FIG. 2.

RAW 264.7 macrophage survival and replication assays with individual SPI2 mutants. (A) Mutants were placed in a background where the luxCDABE operon, under the control of an rpsM promoter, was constitutively expressed. The level of intracellular, gentamicin-protected bacteria was tracked by light production. Individual mutants are indicated on the x axis. The ssrA and ssrB genes were the only genes tested as a double mutant, as they are part of the same SPI2 regulatory unit. Each strain was tested in quadruplicate. The average log₁₀ relative light units (RLU) are reported with normalization to wild-type SL1344 levels and to the levels at 3 h postinfection as described in Materials and Methods. These results are representative of multiple assays. (B) The same strains were assayed by traditional colony counts. Each strain was tested in triplicate, and the average log₁₀ CFU/milliliter are reported after normalization to wild-type SL1344 levels and to the levels at 3 h postinfection. Open circles indicate genes that appear in the macrophage SGL as negatively selected, closed circles indicate genes that are represented in the macrophage microarray data set but did not appear in the SGL, and strains without any symbols are not represented in the microarray data set. Hours postinfection are indicated, mutants are ordered as they appear in the genome, and none of the strains displayed a growth defect in LB. Standard errors are reported, and asterisks indicate a P value of <0.01 as calculated using an unpaired t test comparing each mutant to the wild-type strain for each respective time point.