van Sinderen D,
( 2003 )
Identification of two-component regulatory systems in Bifidobacterium infantis by functional complementation and degenerate PCR approaches.
PMID : 12839803 : DOI : 10.1128/aem.69.7.4219-4226.2003 PMC : PMC165215
Two-component signal transduction systems (2CSs) are widely used by bacteria to sense and adapt to changing environmental conditions. With two separate approaches, three different 2CSs were identified on the chromosome of the probiotic bacterium Bifidobacterium infantis UCC 35624. One locus was identified by means of functional complementation of an Escherichia coli mutant. Another two were identified by PCR with degenerate primers corresponding to conserved regions of one protein component of the 2CS. The complete coding regions for each gene cluster were obtained, which showed that each 2CS-encoding locus specified a histidine protein kinase and an assumed cognate response regulator. Transcriptional analysis of the 2CSs by Northern blotting and primer extension identified a number of putative promoter sequences for this organism while revealing that the expression of each 2CS was growth phase dependent. Analysis of the genetic elements involved revealed significant homology with several distinct regulatory families from other high-G+C-content bacteria. The conservation of the genetic organization of these three 2CSs in other bacteria, including a number of recently published Bifidobacterium genomes, was investigated.
( 2002 )
Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene.
PMID : 11976117 : DOI : 10.1128/aem.68.5.2420-2427.2002 PMC : PMC127544
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.
( 2001 )
New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences.
PMID : 11594590 : DOI : 10.1099/00207713-51-5-1633
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.
( 2001 )
Molecular and biochemical analysis of two beta-galactosidases from Bifidobacterium infantis HL96.
PMID : 11526031 : DOI : 10.1128/aem.67.9.4256-4263.2001 PMC : PMC93155
Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of beta-GalI and beta-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).
Dal Piaz F,
( 2007 )
Genetic and proteomic characterization of rifaximin resistance in Bifidobacterium infantis BI07.
PMID : 17408927 : DOI : 10.1016/j.resmic.2007.02.002
Rifaximin resistance in the probiotic strain Bifidobacterium infantis BI07 was studied to assess the use of an antibiotic-probiotic combination for clinical management of intestinal disorders. A rifaximin-resistant mutant was selected and a 129 bp core region of the rpoB gene was sequenced and compared with the respective sequence of the sensitive clone. A miss-sense mutation of codon 513, producing the substitution of Gln with Arg in the protein sequence, was found. The involvement of metabolic changes associated with rifaximin resistance was also investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. The principal categories of proteins, whose expression levels varied as a consequence of rifaximin resistance, included chaperonins, regulatory factors and metabolic enzymes. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was excluded, as neither structural modifications nor degradation derivates of the drug moiety was identified using liquid chromatography coupled with tandem mass spectrometry.
( 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
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).
de los Reyes-Gavilán CG,
( 2007 )
Induction of alpha-L-arabinofuranosidase activity by monomeric carbohydrates in Bifidobacterium longum and ubiquity of encoding genes.
PMID : 17031615 : DOI : 10.1007/s00203-006-0181-x
Bifidobacterium longum can be isolated from human faeces, some strains being considered probiotics. B. longum NIZO B667 produces an exo-acting alpha-L-arabinofuranosidase, AbfB, previously purified by us, that releases L-arabinose from arabinan and arabinoxylan. This activity was subjected to two-seven-fold induction by L-arabinose, D-xylose, L-arabitol and xylitol and to repression by glucose. Maximum activity was obtained at 48 h incubation except for D-xylose that was at 24 h. High concentrations (200 mM) of L-arabitol also caused repression of the arabinofuranosidase. A unique band of activity showing the same migration pattern as the purified AbfB was found in zymograms of cell free extracts, indicating that the activity was likely due to this sole enzyme. The assessment of the influence of inducers and repressors on the activity of AbfB and on the expression of the abfB gene by real time PCR indicated that regulation was transcriptional. DNA amplifications using a pair of degenerated primers flanking an internal fragment within alpha-L-arabinofuranosidase genes of the family 51 of glycoside hydrolases evidenced that these enzymes are widespread in Bifidobacterium. The aminoacidic sequences of bifidobacteria included a fragment of four to six residues in the position 136-141 that was absent in other microorganisms.
