Metabolic Control of Persister Formation in Escherichia coli Stephanie M. Amato, Mehmet A. Orman, Mark P. Brynildsen  Molecular Cell  Volume 50, Issue 4, Pages 475-487 (May 2013) DOI: 10.1016/j.molcel.2013.04.002 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Diauxic Shift Stimulates Persister Formation (A) E. coli were grown on glucose and a panel of secondary carbon sources. At hourly time points, aliquots of culture were removed, challenged with 5 μg/mL OFL for 4 hr, washed, and plated to measure cfu. To construct the color plot as a function of OD600, cfu/mL values were interpolated from two adjacent measurements to plot persister values at the designated OD600. (B) Diauxic growth (glucose-fumarate, red) results in significant persister formation (p < 0.05), whereas non-diauxic growth does not (p > 0.05) (glucose, black; glucose-fructose, green). (C) Growth on fumarate is not responsible for persister formation in glucose-fumarate media, as evidenced by sole fumarate control (blue). Data are averages of ≥ three independent experiments, error bars indicate data range, and significance was assessed using the null hypothesis that the mean cfu levels in two sample sets were equal. See also Figure S1. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Role of cAMP Addition on Persistence (A–D) At 0.02 OD600, all strains were exposed to either 8 mM cAMP or no treatment. After 2 hr, survival to 5 μg/mL OFL was measured. Wild-type (A) exhibited a significant increase in persisters due to cAMP (p < 0.05) while Δcrp (B) did not. Amino acid supplementation was added to minimal media to allow growth of ΔrelAΔspoT (C) and ΔdksA (D). Wild-type measurements in the same media are provided in Figure S2. Data are averages of ≥ three independent experiments, error bars indicate data range, and significance was assessed using the null hypothesis that the mean cfu levels in two sample sets were equal. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Role of Increased ppGpp from Serine Hydroxamate on Persistence (A) Wild-type at 0.02 OD600 were exposed to either 1 mg/mL SHX, 50 μg/mL CAM, or no treatment. After 2 hr, the survival of the culture to 5 μg/mL OFL was measured. (B and C) Cultures of wild-type (B) and ΔrelAΔspoT (C) strains were exposed to either 1mg/mL SHX or no treatment. aa-supplemented minimal media was used for both wild-type and ΔrelAΔspoT. After 2 hr, the survival of the culture to 5 μg/mL OFL was measured. Data are averages of ≥ three independent experiments, and error bars indicate data range. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Carbon Source Transitions Independent of Growth Arrest Stimulate Persisters through a ppGpp-Dependent Pathway Cells were grown in aa-supplemented minimal media and challenged with 5 μg/mL OFL at 0.02 OD600 and 0.20 OD600, representing growth on glucose and growth after glucose exhaustion, respectively (except for 4.5 mM glucose sample). Wild-type (A) exhibited an increase in persisters after the transition from glucose to fumarate; ΔrelA (B) exhibited a significantly reduced increase in persister formation compared to wild-type (p < 0.05); ΔrelAΔspoT (C) and ΔdksA (D) strains exhibited nearly no increase in persister levels after glucose exhaustion compared to wild-type (p < 0.05). Data are averages of ≥ three independent experiments, error bars indicate data range, and significance was assessed using the null hypothesis that the mutant mean fold change in persisters was equal to the wild-type fold change in persisters. See also Figure S3 and Table S1. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 ppGpp-SpoT Biochemical Network as a Metabolic TA Module (A) Within the ppGpp biochemical network is a double-negative feedback loop (blue). The toxin is ppGpp (red) and the antitoxin is SpoT (green). (B) Blue and red curves are nullclines of equations S15 and S16 (see Supplemental Experimental Procedures). Phase space trajectories are shown in the plane (SpoT, ppGpp). Filled circles indicate stable steady states; open circle indicates an unstable steady state. The initial DksA concentration was 5,000 molecules/cell for each trajectory, and parameters used are given in Tables S3 and S4. (C) PwrbA-gfp GFP distribution at 0.02 OD600 and 0.20 OD600 in glucose-fumarate and glucose media with aa supplementation. (D) PwrbA-gfp persister frequency of sorted cells relative to persister frequency in the whole population was significantly increased in the highest 10% compared to the lowest 10% of the distribution (p < 0.05). (E) PosmE-gfp GFP distribution at 0.02 OD600 and 0.20 OD600 in glucose-fumarate and glucose media with aa supplementation. (F) PosmE-gfp persister frequency of sorted cells relative to persister frequency in the whole population was significantly increased in the highest 10% compared to the lowest 10% of the distribution (p < 0.05). (G) Growth of ΔrelAΔspoT carrying RelA′ and SpoT overexpression plasmids under different induction conditions. (H) Cells were grown 2 hr after RelA′ induction and challenged with 5 μg/mL OFL. Data are averages of ≥ three independent experiments, error bars indicate data range, and significance was assessed using the null hypothesis that the mean persister level relative to the whole population in the lowest 10% was equal to the mean persister level relative to the whole population in the highest 10%. See also Figure S4 and Tables S3–S5. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Nucleoid Proteins Facilitate ppGpp-Dependent Persister Formation (A–E) Cells grown in aa-supplemented minimal media were challenged with 5 μg/mL OFL at 0.02 OD600 and 0.20 OD600. Components of three major NAPs (Δfis [A], ΔhupA [B], ΔhupB [C], ΔihfA [D], ΔihfB [E]) significantly reduced persister formation when compared to wild-type (p < 0.05). Data are averages of ≥ three independent experiments, error bars indicate data range, and significance was assessed using the null hypothesis that the mutant mean fold change in persisters was equal to the wild-type fold change in persisters. See also Figure S5 and Table S2. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Model of Fluoroquinolone Persister Formation from Carbon Source Transitions Upon glucose exhaustion, cAMP levels increase and aa limitation occurs. One or both of these events trigger the activation of the ppGpp-SpoT metabolic TA module. Increased ppGpp leads to increased DksA-dependent repression of RNAP activity. Increased ppGpp also inhibits DNA negative supercoiling, which indicated inhibition of DNA gyrase. FIS, IHF, HU, and SeqA all participate in ppGpp-dependent persister formation and modulate chromosomal negative supercoiling. All components in red increase within the model of persisters, and all components in green decrease within the model. See also Figure S6. Molecular Cell 2013 50, 475-487DOI: (10.1016/j.molcel.2013.04.002) Copyright © 2013 Elsevier Inc. Terms and Conditions