A GENERAL SYSTEM FOR GENERATING UNLABELED GENE REPLACEMENTS IN BACTERIAL CHROMOSOMES

A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistan ce genes. The method is based on the conditional replication of deriva tives of the lactococcal plasmid pWV01, which lacks the repA gene enco ding the replication initiation protein. Replacement vectors can be co nstructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integ ration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is ide ntified in the second step of the procedure by the loss of the lacZ re porter gene present in the delivery vector. The second recombination e vent results either in gene replacement or in restoration of the origi nal copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lact ococcus lactis and Bacillus subtilis as model organisms. The results i ndicate that the method should be applicable to any non-essential gene in numerous bacterial species.