Culture preparation, RNA extraction, and RT-qPCR.

Diamide-treated cultures of B. anthracis Sterne 7702 SR1 (41) and deletion derivatives, ΔspxA1 and ΔspxA2, each harboring either ICEBs1::PspxA2_{(−247/+268)}-lacZ or ICEBs1::PspxA2_(−49/+268)-lacZ (see Table S1 in the supplemental material), were grown in 40 ml LB at 37°C, 200 rpm until mid-exponential phase (OD₆₀₀ of 0.2 to 0.4) and split into two equal volumes. Freshly prepared diamide (0.25 mM diamide; Sigma-Aldrich, St. Louis, MO) was added to one set of cultures and, after incubation for an additional 10 min, the cells were harvested by centrifugation (5,180 × g, 4°C, 10 min), and the cell pellets were frozen at −80°C until use. RNA was purified as previously described (51). The resulting RNA concentrations were measured by UV spectrophotometry. The RNA quality was assessed by measuring the ratio of the absorbance at 260 nm to the absorbance at 280 nm, as well as by visualization in agarose gels. cDNA was prepared from each RNA sample using random primers and Invitrogen SuperScript III RT (Life Technologies) in accordance with the manufacturer's protocol. Triple technical replicates were performed for each quantitative real-time PCR (RT-qPCR) assay, from three independently isolated RNA samples, in a 96-well plate using an ABI Prism Step-One Plus with Step-One Plus (Life Technologies) software version 2.0 sequence detection system. The annealing temperature was 58°C, and extension was performed at 72°C for 1 min for 40 cycles. The primer sequences (see Table S3 in the supplemental material) were designed to specifically amplify within the lacZ gene. The amplification efficiencies were roughly equivalent across all primer sets (E% = 80 to 110). Control reactions (cDNA reactions lacking RT) were performed to verify that no genomic DNA contamination was present (that is, the threshold cycle [C_T] for detection in the control without RT was above 35). Normalization of C_T values was done relative to the signal obtained from reactions amplifying a portion of the rpoB transcript (41). Transcripts (arbitrary units [AU]) were directly reported for RT-qPCR assays of diamide-treated cultures after normalization to the rpoB endogenous control.