Table 1.
Bacterial strains, bacteriophage and plasmids used in this study.
Figure 1.
Methylome determination for B. animalis subsp. lactis CNCM I-2494.
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.
Figure 2.
Schematic representation of the E. coli-bifidobacterial shuttle vectors pDM1 and pDM2.
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.
Figure 3.
Transformation of B. animalis subsp. lactis CNCM I-2494.
pDM1 or pDM2 DNA was isolated from E. coli (grey bars) or B. animalis subsp. lactis CNCM I-2494 (black bars). Data presented are averages of triplicate experiments.
Figure 4.
(A). Restriction analysis of total DNA from B. animalis subsp. lactis CNCM I-2494. Lane1, molecular weight marker X (Roche). Lane 2: Unrestricted total DNA from B. animalis subsp. lactis CNCM I-2494, Lane 3–5 total B. animalis subsp. lactis CNCM I-2494 DNA restricted with lane 3, HindIII; lane 4, AvaII and lane 5, EcoRI. (B) Restriction analysis of plasmid DNA isolated from three representative E. coli Xl1Blue -pWSK29-M.BanLII. Plasmid DNA from E. coli 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.
Figure 5.
Restriction analysis of plasmid DNA.
Restriction analysis of total plasmid DNA isolated from three representative E. coli Xl1Blue-pWSK29-M.BanLI-M.BanLII transformants harbouring pDM1. Lane 1, molecular weight marker X (Roche). Total plasmid DNA from E. coli 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 E. coli Xl1Blue -pWSK29-M.BanLI-M.BanLII + pDM1 are indicated to the left of the figure.
Figure 6.
Transformation efficiency of B. animalis subsp. lactis CNCM I-2494.
Transformation with pDM1 plasmid DNA isolated from CNCM I-2494, E. coli pWSK29, E. coli pWSK29-M.BanLI, E. coli pWSK29-M.BanLII or E. coli pWSK29-M.BanLI-M.BanLII. Data presented are averages of triplicate experiments.