Supplemental Material

This article contains the following supporting material:

• Supplementary Figure 1 - Assessment of the calcium and pH sensitivity of the Mg²⁺-sensitive dye coumarin 343 (C343).

  A: Effect of calcium on the Mg²⁺ responsiveness of C343. Emission spectra of C343 were recorded at varying [MgCl₂] in a buffer containing 50 mM MOPS containing either 2 mM Ca²⁺ (black circles) or 0 mM Ca²⁺ (open squares). The changes in fluorescence intensity (ΔF) at the emission peak are plotted as a function of [MgCl₂]. Curve fitting was used to determine the apparent Kd for Mg²⁺ and the resulting values are shown in the inset. B: Effect of pH on the Mg²⁺ responsiveness of C343. The Mg²⁺-dependence of C343 fluorescence was measured as above and the apparent K_d was determined graphically as above. The calculated K_d values are plotted as a function of pH. Data are means ± SE of 3 separate experiments.

• Supplementary Figure 2 - Time course of induction of phoP::GFP inside macrophages, determined by live cell imaging. RAW264.7 macrophages were infected with S. Typhimurium pMLZ205 for 15 min (MOI of 10). Cells were placed on the heated stage of a confocal microscope and imaged repeatedly for up to 3 h. Short exposures and low excitation intensity were used to minimize photobleaching.

  A: The two leftmost panels show the DIC and corresponding fluorescence image of a macrophage infected with bacteria 30 min prior to image acquisition. Arrows point to the location of bacteria, which fluoresce only weakly at this time. The fluorescence intensity of these bacteria increased progressively over time, as shown. B: Quantification of the fluorescence intensity in the bacteria illustrated in A. Note that saturation of the signal is due to the limited dynamic range of the acquisition system (a CCD camera) and does not reflect completion of MphoP::GFP induction.

• Supplementary Figure 3 - Induction of phoP::GFP in epithelial cells. HeLa and MDCK cells were infected with S. Typhimurium pMLZ205 at a MOI of 10. Cells were harvested, fixed, permeabilized and stained for LPS to quantify the number of bacteria. Flow cytometric analysis of infected cells was as described for macrophages.

  A: Time course of GFP expression in HeLa (squares) and MDCK cells (triangles). B. Time course of GFP expression in HeLa cells infected with S. Typhimurium pMLZ205 and either left otherwise untreated (solid squares) or treated with CcA immediately after infection. phoP::GFP induction was analyzed as in A.