Pan-genome and methylome analysis reveals the diversity of restriction/modification systems in the gut commensal Bifidobacterium breve.

Bifidobacterium breve represents one of the most abundant (bifido)bacterial species in the gastro-intestinal tract of (breast-fed) infants, where their presence is believed to be beneficial. In the present study whole genome sequencing, employing PacBio’s Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variome. Availing of the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BiSeq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, cloning of a selected methyltransferase-encoding gene validated the activity of the corresponding R/M system, and was shown to overcome the barrier they impose to genetic accessibility, thus allowing future genetic manipulation of members of this species. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Following growth, total bifidobacterial DNA was isolated as described by O’Riordan et al. (1998) and then labelled using the Kreatech ULS labelling kit according to the manufacturer’s instructions (Kreatech, Amsterdam, The Netherlands). Labelled total gDNA was hybridised as described in the Agilent manual Two-Color Microarray-Based Gene Expression Analysis (v4.0) (publication no. G4140-90050) to the B. breve UCC2003 microarray slides (feature format: 4 x 44K and 2 x 11 K) overnight at 65 °C (NB. B. breve UCC2003 was used as the reference strain and as a positive control). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data sets were processed as previously described (Garcia De La Nava et al., 2003). Differential signal intensity tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). Data were analysed and visualized using the Multi-experiment Viewer Tmev v.4.9 with hierarchical clustering analysis based on covariance method and complete linkage.