( 2002 )
Bifidobacteria identification based on 16S rRNA and pyruvate kinase partial gene sequence analysis.
PMID : 16887679 : DOI : 10.1016/S1075-9964(03)00025-8
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.
Del Casale A,
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
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.
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
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.
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
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.
van Sinderen D,
( 2004 )
Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses.
PMID : 15466567 : DOI : 10.1128/AEM.70.10.6197-6209.2004 PMC : PMC522111
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.
van Sinderen D,
( 2004 )
Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny.
PMID : 15128574 : DOI : 10.1128/aem.70.5.3110-3121.2004 PMC : PMC404453
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.
Gueneau de Novoa P,
( 2004 )
The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.
PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
( 2003 )
Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification.
PMID : 14602655 : DOI : 10.1128/aem.69.11.6908-6922.2003 PMC : PMC262312
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.
( 2011 )
Bifidobacteria can protect from enteropathogenic infection through production of acetate.
PMID : 21270894 : DOI : 10.1038/nature09646 DOI : 10.1038/nature09646
The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology and pathology. Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated 'omics' approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.
( 2010 )
Immunomodulatory impact of a synbiotic in T(h)1 and T(h)2 models of infection.
PMID : 20929951 : DOI : 10.1177/1753465810379009
The immunomodulatory activity of a synbiotic combination containing three bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium bifidum R0071) and short-chain fructooligosaccharide was examined in two distinct infectious rat models. In the T(h)1 model, Wistar rats were administered the synbiotic combination for 2 weeks prior to challenge with a single oral dose of enterotoxigenic Escherichia coli or vehicle. In the T(h)2 model, pretreated rats were challenged with a single subcutaneous dose of hook worm, Nippostrongylus brasiliensis. Blood samples were collected 3 hours or 4 days postchallenge and serum levels of pro- and anti-inflammatory cytokines were measured. Significant reductions in pro-inflammatory cytokines interleukin (IL)-1�\, IL-1�], IL-6, and tumour necrosis factor (TNF)-�\ were observed in both models suggesting a single, unifying mode of action on an upstream regulator. The N. brasiliensis study also compared the effect of the individual strains to synbiotic. For most of cytokines the combination appeared to average the effect of the individual strains with the exception of IL-4 and IL-10 where there was apparent synergy for the combination. Furthermore, the cytokine response varied by strain. It was concluded that this synbiotic combination of these three microbes could be beneficial in both T(h)1 and T(h)2 diseases.
( 2010 )
Broad conservation of milk utilization genes in Bifidobacterium longum subsp. infantis as revealed by comparative genomic hybridization.
PMID : 20802066 : DOI : 10.1128/AEM.00675-10 PMC : PMC2976205
Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.
( 2008 )
The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome.
PMID : 19033196 : DOI : 10.1073/pnas.0809584105 PMC : PMC2596198 DOI : 10.1073/pnas.0809584105 PMC : PMC2596198
Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.
( 2008 )
Molecular and phenotypic traits of in-vitro-selected mutants of Bifidobacterium resistant to rifaximin.
PMID : 18462927 : DOI : 10.1016/j.ijantimicag.2008.02.002
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.
( 2016 )
Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.
PMID : 27857709 : DOI : 10.3389/fmicb.2016.01747 PMC : PMC5093124
Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation "well defined strains," encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species using plate counting and targeted genotyping. Strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS) of the 16S rRNA sequence using paired-end 2 �� 250 bp Illumina MiSeq technology. Using these methods, we tested the hypothesis that products do contain the quantitative and qualitative list of labeled microbial species. We found that 17 samples (33%) were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58%) of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species, and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to determine the correct taxonomy and quantify the relative amounts of mixed microbial populations in commercial probiotic products.
( 2016 )
Fucosyllactose and L-fucose utilization of infant Bifidobacterium longum and Bifidobacterium kashiwanohense.
