activity increased 31-fold compared with those of its parental strains. The optimal pH for Bbg572 was pH 6.0. Bbg572 was stable at 37-40 degrees C. It hydrolyzed isoflavones, quercetins, and disaccharides with various beta-glucoside linkages. Bbg572 also converted the ginsenosides Rb1 and Rb2. These results suggest that this new beta-glucosidase-positive Bifidobacterium transformant can be utilized for the production of specific aglycone products.
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