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1. Requena  T, Burton  J, Matsuki  T, Munro  K, Simon  MA, Tanaka  R, Watanabe  K, Tannock  GW,     ( 2002 )

Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene.

Applied and environmental microbiology 68 (5)
PMID : 11976117  :   DOI  :   10.1128/aem.68.5.2420-2427.2002     PMC  :   PMC127544    
Abstract >>
Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.
KeywordMeSH Terms
2. Jian  W, Zhu  L, Dong  X,     ( 2001 )

New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences.

International journal of systematic and evolutionary microbiology 51 (Pt 5)
PMID : 11594590  :   DOI  :   10.1099/00207713-51-5-1633    
Abstract >>
The partial 60 kDa heat-shock protein (HSP60) genes of 36 Bifidobacterium strains representing 30 different Bifidobacterium species and subspecies and of the type strain of Gardnerella vaginalis were cloned and sequenced using a pair of universal degenerate HSP60 PCR primers. The HSP60 DNA sequence similarities were determined for the taxa at various ranks as follows: 99.4-100% within the same species, 96% at the subspecies level, and 73-96% (mean 85%) at the interspecies level (and 98% in the case of two groups of closely related species, Bifidobacterium animalis and Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis, whose 165 rRNA sequence similarities are all above 99%). The HSP60 DNA sequence similarities between different Bifidobacterium species and G. vaginalis, a closely related bacterium according to 16S rRNA analysis, ranged from 71 to 79% (mean 75%). Although the topology of the phylogenetic tree constructed using the HSP60 sequences determined was basically similar to that for 16S rRNA, it seemed to be more clear-cut for species delineation, and the clustering was better correlated with the DNA base composition (mol% G+C) than that of the 16S rRNA tree. In the HSP60 phylogenetic tree, all of the high-G+C (55-67 mol%) bifidobacteria were grouped into one cluster, whereas the low-G+C species Bifidobacterium inopinatum (45 mol %) formed a separate cluster with G. vaginalis (42 mol%) and Bifidobacterium denticolens (55 mol%); a Bifidobacterium species of intermediate G+C content formed another cluster between the two. This study demonstrates that the highly conserved and ubiquitous HSP60 gene is an accurate and convenient tool for phylogenetic analysis of the genus Bifidobacterium.
KeywordMeSH Terms
Phylogeny
Sequence Analysis, DNA
3. Fitzgerald  GF,     ( 1999 )

Molecular characterisation of a 5.75-kb cryptic plasmid from Bifidobacterium breve NCFB 2258 and determination of mode of replication.

FEMS microbiology letters 174 (2)
PMID : 10339821  :   DOI  :   10.1111/j.1574-6968.1999.tb13581.x    
Abstract >>
A small cryptic plasmid originating from Bifidobacterium breve NCFB 2258 was cloned and its complete nucleotide sequence determined. pCIBb1 is a circular DNA molecule, 5750 bp in size with a GC composition of 57%. Computer-assisted analysis identified 10 possible open reading frames (ORFs), seven of which could be assigned no function from homology searches. One ORF, rep (380 amino acids), was postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. Demonstration of single-stranded forms of the plasmid in cell lysates that could be specifically degraded by S1 nuclease provided experimental evidence to substantiate a replication mechanism via single-stranded intermediates. Two other ORFs, par (199 amino acids) and an ftsK-like gene (286 amino acids), were assigned putative functions based on the presence of conserved motifs in their deduced proteins.
KeywordMeSH Terms
DNA Replication
4. Ventura  M, Canchaya  C, Zhang  Z, Fitzgerald  GF, van Sinderen  D,     ( 2007 )

Molecular characterization of hsp20, encoding a small heat shock protein of bifidobacterium breve UCC2003.

Applied and environmental microbiology 73 (14)
PMID : 17513584  :   DOI  :   10.1128/AEM.02496-06     PMC  :   PMC1932816    
Abstract >>
Small heat shock proteins (sHSPs) are members of a diverse family of stress proteins that are important in cells to protect proteins under stressful conditions. Genome analysis of Bifidobacterium breve UCC2003 revealed a single sHSP-encoding gene, which was classified as a hsp20 gene by comparative analyses. Genomic surveillance of available genome sequences indicated that hsp20 homologs are not widely distributed in bacteria. In members of the genus Bifidobacterium, this gene appears to be present in only 7 of the 30 currently described species. Moreover, phylogenetic analysis using all available bacterial and eukaryotic sHSP sequences revealed a close relationship between bifidobacterial HSP20 and the class B sHSPs found in members of the division Firmicutes. The results of this comparative analysis and variation in codon usage content suggest that hsp20 was acquired by certain bifidobacteria through horizontal gene transfer. Analysis by slot blot, Northern blot, and primer extension experiments showed that transcription of hsp20 is strongly induced in response to severe heat shock regimens and by osmotic shock.
KeywordMeSH Terms
5. 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.

