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: Two loci, predicted to encode three different R–M systems, were identified from the annotation of the genome sequence of B. breve UCC2003 (S. Leahy. M. O'Connell Motherway, J. Moreno Munoz, G.F. Fitzgerald, D. Higgins and D. van Sinderen, unpubl. results) and designated BbrI, BbrII and BbrIII (Fig. 1A). The G+C content for each system is 58% which is in agreement with the approximately 60% G+C content for bifidobacteria (Ventura et al., 2007). The first gene of the BbrI R–M system, bbrIM, codes for a protein (M.BbrI; 43.2 kDa) with 60% and 53% identity to cytosine‐specific MTases from Clavibacter michiganesis and Photorhabdus luminescens respectively; M.BbrI also contains the six highly conserved motifs characteristic of known 5′‐methylcytosine MTases (Kumar et al., 1984) (Fig. 1B). The cytosine‐specific MTases from C. michiganesis and P. luminescens are known to methylate of the sequence 5′‐GGC(m5)GCC‐3′, which is also the recognition sequence of the BbeI REase identified by Khosaka and colleagues (1982) from B. breve YIT4006. The protein product of the second ORF, bbr0215, exhibits 94% identity to a hypothetical protein encoded by B. longum subsp. longum NCC2705 (Schell et al., 2002). The third gene of the BbrI gene cluster, bbrIR, is separated from bbr0215 by remnants of an insertion sequence element. The bbrIR gene encodes a protein (30 kDa) exhibiting low homology (33%) to various type II R–M system restriction subunits and for this reason it is predicted to represent the restriction component of the BbrI R–M system, probably an isoschizomer of BbeI.
Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology and Department of Food and Nutritional Sciences , National University of Ireland, Cork, Western Road, Cork, Ireland.