Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003

Microb Biotechnol. 2009 May;2(3):321-32. doi: 10.1111/j.1751-7915.2008.00071.x. Epub 2008 Dec 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Bifidobacterium / chemistry
  • Bifidobacterium / enzymology*
  • Bifidobacterium / genetics*
  • DNA Restriction Enzymes / chemistry
  • DNA Restriction Enzymes / genetics*
  • DNA Restriction Enzymes / metabolism
  • Molecular Sequence Data
  • Mutagenesis*
  • Plasmids / genetics*
  • Sequence Alignment
  • Transformation, Bacterial*

Substances

  • Bacterial Proteins
  • DNA Restriction Enzymes