Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003.
Bottom Line: In silico analysis of the Bifidobacterium breve UCC2003 genome predicted two distinct loci, which encode three different restriction/modification systems, each comprising a modification methylase and a restriction endonuclease.Based on sequence homology and observed protection against restriction we conclude that the first restriction endonuclease, designated BbrI, is an isoschizomer of BbeI, the second, BbrII, is a neoschizomer of SalI, while the third, BbrIII, is an isoschizomer of PstI.Expression of each of the B. breve UCC2003 methylase-encoding genes in B. breve JCM 7017 established that BbrII and BbrIII are active and restrict incoming DNA.
Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology and Department of Food and Nutritional Sciences , National University of Ireland, Cork, Western Road, Cork, Ireland.Show MeSH
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Mentions: To determine the individual effect of each R–M system on the transformation frequency of B. breve UCC2003, we first introduced plasmid pAM5, which harbours one PstI, two SalI and three BbeI sites, into B. breve JCM7017 strains harbouring either pNZ8048, pNZ‐M.BbrI, pNZ‐M.BbrII or pNZ‐M.BbrIII. The methylation of the pAM5 DNA at the appropriate sequence in each of the methylase expressing strains was confirmed by restriction analysis (results not shown) prior to introducing 200 ng of each plasmid preparation into B. breve UCC2003 by electroporation. The number of transformants was determined after 48h of anaerobic incubation at 37°C on RCA with tetracycline selection (Fig. 4). pAM5 DNA isolated from JCM7017 expressing M.BbrIII allowed an almost 1000‐fold higher transformation frequency as compared with pAM5 isolated from E. coli or JCM 7017 harbouring pNZ8048. A 10‐ and 5‐fold higher transformation efficiency was observed for pAM5 isolated from JCM7017 expressing M.BbrII and M.BbrI respectively. The transformation frequency obtained with pAM5 DNA isolated from JCM 7017 expressing M.BbrIII was comparable to the transformation frequency obtained with pAM5 plasmid DNA isolated from B. breve UCC2003. However, in the latter case DNA preparations contain just the pAM5 plasmid, while in the former case the DNA preparation would have contained a mixture of pAM5 and pNZ‐M.BbrIII. These results demonstrate that the BbrIII restriction endonuclease (isoschizomer of PstI) is highly active in B. breve UCC2003 and that the activity of this restriction endonuclease appears to represent the main limitation to the genetic accessibility of B. breve UCC2003, at least for plasmid pAM5.
Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology and Department of Food and Nutritional Sciences , National University of Ireland, Cork, Western Road, Cork, Ireland.