Applied and environmental microbiology 73 (8)
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).
KeywordMeSH Terms
6. Mazé  A, O'Connell-Motherway  M, Fitzgerald  GF, Deutscher  J, van Sinderen  D,     ( 2007 )

Identification and characterization of a fructose phosphotransferase system in Bifidobacterium breve UCC2003.

Applied and environmental microbiology 73 (2)
PMID : 17098914  :   DOI  :   10.1128/AEM.01496-06     PMC  :   PMC1796965    
Abstract >>
In silico analysis of the Bifidobacterium breve UCC2003 genome allowed identification of four genetic loci, each of which specifies a putative enzyme II (EII) protein of a phosphoenolpyruvate:sugar phosphotransferase system. The EII encoded by fruA, a clear homologue of the unique EIIBCA enzyme encoded by the Bifidobacterium longum NCC2705 genome, was studied in more detail. The fruA gene is part of an operon which contains fruT, which is predicted to encode a homologue of the Bacillus subtilis antiterminator LicT. Transcriptional analysis showed that the fru operon is induced by fructose. The genetic structure, complementation studies, and the observed transcription pattern of the fru operon suggest that the EII encoded in B. breve is involved in fructose transport and that its expression is controlled by an antiterminator mechanism. Biochemical studies unequivocally demonstrated that FruA phosphorylates fructose at the C-6 position.
KeywordMeSH Terms
7. Ryan  SM, Fitzgerald  GF, van Sinderen  D,     ( 2006 )

Screening for and identification of starch-, amylopectin-, and pullulan-degrading activities in bifidobacterial strains.

Applied and environmental microbiology 72 (8)
PMID : 16885278  :   DOI  :   10.1128/AEM.00257-06     PMC  :   PMC1538741    
Abstract >>
Forty-two bifidobacterial strains were screened for alpha-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.
KeywordMeSH Terms
8. Vaugien  L, Prevots  F, Roques  C,     ( 2002 )

Bifidobacteria identification based on 16S rRNA and pyruvate kinase partial gene sequence analysis.

Anaerobe 8 (1��6��)
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.
KeywordMeSH Terms
9. 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.

Applied and environmental microbiology 71 (12)
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.
KeywordMeSH Terms
10. 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.

Journal of bacteriology 187 (24)
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.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
11. Ventura  M, Lee  JH, Canchaya  C, Zink  R, Leahy  S, Moreno-Munoz  JA, O'Connell-Motherway  M, Higgins  D, Fitzgerald  GF, O'Sullivan  DJ, van Sinderen  D,     ( 2005 )

Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis.

Applied and environmental microbiology 71 (12)
PMID : 16332864  :   DOI  :   10.1128/AEM.71.12.8692-8705.2005     PMC  :   PMC1317369    
Abstract >>
So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus Bifidobacterium. In this report we analyzed three prophage-like elements that are present in the genomes of Bifidobacterium breve UCC 2003, Bifidobacterium longum NCC 2705, and Bifidobacterium longum DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNA(Met) gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the attB sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a B. longum DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among B. breve strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.
KeywordMeSH Terms
12. Margolles  A, Moreno  JA, van Sinderen  D, de Los Reyes-Gavilán  CG,     ( 2005 )

Macrolide resistance mediated by a Bifidobacterium breve membrane protein.

Antimicrobial agents and chemotherapy 49 (10)
PMID : 16189127  :   DOI  :   10.1128/AAC.49.10.4379-4381.2005     PMC  :   PMC1251533    
Abstract >>
A gene coding for a hypothetical membrane protein from Bifidobacterium breve was expressed in Lactococcus lactis. Immunoblotting demonstrated that this protein is located in the membrane. Phenotypical changes in sensitivity towards 21 antibiotics were determined. The membrane protein-expressing cells showed higher levels of resistance to several macrolides.
KeywordMeSH Terms
Drug Resistance, Bacterial
13. Ventura  M, Fitzgerald  GF, van Sinderen  D,     ( 2005 )

Genetic and transcriptional organization of the clpC locus in Bifidobacterium breve UCC 2003.

