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Background  Bifidobacterial genome analysis has provided insights as to how these gut commensals adapt to and persist in the human GIT, while also revealing genetic diversity among members of a given bifidobacterial (sub)species. Bifidobacteria are notoriously recalcitrant to genetic modification, which prevents exploration of their genomic functions, including those that convey (human) health benefits.    Methods  PacBio SMRT sequencing was used to determine the whole genome seqeunces of two  B. longum  subsp.  longum  strains. The  B. longum  pan-genome was computed using PGAP v1.2 and the core  B. longum  phylogenetic tree was constructed using a maximum-likelihood based approach in PhyML v3.0.  M.blmNCII  was cloned in  E. coli  and an internal fragment if  arfB arfB was cloned into pORI19 for insertion mutagenesis.    Results  In this study we present the complete genome sequences of two  Bifidobacterium longum  subsp.  longum  strains. Comparative analysis with thirty one publicly available  B. longum  genomes allowed the definition of the  B. longum  core and dispensable genomes. This analysis also highlighted differences in particular metabolic abilities between members of the  B. longum  subspecies  infantis, longum  and  suis . Furthermore, phylogenetic analysis of the  B. longum  core genome indicated the existence of a novel subspecies. Methylome data, coupled to the analysis of restriction-modification systems, allowed us to substantially increase the genetic accessibility of  B. longum  subsp.  longum  NCIMB 8809 to a level that was shown to permit site-directed mutagenesis.    Conclusions  Comparative genomic analysis of thirty three  B. longum  representatives revealed a closed pan-genome for this bifidobacterial species. Phylogenetic analysis of the  B. longum  core genome also provides evidence for a novel fifth  B. longum  subspecies. Finally, we improved genetic accessibility for the strain  B. longum  subsp.  longum  NCIMB 8809, which allowed the generation of a mutant of this strain.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-015-1968-4) contains supplementary material, which is available to authorized users.

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems