Abstract
Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.
GENOME ANNOUNCEMENT
Bifidobacteria are known to be the first bacteria to colonize the human intestine after birth. Various strains are frequently associated with positive effects on human health (12) and are used as probiotics. The Bifidobacterium genus is comprised of 34 species with considerable phylogenetic heterogeneity. Multilocus sequence typing (4) revealed that Bifidobacterium animalis is phylogenetically distant from other species of the genus, such as B. breve, B. longum, and B. bifidum. Within B. animalis, B. animalis subsp. lactis has drawn particular attention, mainly due to its capacity to grow and survive in milk. Several strains are applied as probiotics in dairy products, and the strain CNCM I-2494 is part of a fermented dairy product commercialized since 1987. Studies have demonstrated the ability of this product to reduce severe colitis in mice (13) and improve gastrointestinal well-being in humans (1, 7).
A total of 34,000 Sanger shotgun sequencing reads (ABI 3700) were realized on cosmid and plasmid libraries and assembled by using Phred-Phrap-Consed. Gap closure by primer walking and Sanger sequencing indicated one circular contig with an average 13.6× coverage. Subsequently, all instances of potential frameshifts and low-quality regions of the sequence were manually inspected and corrected, leading to the complete bona fide genome sequence of 1,943,113 bp.
Open reading frames (ORFs) were predicted using a combination of public (CRITICA) and proprietary similarity-based gene identification algorithms (9). The 1,660 identified ORFs were loaded into the ERGO Genome Analysis Suite (Integrated Genomics, Inc.) for annotation. The annotation was inspected manually and improved where relevant by using sequence similarity tools, such as Pfam, ProSite, ProDom, and COG. For genes without adequate similarity, tools and algorithms associated with the ERGO platform that exploit gene context information, gene fusion events, and regulatory gene clusters were used for function assignment. After manual annotation, functions were assigned to 1,214 ORFs. A genome-wide prediction for protein localization elements was also performed; SignalP v 3.0 (3) predicted 16 proteins carrying signal peptides, and the TMHMM 2.0 algorithm identified the presence of one or more putative transmembrane helices in 396 ORFs (25%). Psort analysis (5) and detection of anchor motifs (LPxTG or WxL) suggested that several proteins can be anchored to the cell wall and potentially play a role in bacterium-host interactions. These include large ORFs encoding predicted proteins homologous to proteins involved in binding to eukaryotic cells. Notably, two of them, a predicted collagen adhesion protein and a predicted fibronectin binding protein, carry a von Willebrand factor type A (vWA) domain, which has been shown to promote adhesion to extracellular matrices (10).
Complete genome sequences of several B. animalis subsp. lactis strains have been reported and compared (2, 6, 8, 11) and have revealed a high genome similarity and almost complete synteny in the subspecies. Comparison of these publically available genome sequences for strain AD011, DSMZ10140, Bl04, and BB12 to the genome sequence of strain CNCM I-2494 indicated several single-nucleotide polymorphisms (69 to 319) and from 622 to 1,227 single-base-pair indels, confirming the high level of homogeneity of B. animalis subsp. lactis.
Nucleotide sequence accession number.
The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 has been deposited in GenBank under accession number CP002915.
Acknowledgments
We thank Integrated Genomics (Chicago, IL) for sequencing efforts, Claudine Médigue (Genoscope, France) for genome comparison expertise, and Eric Johansen and colleagues (Christian Hansen, Denmark) for fruitful discussion. We acknowledge Leyla Boumghar-Bourtchai, Nathalie Goupil-Feuillerat, and Nathan McNulty for their help in genome analysis.
This work was funded by Danone Research.
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