(A) A clone of C. burnetii with a 3.69 kb transposon inserted in the icmL gene (dotI/icmL.1) was isolated after enrichment on medium containing kanamycin. The schematic shows the predicted location and orientation of the transposon in icmL as determined by sequence analysis. The location of probes and restriction sites used to validate the site of transposon insertion by PCR and Southern hybridization are indicated. (B) As indicated, genomic DNA digested with HindIII isolated from the C. burnetii NM strain and the isogenic icmL::Tn mutant was analyzed by Southern hybridization using a probe derived from the icmL gene and a probe derived from the transposon. As predicted from the schematic in panel A, the icmL probe identified a single band of 4.2 kb in the NM strain, and two bands of 3.3 kb and 2.5 kb in the icmL::Tn mutant because of a new HindIII site introduced into the icmL locus by the transposon. When a probe homologous to the transposon was used (Tn probe), a single band was identified in the icmL::Tn mutant, indicating that this strain has a single insertion of the transposon in the chromosome. (C) PCR amplification of genomic DNA from NM and the icmL::Tn mutant using primer sets shown in panel A confirm the predicted location and orientation of the transposon insertion in icmL, and that the transposon is retained by the icmL::Tn strain after introduction of a plasmid encoding icmL (pIcmL) or a plasmid encoding the entire operon harboring icmL (pQC). (D,E) The ability of the icmL::Tn mutant to replicate in HeLa (D) and Vero (E) cells was determined by measuring genomic equivalents (y-axis) at the indicated times after infection (x-axis). There was a 50 to 100-fold increase over 7 days in C. burnetii NM genomic units (black squares). No significant increase in genomic units was detected for the icmL::Tn mutant (white squares) or C. burnetii NM treated with 10 µg/ml chloramphenicol (gray squares). (F,G) The ability of the icmL::Tn mutant to replicate in HeLa (F) and Vero (G) cells was determined by measuring genomic equivalents (y-axis) at the indicated times after infection (x-axis). Replication was observed for C. burnetii NM containing pBlaM (black squares) and the icmL::Tn mutant containing the plasmid pQC (black triangles). The icmL::Tn mutant containing the vector pBlaM (white squares) or for the icmL::Tn mutant containing pIcmL (white triangles). Graphs represent the mean ± SD of at least three independent experiments. (H) Fluorescence micrographs were acquired after infection of HeLa cells for 5 days by the strains of C. burnetii indicated. An anti-Coxiella antibody (green) was used to visualize intracellular bacteria and the nucleus of the host cell was visualized by DAPI staining (blue). Replicating bacteria in large vacuoles were observed for cells infected with C. burnetii NM, C. burnetii NM containing pBlaM and the icmL::Tn mutant containing pQC. Only individual bacteria were observed inside the host cells infected with the icmL::Tn mutant, C. burnetii NM in medium with chloramphenicol, icmL::Tn containing pBlaM and icmL::Tn containing pIcmL (indicated with arrows). These are representative images from at least three independent experiments.