Fig 1.
RsFluc (p)ppGpp-sensitive firefly luciferase reporter.
A, schematic of the (p)ppGpp reporter RsFluc depicting the inducible promoter Phyperspank fused to the (p)ppGpp-senstive riboswitch of D. hafniense ilvE followed by the gene encoding firefly luciferase. B, luminescence (RLU/OD600) of RsFluc (blue; JDB4496), mutant RsFlucmut (green; JDB4631) in wildtype backgrounds and RsFluc in a ∆relA∆sasA∆sasB background (red; JDB4512), C) ppGpp concentration determined by HPLC-MS (black closed circles) and corresponding reporter luminescence corrected for (p)ppGpp0 luminescence by subtraction (blue open circles). D, luminescence per OD600 of RsFluc (black), RsFlucnatA (purple, JDB4730) and RsFluclivK (cyan, JDB4731). Shown are representative examples of at least three biological replicates, each comprising three technical replicates.
Fig 2.
RsFluc reporter response to stimulation of (p)ppGpp synthesis.
Luminescence (RLU/OD600) of: A, RsFluc (black; JDB4496) and RsFlucmut (blue; JDB4631) after amino acid downshift at T60 (dashed line); B, RsFluc (black) and RsFlucmut (blue) after 0 and 60 mins of 50 ng/mL mupirocin treatment. Significance was determined by two-tailed t test: p-value < .05 for RsFluc, no significant difference for RsFlucmut; C, RsFluc grown in defined minimal media containing either 2.5 mg/mL of glucose (black) or 0.5 mg/mL glucose and 2.0 mg/mL arabinose (blue). Respective OD600 measurements are shown in grey and light blue, respectively, and D) RsFluc (black) and a PpurE luciferase promoter fusion in wildtype (orange; JDB4803) and (p)ppGpp0 (purple; JDB4804) backgrounds. Shown are representative examples of at least three biological replicates, each comprising three technical replicates.
Fig 3.
Relative contributions of (p)ppGpp synthetases to dynamics of RsFluc activity.
Luminescence (RLU/OD600) of: A, RsFluc in wildtype (black, JDB4496), ∆relA∆sasA∆sasB (red, JDB4512), ∆sasA (green, JDB4515), ∆sasB (orange, JDB4516), and relA-D264G (blue, JDB4741) backgrounds and B), wildtype (black), ∆sasA (green), ∆sasB (orange), ∆sasA∆sasB (brown, JDB4511), and relA-D264G (blue) strains after amino acid downshift at T60 (dashed line). Shown are representative examples of at least three biological replicates, each comprising three technical replicates.
Fig 4.
RsFluc activity coordinated by allosteric network.
Luminescence (RLU/OD600) of: A, RsFluc in wildtype (black, JDB4496), relA-Y200A (fuschia, JDB4528), and sasB-F42A (blue, JDB4711) strains and B, RsFluc in wildtype (black), relA-Y200A (fuschia), and sasB-F42A (blue) strains after amino acid downshift (dashed line). Shown are representative examples of at least three biological replicates, each comprising three technical replicates.
Fig 5.
RelA hydrolase activity contributes to dynamics of RsFluc activity.
A, schematic of bacitracin-inducible relA constructs. B) luminescence (RLU/OD600) of RsFluc in wildtype (black, JDB4496), ∆relA∆sasA∆sasB with PliaI-relA (blue, JDB4675), ∆relA∆sasA∆sasB with PliaI-relA-D78A (red, JDB4676) and ∆relA∆sasA∆sasB (gray, JDB4512) strains. RelA expression induced by 5 µg/mL bacitracin at T90 (dashed line). Shown is a representative example of at least three biological replicates, each comprising three technical replicates.
Fig 6.
RsFluc activity and amino acid availability.
Luminescence (RLU/OD600) of: A, RsFluc in prototroph (JDB4656) cultured in S7 supplemented with 0.005% (blue), 0.01% (black), and 0.02% (green) casamino acids and B, RsFluc (black) and RsFlucmut (blue) in prototroph background without amino acid supplementation. Shown are representative examples of at least three biological replicates, each comprising three technical replicates.
Fig 7.
Correlation of amino acid biosynthetic gene expression and RsFluc activity.
Luminescence (RLU/OD600) of: A, RsFluc (black, JDB4496), PserA-luc reporter (blue, JDB4759), PilvB-luc reporter (green, JDB4792), and PmetE-luc reporter (red, JBD4798) in the wildtype background and B, reporters as A in the relA-D264G genetic background. Shown are representative examples of at least three biological replicates, each comprising three technical replicates.
Fig 8.
Temporal relationship between protein synthesis and RsFluc activity.
A, luminescence (RLU/OD600) of RsFluc (blue, JDB4496) and growth rate (black). Growth rate/hour (µ) at a given time (t) is defined as log2[OD600(t)-OD600 (t-60)]. B, representative composite (fluorescent, phase) images of cells at TP1 and TP2 (see S11 Fig) in either wildtype (JDB4486) or ∆relA∆sasA∆sasB (‘(p)ppGpp0’; JDB4512) backgrounds. C) and D) quantification of images from B.