The genome of Bifidobacterium pseudocatenulatum IPLA 36007, a human intestinal strain with isoflavone-activation activity
- Ángel Alegría†1,
- Susana Delgado†1,
- Lucía Guadamuro1,
- Ana Belén Flórez1,
- Giovanna E Felis2,
- Sandra Torriani2 and
- Baltasar Mayo1, 3Email author
© Alegría et al.; licensee BioMed Central Ltd. 2014
Received: 23 June 2014
Accepted: 16 July 2014
Published: 23 July 2014
Bifidobacterium species, including Bifidobacterium pseudocatenulatum, are among the dominant microbial populations of the human gastrointestinal tract. They are also major components of many commercial probiotic products. Resident and transient bifidobacteria are thought to have several beneficial health effects. However, our knowledge of how these bacteria interact and communicate with host cells remains poor. This knowledge is essential for scientific support of their purported health benefits and their rational inclusion in functional foods.
This work describes the draft genome sequence of Bifidobacterium pseudocatenulatum IPLA 36007, a strain isolated as dominant from the feces of a healthy human. Besides several properties of probiosis, IPLA 36007 exhibited the capability of releasing aglycones from soy isoflavone glycosides. The genome contains 1,851 predicted genes, including 54 genes for tRNAs and fie copies of unique 16S, 23S and 5S rRNA genes. As key attributes of the IPLA 36007 genome we can mention the presence of a lysogenic phage, a cluster encoding type IV fimbriae, and a locus encoding a clustered, regularly interspaced, short, palindromic repeat (CRISPR)-Cas system. Four open reading frames (orf s) encoding β-glucosidases belonging to the glycosyl hydrolase family 3, which may act on isoflavone glycosides, were encountered. Additionally, one gene was found to code for a glycosyl hydrolase of family 1 that might also have β-glucosidase activity.
The availability of the B. pseudocatenulatum IPLA 36007 genome should allow the enzyme system involved in the release of soy isoflavone aglycones from isoflavone glycosides, and the molecular mechanisms underlying the strain’s probiotic properties, to be more easily understood.
Bifidobacterium species are majority bacteria among those inhabiting the gastrointestinal tract (GIT) of animals and humans. They play important roles in maintaining human health via the digestion of foods, production of essential vitamins, and metabolization of endogenous and exogenous compounds, as well as by preventing the colonization and/or overgrowth of pathogens in the GIT . Molecular analyses have shown that members of the Bifidobacterium catenulatum group (which includes B. catenulatum and B. pseudocatenulatum) are abundant in fecal samples from adult humans [2, 3]. B. pseudocatenulatum strains have a number of probiotic properties, such as the possession of antinutrient-degrading enzymes , the ability to bind mutagenic aromatic amines , and the capacity to reduce cholesterol levels . However, compared to other bifidobacterial species, the genome of B. pseudocatenulatum has been very little explored. The Genomes Online Database (GOLD) (http://www.genomesonline.org) only contains the draft sequence of a single strain, B. pseudocatenulatum DSM 20438 (Gi02660), plus recently released incomplete sequences of five other strains (D2CA; TSDC19.1-1.1; TSDC19.1-1.2; TSDC19.1-1.3; and TSDC17.2-1.1). Sequence analysis of additional B. pseudocatenulatum strains would provide greater insight into the intra-specific variation of this species, and supply information on the genetics that underlay strain-specific capabilities. Recently, B. pseudocatenulatum has been used as a cloning host for the expression of natural  and synthetic  genes. Genomic analyses of strains of this species might allow the confident use of this bacterium in other biotechnological applications.
Bifidobacterial strains have been shown to be involved in the conversion of isoflavone glycosides into aglycones [9, 10], a key step in making isoflavones bioavailable and harnessing their estrogenic activity . Indeed, the genomes of sequenced bifidobacteria show an impressive array of genes coding for glycosyl hydrolases, including β-glucosidases, which are thought to be involved in the release of aglycones from dietary polyphenols such as soy isoflavones [12, 13]. However, the enzyme(s) involved in the hydrolysis of soy isoflavone glycosides remain(s) mostly unknown. So far, a β-glucosidase from Bifidobacterium animalis subsp. lactis has been shown to possess aglycone-releasing activity from isoflavones by cloning and expression of its encoding gene in Bifidobacterium bifidum.
The present work provides a draft genome sequence for B. pseudocatenulatum IPLA 36007, an intestinal human strain able to release aglycones from the soy isoflavone glycosides daidzin and genistin. This capability endows it with properties of interest in terms of its use in functional foods.
