Fig. 5. Preserving specific protease substrates during low Mg²–triggered slow growth state speeds the return to the growth state.

(A) Growth curves of wild-type (EG13918) clpS (JY881), and phoP (MS7953s) S. Typhimurium expressing PhoP-HA and wild-type S. Typhimurium expressing PhoP(L4P)-HA (JY992) under low-Mg²⁺ conditions after 3 h growth followed by 3 h treatment with chloramphenicol (Cm) to induce growth arrest or with no Cm treatment. (B) Quantification of ATP amount and (C) immunoblotting and quantification of protease substrate PhoP-HA in the indicated strains grown low-Mg²⁺ conditions for 3 h, followed by 3 h of treatment with or without Cm. GroEL is a loading control. (D) Growth curves of wild-type (14028s) and rpoS (EG18022) S. Typhimurium under Mg²⁺-replete conditions after washing antibiotics from 6 h samples in (A) and (B). (E) Quantification of ATP and (F) immunoblotting and quantification of the protease substrate RpoS in strains 14028s and EG18022 grown under low-Mg²⁺ conditions for 3 h, followed by 3 h treatment with or without Cm. OmpA is a loading control. Growth curves (A and D) show the average of 4 independent experiments, and error bars represent SDs. ATP amounts (B and E) were calculated with normalization to luminescence by OD₆₀₀, and are the average of independent experiments. Western blotting (C and F) was performed with antibodies directed to the HA peptide, GroEL, or OmpA, and densitometry graphs show the average and SD for protein amounts in different strains relative to those in the untreated bacteria at 3 h from 3 independent experiments. Unpaired Student’s t tests were performed between untreated samples at 6 h with the other combinations; **P < 0.01 and ***P < 0.001.