Figure 3.
Essentiality of pyrG In Vitro and Ex Vivo
(A) Ten microliters of a pyrG cKD mutant suspension containing about 105 cfu were spotted at the indicated dilutions on Middlebrook 7H10 plates (±500 ng/ml ATc).
(B) Bacteria were grown in 7H9 medium (±500 ng/ml ATc) and diluted 1:10 in fresh media (±500 ng/ml ATc) every 3 days. OD540 nm was recorded and used to compile the growth curves. Each experiment was repeated at least twice. Gray line, pyrG conditional mutant grown without ATc; black line, pyrG conditional mutant grown with ATc.
(C) Growth of pyrG conditional mutant and its parental strain (control) in THP-1-derived macrophages at an MOI of 1:20 (bacteria/macrophage). The results are expressed as cfu per well. The reported values represent the average and the SE obtained from two parallel independent infections. The experiment was repeated twice using independent bacterial inocula and THP-1 cultures. ATc (200 ng/ml) was added or not to the cell culture medium. Circles, pyrG conditional mutant plus ATc; diamonds, control plus ATc; triangles, pyrG conditional mutant, no ATc.