Materials and methods
Isolation and DNA preparation
B. pseudocatenulatum IPLA 36007 was isolated among the dominant bacteria from fecal samples of a healthy human, in a study approved by The Ethic Committee of the Asturias Principality, Spain . The strain was grown anaerobically at 37°C in MRS medium (Merck, Darmstadt, Germany) supplemented with 0.25% cysteine (Merck). Genomic DNA was extracted and purified from pure cultures using the GenElute™ Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, Miss., USA) following the manufacturer’s instructions for extracting DNA from Gram-positive bacteria. The concentration and quality of the DNA was measured using an Epoch microvolume spectrophotometer (BioTek Instruments, Winooski, Vt., USA).
Aglycone releasing-activity from isoflavone glycosides
Strains were incubated anaerobically in a MRS basal medium without dextrose and supplemented with 2% cellobiose and 100 μM daidzin or ginistin (Sigma-Aldrich) at 37°C for 24 h. One ml cultures were centrifuged and the cells suspended in the same volume of 0.1 M sodium acetate buffer pH 4.1. Isoflavones and derivatives were then extracted with ethyl acetate (Sigma-Aldrich). The organic phase was evaporated and the dried pellet suspended in 100 μl of methanol. Five 5 μl were used for analysis by TLC in silica gel 60 F254 plates (Merck). Isoflavones were separated in a toluene:acetone (2:1) solvent system, revealed by UV light at 365 nm in a transilluminator and visualized with an ImageQuant 350 (GE Healthcare Bio-Sciences, Buckinghamshire, UK).
Genome sequencing, assembly and annotation
A genomic library of 0.5 kbp was constructed and paired-end sequenced (approximately 155-fold coverage) using a HiSeq 1000 System sequencer (Illumina, Inc., San Diego, CA, USA). Quality-filtered reads were assembled in contigs using Velvet software v.1.2.10. (https://www.ebi.ac.uk/~zerbino/velvet/). Gaps within the contigs were closed by direct sequencing of amplicons obtained by PCR with oligonucleotide primers designed to anneal in the flanking regions. The genome was annotated with the RAST annotation system (http://rast.nmpdr.org/) and the NCBI Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The KEGG Pathway (http://www.genome.jp/kegg/pathway.html), Uniprot (http://www.uniprot.org) and COG (http://www.ncbi.nlm.nih.gov/COG) databases were consulted for description of specific genes and proteins. If required, DNA and deduced protein sequences were individually subjected to BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multi-blast protein comparisons were performed with the CLC Bioinformatics Database software package (CLC bio, Aarhus, Denmark).
Nucleotide sequence accession numbers
The results of this Whole Genome Shotgun project have been deposited in the GenBank database under accession number JEOD00000000. The version described in this paper is JEOD01000000.
Results and discussion
Key features of the B. pseudocatenulatum IPLA 36007 genome
Size of the genome
G + C content
(from 203 to 548,016 bp long)
Open reading frames (ORFs)
Coding sequences (CDS)
5 rRNA operons, 54 tRNA, 1 ncRNA
Plasmid free strain
22 repeats, 21 spacers, 8 CDS
Glycosyl-hydrolases and glycosyl transferases
5 glycosyl hydrolases family_3, 1 glycosyl hydrolase family_1
This strain was demonstrated plasmid-free (data not shown), and, in agreement, no plasmid-associated genes were found. However, one integrated phage of around 43.5 kbp was recorded. The lysogenic phage region consisted of 76 CDS, and included a gene encoding a retron-type RNA-directed DNA polymerase typical of group II introns . Phage-related sequences have been described in the genome of 22 strains of different bifidobacteria species, but only fragmentary information exists with regard to their functionality . As in other bacteria, bifidobacterial prophages have been shown to possess classical modular genomic organization in which the DNA lysogeny module and the DNA packaging region are the most highly conserved.
The availability of the genome of the B. pseudocatenulatum IPLA 36007 strain should allow the enzymes involved in the release of soy isoflavone aglycones from isoflavone glycosides to be known. This is essential for the rational use of IPLA 36007 as a probiotic in functional foods. The ability of IPLA 36007 to colonize the GIT could be exploited to deliver the aglycone-releasing activity straightway into the human intestine. Comparison of sequences from different sequenced strains would provide greater insights into the genetic variation within this species. It will further allow the core genome and pangenome of B. pseudocatenulatum to be identified, while contributing towards defining the gene set required to be competitive in the human GIT.
This study was supported by a project from the Spanish Ministry of Economy and Competitiveness (Ref. AGL2011-24300). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). A.B. Flórez and A. Alegría were supported by a contract of the JAE Program from CSIC and a short-term fellowship from FEMS, respectively.
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