Applied and environmental microbiology 71 (10)
PMID : 16204550  :   DOI  :   10.1128/AEM.71.10.6282-6291.2005     PMC  :   PMC1266005    
Abstract >>
A homolog of the clpC ATPase gene was identified in the genome of Bifidobacterium breve UCC 2003. Since this gene is very well conserved among eubacteria, we employed a PCR-based approach using primers based on highly conserved regions of ClpC proteins in order to identify homologous genes in other bifidobacterial species. Analysis by slot blot, Northern blot, and primer extension experiments showed that transcription of clpC is induced in response to moderate heat shock regimes. Moreover, we identified in the genome sequence of B. breve UCC 2003 a gene, designated clgR, which is predicted to encode a transcriptional regulator involved in regulation of the bifidobacterial clpC gene. The role of this protein in the regulation of B. breve UCC 2003 clpC gene expression was investigated by performing gel retardation experiments. We show that a biologically active ClgR molecule requires one or more proteinaceous coactivators to assist in the specific binding of ClgR to the clpC promoter region.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription, Genetic
14. Ventura  M, Kenny  JG, Zhang  Z, Fitzgerald  GF, van Sinderen  D,     ( 2005 )

The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction.

Microbiology (Reading, England) 151 (Pt 9)
PMID : 16151199  :   DOI  :   10.1099/mic.0.28176-0    
Abstract >>
The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.
KeywordMeSH Terms
15. 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.

Applied and environmental microbiology 71 (1)
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.
KeywordMeSH Terms
Adenosine Triphosphatases
Bacterial Proteins
Gene Expression Regulation, Bacterial
Molecular Chaperones
Operon
16. Ryan  SM, Fitzgerald  GF, van Sinderen  D,     ( 2005 )

Transcriptional regulation and characterization of a novel beta-fructofuranosidase-encoding gene from Bifidobacterium breve UCC2003.

Applied and environmental microbiology 71 (7)
PMID : 16000751  :   DOI  :   10.1128/AEM.71.7.3475-3482.2005     PMC  :   PMC1169055    
Abstract >>
An operon involved in fructooligosaccharide breakdown was identified in the genome of Bifidobacterium breve UCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a beta-fructofuranosidase. Active transcription of the operon was observed when B. breve UCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The beta-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37 degrees C, respectively, and there was a dependence on bivalent cations, particularly manganese. The Km and Vmax values for sucrose hydrolysis were determined to be 25 +/- 2 mM and 24 +/- 3 micromol min(-1) mg(-1), respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the beta(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the beta(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription, Genetic
17. Ventura  M, Canchaya  C, Zink  R, Fitzgerald  GF, van Sinderen  D,     ( 2004 )

Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses.

Applied and environmental microbiology 70 (10)
PMID : 15466567  :   DOI  :   10.1128/AEM.70.10.6197-6209.2004     PMC  :   PMC522111    
Abstract >>
The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes, including the GroEL and GroES proteins. The groES and groEL genes are highly conserved among eubacteria and are typically arranged as an operon. Genome analysis of Bifidobacterium breve UCC 2003 revealed that the groES and groEL genes are located in different chromosomal regions. The heat inducibility of the groEL and groES genes of B. breve UCC 2003 was verified by slot blot analysis. Northern blot analyses showed that the cspA gene is cotranscribed with the groEL gene, while the groES gene is transcribed as a monocistronic unit. The transcription initiation sites of these two mRNAs were determined by primer extension. Sequence and transcriptional analyses of the region flanking the groEL and groES genes of various bifidobacteria revealed similar groEL-cspA and groES gene units, suggesting a novel genetic organization of these chaperones. Phylogenetic analysis of the available bifidobacterial groES and groEL genes suggested that these genes evolved differently. Discrepancies in the phylogenetic positioning of groES-based trees make this gene an unreliable molecular marker. On the other hand, the bifidobacterial groEL gene sequences can be used as an alternative to current methods for tracing Bifidobacterium species, particularly because they allow a high level of discrimination between closely related species of this genus.
KeywordMeSH Terms
Genes, Bacterial
18. Ventura  M, Canchaya  C, van Sinderen  D, Fitzgerald  GF, Zink  R,     ( 2004 )

Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny.

