title: Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis.
abstract: A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH260PZ, Mid Lothian, Scotland, United Kingdom.
authors.forename: Caray A
authors.initials: CA
authors.lastname: Walker
authors.forename: Willie
authors.initials: W
authors.lastname: Donachie
authors.forename: David G E
authors.initials: DG
authors.lastname: Smith
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fontaine
mesh_terms: D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016925:Corynebacterium pseudotuberculosis / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial