Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome

Directed integration of heterologous DNA into the insertion sequence IS13869 of Brevibacterium lactofermentum has been achieved by using integrative vectors that contain a 600 bp fragment from IS13869 and a kanamycin resistance cartridge with unique PacI, SwaI and PmeI sites. The incorporation of these restriction sites for rare-cutting enzymes, which recognize octanucleotide sequences, allowed rapid mapping of the location of plasmid insertion into the chromosome. Mapping by pulsed-field gel electrophoresis and hybridization with an IS13869 probe showed four types of transformants. In three of these types, integration of the vector occurred at each of three different sites in the chromosome. These vectors have the advantage of increasing the number of integration events due to the presence of multiple targets in the genome.