TARGETED INTEGRATION OF FOREIGN GENES INTO REPETITIVE SEQUENCES OF THE BREVIBACTERIUM-LACTOFERMENTUM CHROMOSOME

Directed integration of heterologous DNA into the insertion sequence I S13869 of Brevibacterium lactofermentum has been achieved by using int egrative vectors that contain a 600 bp fragment from IS13869 and a kan amycin resistance cartridge with unique PacI, SwaI and PmeI sites. The incorporation of these restriction sites for rare-cutting enzymes, wh ich recognize octanucleotide sequences, allowed rapid mapping of the l ocation of plasmid insertion into the chromosome. Mapping by pulsed-fi eld 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 integra tion events due to the presence of multiple targets in the genome.