Cultivation and Storage of C. burnetii

Bacterial strains used in this study are listed in Table S1. The C. burnetii Nine Mile phase II (RSA439, clone 4, NMII) strain was used for this work. NMII C. burnetii and genetic transformants were axenically cultured in ACCM‐D as previously described (Sandoz et al., 2014 (link)), with the addition of 4‐hydroxyphenylpyruvic acid (4‐HPP) for tyrosine‐based nutritional selection. For storage, bacteria were pelleted following 7 days of growth, washed three times in phosphate‐buffered saline (PBS; 1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4, pH 7.4), and then suspended in a freezing medium (ACCM‐D containing 10% dimethyl sulfoxide) and frozen at −80°C. E. coli Stellar (Takara) and E. coli PIR2 (Invitrogen) cells were used for recombinant DNA procedures and cultivated in Luria‐Bertani (LB) broth or terrific broth. THP‐1 macrophages (TIB‐202; ATCC) and African green monkey kidney (Vero) cells (CCL‐81; ATCC) were maintained in an RPMI‐1640 medium containing 10% FBS at 37°C and 5% CO₂. C. burnetii replication in host cells or in ACCM‐D was measured by quantitative PCR of genome equivalents (GE) as previously described (Howe et al., 2010 (link); Omsland et al., 2009 (link)) using a probe specific to groEL.