Applied and environmental microbiology 70 (5)
PMID : 15128574  :   DOI  :   10.1128/aem.70.5.3110-3121.2004     PMC  :   PMC404453    
Abstract >>
The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.
KeywordMeSH Terms
Operon
19. MacConaill  LE, Fitzgerald  GF, Van Sinderen  D,     ( 2003 )

Investigation of protein export in Bifidobacterium breve UCC2003.

Applied and environmental microbiology 69 (12)
PMID : 14660341  :   DOI  :   10.1128/aem.69.12.6994-7001.2003     PMC  :   PMC309956    
Abstract >>
The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.
KeywordMeSH Terms
20. 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.

Applied and environmental microbiology 69 (11)
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.
KeywordMeSH Terms
21. Suzuki  R, Katayama  T, Kim  BJ, Wakagi  T, Shoun  H, Ashida  H, Yamamoto  K, Fushinobu  S,     ( 2010 )

Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

The Journal of biological chemistry 285 (44)
PMID : 20739284  :   DOI  :   10.1074/jbc.M110.156281     PMC  :   PMC2962526    
Abstract >>
Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (�\,�]-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.
KeywordMeSH Terms
22. Macouzet  M, Lee  BH, Robert  N,     ( 2010 )

Genetic and structural comparison of linoleate isomerases from selected food-grade bacteria.

Journal of applied microbiology 109 (6)
PMID : 20825518  :   DOI  :   10.1111/j.1365-2672.2010.04844.x    
Abstract >>
To advance the understanding of the molecular mechanisms underlying the capacity of bifidobacteria and lactic bacteria to convert linoleic acid (LA) into conjugated linoleic acid (CLA) by the linoleate isomerase (LI). The potential LI enzymes of selected Lactobacillus, Bifidobacterium and Leuconostoc strains were compared at the genetic, amino acid sequence and functional levels. Genetic analysis was achieved by insertional mutagenesis and hybridization studies using a Lact. reuteri LI probe. Biotransformation studies monitored by gas chromatography showed that Bif. breve is a major CLA producer after the reference Lact. reuteri strain. The putative Bif. breve LI gene was PCR isolated and sequenced. The putative LI gene identified in this study seems essential for bacterial growth. Comparative studies indicate that the deduced protein is membrane bound and reveal the presence of several highly conserved domains among a wide range of Gram-positive bacteria. Given the multiple health benefits of CLA, the capability of some bacteria to convert LA into CLA is of great relevance. Nevertheless, the yields of CLA remain low, and the regulation of the process is far from being understood. A deeper knowledge of this capacity by the genetic studies is revealing the identity of the LI and will eventually contribute to its control.
KeywordMeSH Terms
23. Suzuki  R, Kim  BJ, Shibata  T, Iwamoto  Y, Katayama  T, Ashida  H, Wakagi  T, Shoun  H, Fushinobu  S, Yamamoto  K,     ( 2010 )

Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve.

Acta crystallographica. Section F, Structural biology and crystallization communications 66 (Pt 8)
PMID : 20693675  :   DOI  :   10.1107/S1744309110023845     PMC  :   PMC2917298    
Abstract >>
The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl2, 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a=b=174.8, c=163.8 A, and diffracted to beyond 1.7 A resolution.
KeywordMeSH Terms
24. Turroni  F, Foroni  E, O'Connell Motherway  M, Bottacini  F, Giubellini  V, Zomer  A, Ferrarini  A, Delledonne  M, Zhang  Z, van Sinderen  D, Ventura  M,     ( 2010 )

Characterization of the serpin-encoding gene of Bifidobacterium breve 210B.

