Constructing unmarked gene deletions in Legionella pneumophila

The ability to construct recombinant alleles efficiently in strains of interest, particularly unmarked deletions that reduce the potential for polar effects, is essential to studies of both pathogenesis and basic bacterial physiology. Here we describe a three-phase approach for generating unmarked deletions in Legionella pneumophila by constructing a mutant allele in E. coli using λ-Red recombination, so-called recombineering; transferring the allele onto the L. pneumophila chromosome by natural transformation; and then removing the selectable marker by utilizing the Flp site-specific recombinase. This strategy can decrease the amount of clone screening required while also increasing the percentage of the time the desired allele is obtained on the first attempt. The approach is particularly suited for constructing multiple unmarked deletions in a single strain in fewer steps than traditional methods.