PMID : 27782805 : DOI : 10.1186/s12866-016-0867-4 PMC : PMC5080750
Human milk oligosaccharides (HMOs) are one of the major glycan source of the infant gut microbiota. The two species that predominate the infant bifidobacteria community, Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, possess an arsenal of enzymes including �\-fucosidases, sialidases, and �]-galactosidases to metabolise HMOs. Recently bifidobacteria were obtained from the stool of six month old Kenyan infants including species such as Bifidobacterium kashiwanohense, and Bifidobacterium pseudolongum that are not frequently isolated from infant stool. The aim of this study was to characterize HMOs utilization by these isolates. Strains were grown in presence of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3'-sialyl-lactose (3'-SL), 6'-sialyl-lactose (6'-SL), and Lacto-N-neotetraose (LNnT). We further investigated metabolites formed during L-fucose and fucosyllactose utilization, and aimed to identify genes and pathways involved through genome comparison. Bifidobacterium longum subsp. infantis isolates, Bifidobacterium longum subsp. suis BSM11-5 and B. kashiwanohense strains grew in the presence of 2'-FL and 3'- FL. All B. longum isolates utilized the L-fucose moiety, while B. kashiwanohense accumulated L-fucose in the supernatant. 1,2-propanediol (1,2-PD) was the major metabolite from L-fucose fermentation, and was formed in equimolar amounts by B. longum isolates. Alpha-fucosidases were detected in all strains that degraded fucosyllactose. B. longum subsp. infantis TPY11-2 harboured four �\-fucosidases with 95-99 % similarity to the type strain. B. kashiwanohense DSM 21854 and PV20-2 possessed three and one �\-fucosidase, respectively. The two �\-fucosidases of B. longum subsp. suis were 78-80 % similar to B. longum subsp. infantis and were highly similar to B. kashiwanohense �\-fucosidases (95-99 %). The genomes of B. longum strains that were capable of utilizing L-fucose harboured two gene regions that encoded enzymes predicted to metabolize L-fucose to L-lactaldehyde, the precursor of 1,2-PD, via non-phosphorylated intermediates. Here we observed that the ability to utilize fucosyllactose is a trait of various bifidobacteria species. For the first time, strains of B. longum subsp. infantis and an isolate of B. longum subsp. suis were shown to use L-fucose to form 1,2-PD. As 1,2-PD is a precursor for intestinal propionate formation, bifidobacterial L-fucose utilization may impact intestinal short chain fatty acid balance. A L-fucose utilization pathway for bifidobacteria is suggested.
( 2016 )
Functional characterization of a novel �]-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697 on structurally diverse fructans.
PMID : 27086652 : DOI : 10.1111/jam.13154
In this study, we describe the isolation of a gene encoding a novel �]-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697, and the characterization of the enzyme, the second one found in this strain, significantly different in primary sequence to the already reported bifidobacterial �]-fructofuranosidases. The gene, found through genome-mining was expressed in Escherichia coli C41(DE3). The recombinant enzyme (B.longum_l1) has a molecular weight of 75 kDa, with optimal activity at 50�XC, pH 6�P0-6�P5, and a remarkable stability with a half-life of 75�P5 h at 50�XC. B.longum_l1 has a wide specificity for fructans, hydrolysing all substrates through an exo-type mechanism, including Oligofructose P95 (�]2-1 fructooligosaccharides (FOS), DP 2-8), Raftilose Synergy 1(�]2-1 FOS & inulin, DP 2-60), Raftiline HP (inulin, DP 2-60), bacterial inulin (3000 kDa) and levan (8�P3 & 3500 kDa), Agave fructans (mixed fructans, DP 3-29) and levan-type FOS (�]2-6 FOS, DP 2-8), with the highest relative activity and turnover number found for levan-type FOS. The apparent affinity of the enzyme for levan-type FOS and Oligofructose P95 was found to be 9�P2 and 4�P6 mmol l(-1) (Km) with a specific activity of 908 and 725 �gmol min(-1) mg(-1) of protein (k2), respectively, more than twice the activity for sucrose. B.longum_l1 is a wide substrate specificity enzyme, which may contribute to the competitiveness and persistence of this strain in the colon. The bifidobacterial �]-fructofuranosidase activity was evaluated with a wide variety of substrates including noncommercial fructans, such as levan-type and mixed agave fructans. Its activity on these substrates certainly strengthens their commercial prebiotic character and contributes to the understanding of bifidobacteria stimulation by structurally diverse fructans.