Applied and environmental microbiology 76 (10)
PMID : 20348296  :   DOI  :   10.1128/AEM.02938-09     PMC  :   PMC2869134    
Abstract >>
Members of the serpin (serine protease inhibitor) superfamily have been identified in higher multicellular eukaryotes, as well as in bacteria, although examination of available genome sequences has indicated that homologs of the bacterial serpin-encoding gene (ser) are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least 5, and perhaps up to 9, of the 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria produce serpins that form a separate clade. We characterized the ser(210B) locus of Bifidobacterium breve 210B, which encompasses a number of genes whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, microarray, reverse transcription-PCR (RT-PCR), and quantitative real-time PCR (qRT-PCR) analyses revealed that a 3.5-kb polycistronic mRNA encompassing the ser(210B) operon with a single transcriptional start site is strongly induced following treatment of B. breve 210B cultures with some proteases. Interestingly, transcription of other bifidobacterial ser homologs appears to be triggered by different proteases.
KeywordMeSH Terms
Models, Molecular
25. Kim  BJ, Kim  HY, Yun  YJ, Kim  BJ, Kook  YH,     ( 2010 )

Differentiation of Bifidobacterium species using partial RNA polymerase {beta}-subunit (rpoB) gene sequences.

International journal of systematic and evolutionary microbiology 60 (Pt 12)
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.
KeywordMeSH Terms
Phylogeny
26. Kiwaki  M, Sato  T,     ( 2009 )

Antimicrobial susceptibility of Bifidobacterium breve strains and genetic analysis of streptomycin resistance of probiotic B. breve strain Yakult.

International journal of food microbiology 134 (3)
PMID : 19616336  :   DOI  :   10.1016/j.ijfoodmicro.2009.06.011    
Abstract >>
The minimum inhibitory concentrations (MICs) of 17 antimicrobials for 26 Bifidobacterium breve strains of various origins were determined by broth microdilution. MIC distributions for 17 antimicrobials except streptomycin and tetracycline were unimodal for all strains tested, whereas bimodal distributions were observed for streptomycin and tetracycline. The probiotic strain B. breve strain Yakult showed intrinsic susceptibility to all antimicrobials except streptomycin to which the strain showed an atypically higher MIC of >256 microg/ml. Because this strain is a commercial strain, which is often ingested by many consumers on a daily basis, it is very important to determine the genetic basis for streptomycin resistance of this strain. Molecular analysis revealed that a mutation of the rpsL gene for ribosomal protein S12 was responsible for this streptomycin resistance. The resistance of B. breve strain Yakult to streptomycin, therefore, is caused by a chromosomal mutation and very unlikely to be transferred to other microorganisms.
KeywordMeSH Terms
Mutation
27. Shkoporov  AN, Efimov  BA, Khokhlova  EV, Steele  JL, Kafarskaia  LI, Smeianov  VV,     ( 2008 )

Characterization of plasmids from human infant Bifidobacterium strains: sequence analysis and construction of E. coli-Bifidobacterium shuttle vectors.

Plasmid 60 (2)
PMID : 18652842  :   DOI  :   10.1016/j.plasmid.2008.06.005    
Abstract >>
A survey of infant fecal Bifidobacterium isolates for plasmid DNA revealed that a significant portion of the strains, 17.6%, carry small plasmids. The majority of plasmid-harboring strains belonged to the Bifidobacterium longum/infantis group. Most of the plasmids could be assigned into two groups based on their sizes and the restriction profiles. Three plasmids, pB44 (3.6 kb) from B. longum, pB80 (4.9 kb) from Bifidobacterium bifidum, and pB21a (5.2kb) from Bifidobacterium breve were sequenced. While the former two plasmids were found to be highly similar to previously characterized rolling-circle replicating pKJ36 and pKJ56, respectively, the third plasmid, pB21a, does not share significant nucleotide homology with known plasmids. However, it might be placed into the pCIBb1-like group of bifidobacterial rolling-plasmids based on the homology of its Rep protein and the overall molecular organization. Two sets of Escherichia coli-Bifidobacterium shuttle vectors constructed based on pB44 and pB80 replicons were capable of transforming B. bifidum and B. breve strains with efficiency up to 3x10(4)cfu/microg DNA. Additionally, an attempt was made to employ a broad host range conjugation element, RP4, in developing of E. coli-Bifidobacterium gene transfer system.
KeywordMeSH Terms
28. Zhao  H, Lu  L, Xiao  M, Wang  Q, Lu  Y, Liu  C, Wang  P, Kumagai  H, Yamamoto  K,     ( 2008 )

Cloning and characterization of a novel alpha-galactosidase from Bifidobacterium breve 203 capable of synthesizing Gal-alpha-1,4 linkage.

