Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria.

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta.