Replacement Recombination in<i>Coryneform</i>Bacteria: High Efficiency Integration Requirement for Non-methylated Plasmid DNA

Abstract

We have developed a versatile system that permits efficient manipulation of coryneform bacteria by gene disruption and replacement. Circumventing the methyl-specific restriction system in certain of these bacteria by using non-methylated plasmid DNA was necessary. Integrative plasmid DNA containing an in vitro disrupted copy of the Brevihacterium ftavum thr B gene was introduced into B. ftavum at an efficiency of 2.4 × 102 integrants/μg. Integration occurred by homologous recombination either via a Campbell-like mechanism or via a double crossover event. Integrants were stable for over 32 generations.