FEMS microbiology letters 285 (2)
PMID : 18564335  :   DOI  :   10.1111/j.1574-6968.2008.01246.x    
Abstract >>
A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.
KeywordMeSH Terms
29. 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.

International journal of antimicrobial agents 31 (6)
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.
KeywordMeSH Terms
30. Matsuki  T, Yahagi  K, Mori  H, Matsumoto  H, Hara  T, Tajima  S, Ogawa  E, Kodama  H, Yamamoto  K, Yamada  T, Matsumoto  S, Kurokawa  K,     ( 2016 )

A key genetic factor for fucosyllactose utilization affects infant gut microbiota development.

Nature communications 7 (N/A)
PMID : 27340092  :   DOI  :   10.1038/ncomms11939     PMC  :   PMC4931012    
Abstract >>
Recent studies have demonstrated that gut microbiota development influences infants' health and subsequent host physiology. However, the factors shaping the development of the microbiota remain poorly understood, and the mechanisms through which these factors affect gut metabolite profiles have not been extensively investigated. Here we analyse gut microbiota development of 27 infants during the first month of life. We find three distinct clusters that transition towards Bifidobacteriaceae-dominant microbiota. We observe considerable differences in human milk oligosaccharide utilization among infant bifidobacteria. Colonization of fucosyllactose (FL)-utilizing bifidobacteria is associated with altered metabolite profiles and microbiota compositions, which have been previously shown to affect infant health. Genome analysis of infants' bifidobacteria reveals an ABC transporter as a key genetic factor for FL utilization. Thus, the ability of bifidobacteria to utilize FL and the presence of FL in breast milk may affect the development of the gut microbiota in infants, and might ultimately have therapeutic implications.
KeywordMeSH Terms
31. Bottacini  F, O'Connell Motherway  M, Casey  E, McDonnell  B, Mahony  J, Ventura  M, van Sinderen  D,     ( 2015 )

Discovery of a conjugative megaplasmid in Bifidobacterium breve.

Applied and environmental microbiology 81 (1)
PMID : 25326305  :   DOI  :   10.1128/AEM.02871-14     PMC  :   PMC4272746    
Abstract >>
Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.
KeywordMeSH Terms
Plasmids
32. Yi  SH, Alli  I, Park  KH, Lee  B,     ( 2011 )

Overexpression and characterization of a novel transgalactosylic and hydrolytic �]-galactosidase from a human isolate Bifidobacterium breve B24.

New biotechnology 28 (6)
PMID : 21888996  :   DOI  :   10.1016/j.nbt.2011.07.006    
Abstract >>
After the complete gene of a �]-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant �]-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the �]-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45�XC could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45�XC for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant �]-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing.
KeywordMeSH Terms
33.     ( 1996 )

Purification and characterization of beta-D-glucosidase (beta-D-fucosidase) from Bifidobacterium breve clb acclimated to cellobiose.

Bioscience, biotechnology, and biochemistry 60 (2)
PMID : 9063964  :  
Abstract >>
The beta-D-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only beta-D-glucosidase activity but also beta-D-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47,000-48,000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45 degrees C, respectively. The enzyme was stable up to 40 degrees C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the Km values for p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-fucoside were 1.3 mM and 0.7 mM, respectively. This enzyme had also transferase activity for the beta-D-fucosyl group but not for the beta-D-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of beta-D-glucosidase from other bacteria, actinomycetes, and plants.
KeywordMeSH Terms
34.     ( 1997 )

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine.

FEMS microbiology letters 154 (2)
PMID : 9311137  :   DOI  :   10.1111/j.1574-6968.1997.tb12670.x    
Abstract >>
The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.
KeywordMeSH Terms
Genes, Bacterial
35.     ( 1996 )

Cloning and nucleotide sequence of the beta-D-glucosidase gene from Bifidobacterium breve clb, and expression of beta-D-glucosidase activity in Escherichia coli.

