Positive-negative KG cassettes for construction of multi-gene deletions using a single drug marker

Positive-negative KG cassettes were developed in order to create a number of independent deletion mutations on the bacterial chromosome using a single drug marker. These cassettes consist of a kanamycin-resistant (Km(R)) gene for positive screening and a galactokinase gene (galK) for negative screening. Both genes are in an operon driven by the native Km(R) promoter and are flanked by identical fragments of yeast chromosomal DNA approximately one kb in size. An internal region of a cloned target gene of a bacterium is replaced with a cassette, which is then transformed into the bacterium. The intact gene on the chromosome is replaced with the mutated gene by homologous recombination. From the Km(R) cells thus obtained, those cells which lose both Km(R) and galK genes by homologous recombination between the identical yeast DNA fragments are subsequently screened on plates containing 2-deoxygalactose, a non-metabolizable analogue of galactose. This method was applied to isolate a triple-deletion mutant of pkn3, pkn1, and pkn11 from Myxococcus xanthus.