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. 2018 Jan 1;32(1):79–92. doi: 10.1101/gad.309245.117

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© 2018 Pontes and Groisman; Published by Cold Spring Harbor Laboratory Press

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Figure 6. — Translation recycles intracellular Pi from ATP, preventing a Pi starvation response. (A) Intracellular ATP levels of wild-type Salmonella (14028s) lacking or harboring either pUHE-ATPase (pATPase) or pUHE-21 (pVector). (B) mRNA amounts of the phoB, pstS, and thrS genes produced by wild-type Salmonella (14028s) lacking or harboring either pATPase or pVector. (C) mRNA amounts of the pstS and thrS genes produced by wild-type (14028s) and phoB (EG9054) Salmonella. For A–C, cells were grown in MOPS containing 10 mM MgCl₂ and 2 mM K₂HPO₄ for 2 h followed by 30 min of treatment with 1 mM IPTG (when harboring pATPase or pVector) followed by 30 min of treatment with 25 µg/mL chloramphenicol. mRNA amounts were normalized to those of the ompA gene. (D) Western blot analysis of extracts prepared from wild-type (14028s) and phoB-HA (MP1429) Salmonella and phoB-HA (MP1429) Salmonella harboring either pATPase or pVector following separation on Phos-tag SDS-PAGE to detect PhoB-HA and PhoB-HA-P. Cells were grown in MOPS containing 10 mM MgCl₂ and 200 µM K₂HPO₄ for 2 h and treated for 35 min with 1 mM IPTG followed by chloramphenicol as in A–C. Error bars represent the standard deviations. Graphs are representative of at least three independent experiments with a total of at least six biological replicates. See also Supplemental Figures S3 and S4.