Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003.
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. By exploiting knowledge on restriction/modification in B. breve UCC2003 we successfully increased the transformation efficiency to a level that allows the reliable generation of mutants by homologous recombination using a non-replicative plasmid.
Alimentary Pharmabiotic Centre, Department of Microbiology and Department of Food and Nutritional Sciences , National University of Ireland, Cork, Western Road, Cork, Ireland.
Mary
M
O'Connell Motherway
Jonathan
J
O'Driscoll
Gerald F
GF
Fitzgerald
Douwe
D
Van Sinderen
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001644:Bifidobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics*; D016415:Sequence Alignment; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004262:DNA Restriction Enzymes
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