Fig 2.

ToxRS regulates the expression of T3SS-1 genes and leuO (VP0350). (A) The expression levels of two genes encoding T3SS-1 substrates (VP1680 and VP1656) and two genes encoding T3SS structural proteins (VP1694 and VP1695) were examined in the ΔtoxRS and isogenic wild-type strains. An unpaired Student's t test was used to compare differences in gene expression in the ΔtoxRS strain to expression in wild-type cells. *, P < 0.05. (B) The expression of gene VP0350, which encodes a LysR-family transcriptional regulator, was examined in the ΔtoxRS and wild-type strains and was grown under standard conditions as well as ToxR-inducing conditions (pH 5). An unpaired Student's t test was used to compare differences in VP0350 expression under acid conditions and in the ΔtoxRS strain to expression in wild-type cells grown in LB, 3% NaCl, pH 7. *, P < 0.05; ***, P < 0.001. (C) The expression of VP1680, which encodes the T3SS-1 effector protein VopQ, was examined in the ΔtoxRS, ΔVP0350, and WBW0350 strains. An unpaired Student's t test was used to compare differences in VP1680 expression in the ΔtoxRS strain, ΔVP0350, and WBW0350 strains to expression in the wild type. *, P < 0.05. WBW0350 is a ΔtoxRS strain that ectopically expressed VP0350. Bars represent the relative expression of the given gene normalized to the 16S rRNA gene and compared to expression in wild-type cells grown in LB, 3% NaCl, pH 7. RNA was extracted for each condition on two separate occasions, and qPCR was run in duplicate for each sample. Error bars indicate standard deviations.