A Simple Allelic Exchange Method for Efficient Seamless Knockout of Up to 34-kbp-Long Gene Cassettes in Pseudomonas

Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp., based on a traditional allelic exchange strategy. The upstream and downstream sequences of the target gene cluster to be deleted were amplified using primers with 5'-end sequences identical to the multiple cloning sites of a suicide plasmid (mutant allele insert vector). The sequences were then fused with the linearized suicide plasmid in one step via seamless cloning. The resulting allelic exchange vector (recombinant plasmid) was introduced from the donor strain (Escherichia coli SM 10) into recipient cells (Pseudomonas putida, P. composti, and P. khazarica) via conjugation. Single-crossover merodiploids (integrates the vector into host chromosome by homologous recombination) were screened based on antibiotic resistance conferred by the plasmid, and double-crossover haploids (deleting the target gene clusters and inserted alien plasmid backbone) were selected using sucrose-mediated counterselection. Unlike other approaches, the method described herein introduces no selective marker genes into the genomes of the knockout mutants. Using our method, we successfully deleted polysaccharide-encoding gene clusters in P. putida, P. composti, and P. khazarica and generated four mutants with single-gene cassette deletions up to 18 kbp and one mutant with double-gene cassette deletion of approximately 34 kbp. Collectively, our results indicate that this method is ideal for the deletion of long genetic sequences, yielding seamless mutants of various Pseudomonas spp.