Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve

Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastrointestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio 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 genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) 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, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species.</p

Identification of Restriction-Modification Systems of <i>Bifidobacterium animalis</i> subsp. <i>lactis</i> CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis

<div><p><i>Bifidobacterium animalis</i> subsp. <i>lactis</i> CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.</p></div

Transformation efficiency of <i>B. animalis subsp. lactis</i> CNCM I-2494.

<p>Transformation with pDM1 plasmid DNA isolated from CNCM I-2494, <i>E. coli</i> pWSK29, <i>E. coli</i> pWSK29-M.BanLI, <i>E. coli</i> pWSK29-M.BanLII or <i>E. coli</i> pWSK29-M.BanLI-M.BanLII. Data presented are averages of triplicate experiments.</p

Transformation of <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494.

<p>pDM1 or pDM2 DNA was isolated from <i>E. coli</i> (grey bars) or <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494 (black bars). Data presented are averages of triplicate experiments.</p

Figure 4

<p><b>(A)</b>. Restriction analysis of total DNA from <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494. Lane1, molecular weight marker X (Roche). Lane 2: Unrestricted total DNA from <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494, Lane 3–5 total <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494 DNA restricted with lane 3, HindIII; lane 4, AvaII and lane 5, EcoRI. <b>(B)</b> Restriction analysis of plasmid DNA isolated from three representative <i>E. coli</i> Xl1Blue -pWSK29-M.BanLII. Plasmid DNA from <i>E. coli</i> Xl1Blue -pWSK29-M.BanLII restricted with PstI and XbaI, lanes 2–4 (expected and obtained, product sizes of PstI +XbaI digests are 5420 bp and 1605 bp), or AvaII, lanes 5–7.</p

Methylome determination for <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494.

<p>Methylated motifs from the methylome analysis are depicted in Panel A. Methylated motifs are highlighted in yellow (5mC) or orange (6mA). Panel B, (I) and (II) illustrate the MTase specificities determined from the genomic positions detected as methylated. The methylated positions are indicated by red text with an asterisk.</p

Bacterial strains, bacteriophage and plasmids used in this study.

<p>Bacterial strains, bacteriophage and plasmids used in this study.</p

Restriction analysis of plasmid DNA.

<p>Restriction analysis of total plasmid DNA isolated from three representative <i>E. coli</i> Xl1Blue-pWSK29-M.BanLI-M.BanLII transformants harbouring pDM1. Lane 1, molecular weight marker X (Roche). Total plasmid DNA from <i>E. coli</i> Xl1Blue -pWSK29-M.BanLI-M.BanLII + pDM1 restricted with Sal1, lanes 2–4 or AvaII, lanes 5–7. The sizes of the expected and obtained SalI restriction fragments for pWSK29-M.BanLI-M.BanLII component of the total plasmid preparation are 7019bp and 1353bp (and indicated in red) while for pDM1 the expected restriction fragment sizes are 3801bp and 2565bp (and indicated in blue). The expected SalI digest restriction fragment sizes for the total plasmid complement of <i>E. coli</i> Xl1Blue -pWSK29-M.BanLI-M.BanLII + pDM1 are indicated to the left of the figure.</p

Schematic representation of the <i>E. coli</i>-bifidobacterial shuttle vectors pDM1 and pDM2.

<p>pDM1 is a derivative of pAM5 where the tetracycline resistance gene has been replaced with the spectinomycin resistance cassette. pDM2 is a derivative of the plasmid pDG7 where the spectinomycin resistance cassette was cloned in the unique EcoR1 and HindIII sites of pDG7.</p