FIG. 1.

After antibiotic resistance cassettes are efficiently cured from the L. pneumophila chromosome, plasmids expressing the Flp recombinase can be segregated from the host strain. (A) Flp-mediated excision of a cat cassette was examined in a letA::FRT-cat-FRT strain using pMMBFlp and IPTG induction in broth and then plating on medium with and without chloramphenicol (see Table S1 in the supplemental material). Data represent means ± standard errors of the means (SEMs) of 3 experiments. (B) Loss of Flp-encoding plasmids with either an RSF1010 (pMMBFlp) or ColE1 (pBSFlp) origin of replication. For cultures that were selected for plasmid loss with sucrose, overnight cultures were first grown in the absence of sucrose and then exponential-phase cultures were normalized, resuspended, and grown overnight in ACES-buffered yeast extract broth with thymidine plus 5% sucrose without antibiotics. After cultures were plated on media with and without gentamicin, the presence of the resistance cassette carried on the vector was scored. Data represent means ± SEMs of ≥3 experiments.