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 expected phenotypic consequences of the created gene disruptions in galA and apuB, strains B. breve UCC2003, and individual representatives of B. breve UCC2003 mutants generated by insertion of pORI19‐tet‐G744 or pORI19‐tet‐G476, designated here as UCC2003‐galA‐476 and UCC2003‐galA‐744, respectively, were analysed for their ability to grow on galactan as the sole carbohydrate source (Fig. 6A). Similarly, B. breve UCC2003 and a derivative with an integrated pORI19‐tet‐apuB (designated UCC2003‐apuB‐939) were analysed for the ability to grow on starch, amylopectin, glycogen or pullulan as the sole carbohydrate source (Fig. 6B). In contrast to the wild‐type B. breve UCC2003, the B. breve UCC2003‐galA‐476 or UCC2003‐galA‐744 mutant strains failed to grow on potato galactan, while comparable growth of the parent and galA mutant strains was observed when glucose was the sole carbohydrate source. In a similar manner it was shown that B. breve UCC2003‐apuB‐939 failed to grow on starch, amylopectin, glycogen or pullulan, which contrasted with observed good growth on these substrates by the parent strain. Comparable growth for parent and mutant strains was observed when glucose was used as the sole carbohydrate source. These results confirm that the chromosomal plasmid integrations in UCC2003 cause a demonstrable phenotype and clearly illustrate the importance of the extracellular enzymes specified by galA and apuB in the metabolism of specific high‐molecular‐weight polysaccharides by B. breve UCC2003.
Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology and Department of Food and Nutritional Sciences , National University of Ireland, Cork, Western Road, Cork, Ireland.