## 2 Materials and Methods
### Biological Validation Assays  
RelA Mutagenesis: The ASKA(-) clone JW2755-AM, containing an _E. coli_ W3110 RelA in the PCA24N vector, was used for mutagenesis. A 1144-bp _Psil/Nsil_ fragment was replaced by a synthetic construct. This construct contained 2 designated changes. First, a single nucleotide silent substitution (769 C/A) introduced at an _Xbal_ restriction site, as described in [43; 44]. Second, a TA/GC substitution at position 1027 replaced TAC (Y-310) with GCC (A-310). The 365-bp region between _Xbal_ (769) and _Nsil_ (1144) contains the RelA active center and can be easily swapped with a synthetic construct to replace any of the tested amino acids. This method was applied to introduce the Y/A-319 mutation. A 365-bp _Xbal/Nsil_ fragment was replaced by a synthetic fragment with TAT-Y319 (position 1053) replaced with a GCC-A319 codon, as described in [45]. All mutations were confirmed by [PERSON] DNA sequencing. RelA Protein Purification: The functional RelA enzyme and its Y/A-319 and Y/A-310 mutants were purified from host cell AG1 strains carrying the pJW2755-AM, pJEK2020-43, and pJEK2020-20 plasmids, respectively. One liter of LB broth was inoculated with 20 mL of overnight culture (OD\({}_{600}\) = 0.9) and grown for 4 h (OD\({}_{600}\) = 0.8) before induction with 1.5 mM IPTG for 4 h. Cultures were spun down, washed with phosphate-buffered saline (PBS), and resuspended in lysis buffer (50 mM NaH\({}_{2}\)PO\({}_{4}\), 300 mM NaCl, 10 mM imidazole, pH 8.0) for lysing. To that resuspension, 1 \(\mu\)L/mL ThermoFisher Halt(tm) Protease Inhibitor Cocktail (100\(\times\)) was added without EDTA and cells were lysed with sonication on ice (cycles of 10 s on and 10 s off for a total of 3 min of sonication, 2\(\times\)). Lysates were spun down to remove cellular debris using a Sorvall RT7 Plus tabletop centrifuge (3.3 g, 15 min). Millipore Sigma PureProteome(tm) Nickel Magnetic Beads were used according to a modification of the manufacturer's instructions. Supernatant was placed in 200 \(\mu\)L of nickel affinity beads for a period of 30 min. Beads were captured on a magnetic rack and the supernatant was removed. Beads were then washed 4\(\times\) with wash buffer (50 mM NaH\({}_{2}\)PO\({}_{4}\), 300 mM NaCl, 10 mM imidazole, pH 8.0). RelA was eluted twice using 300 mM imidazole elution buffer (50 mM NaH\({}_{2}\)PO\({}_{4}\), 300 mM NaCl, 300 mM imidazole, pH 8.0) and a final elution using 500 mM imidazole elution buffer (50 mM NaH\({}_{2}\)PO\({}_{4}\), 300 mM NaCl, 500 mM imidazole, pH 8.0). An SDS-page gel was run to confirm the presence and purity of RelA. Imidazole buffer was exchanged for PBS buffer and RelA was concentrated using Amicon(tm) Ultra-4 Centrifugal Filter Unit 30 kDa (MilliporeSigma, Burlington, MA, USA) nominal molecular weight limit. Nanodrop showed an average concentration of 1 mg/mL with a 260:280 ratio -0.73. Fluorescent Reporter RelA Activity Assay: The plasmid pAG001 (ampicillin 100 \(\mu\)g/mL), carrying a _//fp_ fluorescent protein gene driven by the P_rps_/ promoter, was used to detect the intracellular ppGpp concentrations, as published [46]. To validate the assay, this reporter plasmid, which is based on the broad host range RK2 minimal replicon, was introduced into _E. coli_ K12 CF1648, and its _relA_ mutants--(CF1652) [19], and AG1 (_relA1_) (NBRP Japan). To analyze the effect of overexpression of RelA and the Y/A310, and Y/A319 substitutions, ASKA plasmid pJW2755-AM [47] (chloramphenicol 25 \(\mu\)g/mL) and its derivatives--pJEK2020-20 with the Y/A310 mutation and pJEK2020-43 with theY/A319 mutation--were extracted using the ThermoFisher Plasmid Mini DNA Extraction Kit, and transformed into AG1 pAG001 strain (ampicillin 100 \(\upmu\)g/mL, chloramphenicol 25 \(\upmu\)g/mL). For the fluorescent RelA activity assay, overnight cultures of the selected strains were diluted 1:100 in fresh LB medium and 200 \(\upmu\)L aliquots were placed into 96-well plates (Costar).