Overcoming the Restriction Barrier to Plasmid Transformation and Targeted Mutagenesis in Bifidobacterium Breve UCC2003

Overview

Journal Microb Biotechnol

Specialties Biotechnology
Microbiology

Date 2011 Jan 26

PMID 21261927

Citations 72

Authors

Mary OConnell Motherway

Jonathan ODriscoll

Gerald F Fitzgerald

Douwe Van Sinderen

Affiliations

Soon will be listed here.

Abstract

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.

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