Induction of ESX-5 genes by phosphate limitation requires RegX3. Wild-type M. tuberculosis Erdman (WT), ΔregX3, and ΔregX3 pNDregX3 were cultured in Pi-free 7H9 medium for 96 hours. RNA was extracted at 0, 24, 48, 72 and 96 hours. Abundance of the esxN, espG5, eccD5, eccB5, ppe41, modD, esxB, and espA transcripts relative to 16S rRNA was determined by quantitative RT-PCR. Data shown are the means +/− standard deviations of three independent experiments. Asterisks indicate statistically significant differences in transcript abundance between WT and ΔregX3 or ΔregX3 pNDregX3 and ΔregX3: * P < 0.05; ** P < 0.005