( 2015 )
Subspeciation of Bifidobacterium longum by multilocus approaches and amplified fragment length polymorphism: Description of B. longum subsp. suillum subsp. nov., isolated from the faeces of piglets.
PMID : 26007614 : DOI : 10.1016/j.syapm.2015.05.001
The species Bifidobacterium longum is currently divided into three subspecies, B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis. This classification was based on an assessment of accumulated information on the species' phenotypic and genotypic features. The three subspecies of B. longum were investigated using genotypic identification [amplified-fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and multilocus sequence typing (MLST)]. By using the AFLP and the MLSA methods, we allocated 25 strains of B. longum into three major clusters corresponding to the three subspecies; the cluster comprising the strains of B. longum subsp. suis was further divided into two subclusters differentiable by the ability to produce urease. By using the MLST method, the 25 strains of B. longum were divided into eight groups: four major groups corresponding to the results obtained by AFLP and MLSA, plus four minor disparate groups. The results of AFLP, MLSA and MLST analyses were consistent and revealed a novel subspeciation of B. longum, which comprised three known subspecies and a novel subspecies of urease-negative B. longum, for which the name B. longum subsp. suillum subsp. nov. is proposed, with type strain Su 851(T)=DSM 28597(T)=JCM 19995(T).
( 2014 )
Insights into the binding specificity and catalytic mechanism of N-acetylhexosamine 1-phosphate kinases through multiple reaction complexes.
PMID : 24816108 : DOI : 10.1107/S1399004714004209
Utilization of N-acetylhexosamine in bifidobacteria requires the specific lacto-N-biose/galacto-N-biose pathway, a pathway differing from the Leloir pathway while establishing symbiosis between humans and bifidobacteria. The gene lnpB in the pathway encodes a novel hexosamine kinase NahK, which catalyzes the formation of N-acetylhexosamine 1-phosphate (GlcNAc-1P/GalNAc-1P). In this report, seven three-dimensional structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP from both Bifidobacterium longum (JCM1217) and B. infantis (ATCC15697) were solved at resolutions of 1.5-2.2 ?. NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The NahK structures presented here represent the first multiple reaction complexes of the enzyme. This structural information reveals the molecular basis for the recognition of the given substrates and products, GlcNAc/GalNAc, GlcNAc-1P/GalNAc-1P, ATP/ADP and Mg(2+), and provides insights into the catalytic mechanism, enabling NahK and mutants thereof to form a choice of biocatalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.
( 2012 )
Endo-�]-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins.
PMID : 22745059 : DOI : 10.1074/mcp.M112.018119 PMC : PMC3434770
Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-�]-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(�\1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.
( 2012 )
1,3-1,4-�\-L-fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains.
PMID : 22451675 : DOI : 10.1074/jbc.M111.333781 PMC : PMC3351332
�\-L-fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially �\-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an �\-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Gal�]1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an �\-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-�\-L-fucosidase (EC 126.96.36.199) and �\-L-fucosidase (EC 188.8.131.52) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.
( 2012 )
Bifidobacterium longum subsp. infantis ATCC 15697 �\-fucosidases are active on fucosylated human milk oligosaccharides.
PMID : 22138995 : DOI : 10.1128/AEM.06762-11 PMC : PMC3264123
Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 �\-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 �\-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.
( 1997 )
Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine.
PMID : 9311137 : DOI : 10.1111/j.1574-6968.1997.tb12670.x
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.
( 2013 )
Genetic diversity of bile salt hydrolases among human intestinal bifidobacteria.
PMID : 23591474 : DOI : 10.1007/s00284-013-0362-1 PMC : PMC3722454
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.
( 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
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.