Bioscience, biotechnology, and biochemistry 60 (12)
PMID : 8988633  :  
Abstract >>
Genomic DNA encoding a beta-D-glucosidase (EC 3.2.1.21), which has beta-D-fucosidase activity, was cloned from Bifidobacterium breve clb. We sequenced a 1.9-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51,513 Da. A putative ribosome binding site was found 5 bp upstream of the initiation codon. The amino acid sequence of this beta-D-glucosidase from Bifidobacterium breve clb had 46% identity with that of beta-glucosidase from Microbispore bispore. The enzyme of Bifidobacterium breve clb was expressed in Escherichia coli. A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more beta-D-glucosidase activity than that from Bifidobacterium breve clb. The recombinant enzyme was purified to homogeneity from cell-free extracts of the recombinant strain using 4 column chromatographies. The recovery of enzyme from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve clb. The enzymatic properties were similar to those of Bifidobacterium breve clb. For application of this recombinant enzyme, we attempted to synthesize a disaccharide that seemed to be specifically assimilated by Bifidobacteria using the condensation activity of the enzyme.
KeywordMeSH Terms
36. Martínez  N, Luque  R, Milani  C, Ventura  M, Bañuelos  O, Margolles  A,     ( 2018 )

A Gene Homologous to rRNA Methylase Genes Confers Erythromycin and Clindamycin Resistance in Bifidobacterium breve.

Applied and environmental microbiology 84 (10)
PMID : 29500262  :   DOI  :   10.1128/AEM.02888-17     PMC  :   PMC5930361    
Abstract >>
Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island.IMPORTANCEBifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains.
KeywordMeSH Terms
Bifidobacterium
Bifidobacterium breve
clindamycin resistance
erythromycin resistance
microbiota
probiotics
37. Sotoya  H, Shigehisa  A, Hara  T, Matsumoto  H, Hatano  H, Matsuki  T,     ( 2017 )

Identification of genes involved in galactooligosaccharide utilization in Bifidobacterium breve strain YIT 4014T.

Microbiology (Reading, England) N/A (N/A)
PMID : 28920844  :   DOI  :   10.1099/mic.0.000517    
Abstract >>
Galactooligosaccharides (GOS) are mixed oligosaccharides that are mainly composed of galactosyllactoses (GLs), which include 3'-GL, 4'-GL, and 6'-GL. Data from numerous in vitro and in vivo studies have shown that GOS selectively stimulate the growth of bifidobacteria. Previously, we identified the gene locus responsible for 4'-GL utilization, but the selective routes of uptake and catabolism of 3'- and 6'-GL remain to be elucidated. In this study, we used differential transcriptomics to identify the utilization pathways of these GLs within the Bifidobacterium breve YIT 4014T strain. We found that the BBBR_RS 2305-2320 gene locus, which includes a solute-binding protein (SBP) of an ATP-binding cassette (ABC) transporter and �]-galactosidase, were up-regulated during 3'- and 6'-GL utilization. The substrate specificities of these proteins were further investigated, revealing that �]-galactosidase hydrolyzed both 3'-GL and 6'-GL efficiently. Our surface plasmon resonance results indicated that the SBP bound strongly to 6'-GL, but bound less tightly to 3'-GL. Therefore, we looked for the other SBPs for 3'-GL and found that the BBBR_RS08090 SBP may participate in 3'-GL transportation. We also investigated the distribution of these genes in 17 bifidobacterial strains, including 9 B. breve strains, and found that the �]-galactosidase genes were present in most bifidobacteria. Homologues of two ABC transporter SBP genes were found in all B. breve strains and in some bifidobacteria that are commonly present in the human gut microbiota. These results provide insights into the ability of human-resident bifidobacteria to utilize the main component of GOS in the gastrointestinal tract.
KeywordMeSH Terms
38. Freitas  AC, Hill  JE,     ( 2017 )

Quantification, isolation and characterization of Bifidobacterium from the vaginal microbiomes of reproductive aged women.

Anaerobe 47 (N/A)
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.
KeywordMeSH Terms
Antibiotic
Bifidobacterium
Fermentation pattern
Hydrogen peroxide
Lactic acid
Vaginal microbiome
Antibiotic
Bifidobacterium
Fermentation pattern
Hydrogen peroxide
Lactic acid
Vaginal microbiome
Bacterial Load
Microbiota
39.     ( 2013 )

Genetic diversity of bile salt hydrolases among human intestinal bifidobacteria.

Current microbiology 67 (3)
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.
KeywordMeSH Terms
Genetic Variation
40.     ( 2013 )

Bifidobacterial succession and correlation networks in a large unselected cohort of mothers and their children.

Applied and environmental microbiology 79 (2)
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.
KeywordMeSH Terms
Biodiversity
Genetic Variation

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