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 pknII from Myxococcus xanthus