FIG 8.

The C. burnetii dif site and XerL can functionally replace endogenous dif/Xer in L. pneumophila. (A) Sequence alignment of the L. pneumophila dif site with the plasmid and chromosomal dif sites of Coxiella burnetii. The gray arrows indicate the inverted repeat portion of the sequence. (B) Circos plot showing GC skew across the chromosome of several C. burnetii strains. Each ring corresponds to one genome, and the black lines indicate the position of the dif site. C. burnetii strains from inside to outside: 3262, Heizburg, Dugway FJ108-111, RSA 493, CbuG_Q212, Schperling, and CbuK_Q154. (C) Strains were generated in which the endogenous L. pneumophila dif was replaced by the C. burnetii chromosomal dif site (dif_{Cb_chr}) or plasmid dif site (dif_{Cb_pl}) and the endogenous XerL was replaced by C. burnetii XerL or a predicted catalytic point mutant (Y389F). Colony sizes for each strain were quantified after 4 days of growth at 37°C on CYET spread plates. Each colony radius is plotted as a separate data point, with the geometric mean shown by the purple line. (D) Intramolecular recombination assays in Lp02 Δlpg1867 strains containing a kanamycin reporter cassette flanked by the C. burnetii chromosomal dif site (dif_{Cb_chr}; left panel) or C. burnetti plasmid dif site (dif_{Cb_pl}; right panel). After electroporation with the indicated expression plasmids, the transformants were plated on selective and nonselective media to quantify “uninduced” excision. For the “induced” samples, the transformants underwent an additional overnight incubation with 100 ng/mL anhydrotetracycline prior to plating on selective and nonselective media. (E) Intermolecular recombination assay in Lp02 strains containing C. burnetii dif/Xer components. As indicated below the plot, the nonreplicative plasmids contained a sequence corresponding to the dif site from the C. burnetii chromosome (Cb_chr) or plasmid (Cb_pl). These were transformed into strains encoding the C. burnetii XerL (wild type or mutant) and containing the Cb_chr or Cb_pl dif in place of the wild-type L. pneumophila dif on the chromosome. Wild-type L. pneumophila Lp02 strains transformed with a plasmid containing the LME-1 att site or L. pneumophila dif site were included as positive controls. Each dot shows the value obtained from a single experiment, and each horizontal line represents the mean of 3 independent experiments. The Cb_chr* dif site is a truncated version of the chromosomal dif site, with truncations made to mimic the shorter 22-bp LME-1 att site version of dif_Lp.