B420 and Bi-07 are strains sold commercially as probiotics due to their ability to confer health benefits to humans (10
). The whole-genome sequence for each B. animalis
strain was obtained by utilizing FLX Titanium 454 sequencing (15
). A total of 77,519 and 81,767 reads were generated for B420 and Bi-07, respectively, with an average coverage of 18×. Genome assemblies were scaffolded against strain Bl-04 using NGen (DNAStar, Madison, WI) software and subsequently inspected for quality using SeqMan Pro (DNAStar, Madison, WI). Gaps in the sequences were closed by PCR followed by Sanger sequencing. Both genomes were annotated using RAST (2
) and manually curated. Closed genomes were aligned against the previously sequenced genomes of B. animalis
Bl-04, DSMZ 10140, AD011, V9, BLC1, BB-12, and CNCM I-2494 using progressiveMauve (3
). The alignment highlighted several conspicuous ~5- to 30-bp deletions in both the CNCM I-2494 and BB-12 strains which occur mostly in intergenic regions that may be difficult to sequence or assemble correctly.
In order to determine the overall differences in the two new sequences, alignment and single nucleotide polymorphism (SNP) analysis were done using progressiveMauve (7
). Previously, an SNP detection method was derived from differences identified in strains Bl-04 and DSMZ 10140 (3
) that determined genetic differences in strains across 35 loci. Overall, we identified 9 never-reported SNPs in Bi-07 and one new SNP in B420 compared to the type strain DSMZ 10140. These novel SNPs complement and confirm the two previously defined groups that diverge most notably by an indel in the clustered regularly interspaced short palindromic repeat (CRISPR) locus and provide a new sequence from each phylogenetic branch. From the newly reported SNPs, a single nonsynonymous mutation was detected in Bi-07 which substitutes a glycine for arginine in a putative β-xylosidase gene. The remaining SNPs were either synonymous substitutions or found in intergenic regions.
The sequences of the commercialized strains B420 and Bi-07 were used to evaluate the qualified presumption of safety (QPS) requirements by the European Food Safety Authority (EFSA) under the qualifications that strains must not harbor acquired antimicrobial resistance genes to clinically relevant antimicrobials (8
). The presence of a tetracycline resistance gene, tetW
, flanked by a putative transposase gene in B. animalis
was confirmed for both strains, although phenotypically, Bi-07 tested below the EFSA breakpoint (9
). Comparative genomics revealed that there were no differences in the sequence of the tetW
resistance gene or within 5 kb of the gene. While regulation of the tetW
gene in B. animalis
is still unknown, no genetic change potentially involved in differential regulation was observed (1
). These genomes confirm synteny and the high level of genomic sequence conservation within this species and set the stage for a refined typing scheme.
Nucleotide sequence accession numbers.
The complete genome sequences of B. animalis subsp. lactis strains B420 and Bi-07 have been deposited in GenBank under the following numbers: for B420, CP003497, and for Bi-07, CP003498.