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: In order to verify the prediction that M.BbrI, M.BbrII and M.BrrIII represent distinct MTases that protect, based on their similarities to characterized R–M systems, DNA sequences cut by BbeI, SalI and PstI, respectively, genomic DNA of B. breve UCC2003 was restricted with these enzymes and analysed by agarose gel electrophoresis. The results obtained showed that B. breve UCC2003 genomic DNA is protected from restriction with BbeI and PstI, but not SalI (Fig. 3A).
Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology and Department of Food and Nutritional Sciences , National University of Ireland, Cork, Western Road, Cork, Ireland.