## 2 Materials and Methods
### Biological Validation Assays
The plates were placed in a Tecan Infinite M200 Pro Microplate Reader with a programmed growth cycle (18 h, 37 \({}^{\circ}\)C, orbital rotation 3.5). Cell density was measured at OD\({}_{600}\) and YFP fluorescence activity was detected with 505 nm/535 nm (excitation/emission). Enzymatic activity was measured as Relative Fluorescence Units (RFU-YFP/OD\({}_{600}\)). In vitro (p)ppGpp quantification: In vitro (p)ppGpp quantification was carried out using techniques similar to those previously reported in the literature [25; 26; 28; 48]. The RelA enzyme was purified, as described above. Roughly 0.4 \(\upmu\)g of RelA protein was added to a 1.5 mL microcentrifuge tube containing a reaction mix composed of 1 \(\times\) PBS, 5 mM MgCl\({}_{2}\), 0.5 mM ATP, 0.5 mM GTP, 0.5 mM GDP, and 20 \(\upmu\)Ci[\(\gamma\)-32P]ATP (3000 Ci/mmol; PerkinElmer, Waltham, MA, USA) and varying concentrations of the compound of interest. These reactions were incubated at 37 \({}^{\circ}\)C for 1 h. The reactions were stopped by the addition of 5 \(\upmu\)L formic acid (88%). The reaction mixtures were then spotted on a stationary-phase polyethyleneimine (PEI)-cellulose TLC plate (Sorbent Technologies, Norcross, GA, USA) using potassium phosphate monobasic (1.5 M) as the mobile-phase. The plates were then dried, and the radiation levels were read using a Molecular Dynamics Storage Phosphor Screen. A Molecular Dynamics Storm 840 Phosphor imager Scanner was used to read the phosphor screen and ImageJ was used to process the images. In vivo (p)ppGpp Quantification: In vivo (p)ppGpp quantification was carried out using techniques similar to those previously reported in the literature [25; 26; 28; 48]. One milliliter of overnight cell culture of _E. coli_ C was placed in 1.5 mL microcentrifuge tubes and pelleted. To this pellet, 50 \(\upmu\)L of a reaction mixture containing 20 \(\upmu\)Ci orthophosphorhic acid and 40 \(\upmu\)M serine hydroxamate in 1\(\times\) MOPS minimal medium was added. The cell pellet was resuspended by gentle vortexing and placed in an incubator for 1 h. Cell growth arrest and cell lysis were completed by the addition of 15 \(\upmu\)L formic acid (88%). The lysate was then centrifuged to remove any insoluble components and the supernatant was spotted on a stationary-phase PEI-cellulose TLC plate. Plates were processed and analyzed as described above. Biofilm dispersal assays: For biofilm formation on polystyrene surfaces, flat-bottom 96-well microtiter plates (Corning Inc., Corning, NY, USA) were used. Two hundred microliters of bacterial culture (100\(\times\) diluted overnight culture; approximately 10\({}^{7}\) cells) in fresh LB medium was added to each well. These were allowed to grow for 24 h. The planktonic cells and medium were then aspirated, and the plates were washed twice with 1\(\times\) PBS, aspirating and discarding the PBS wash each time. Fresh LB with hit compounds was added to the biofilm wells. These cultures were then allowed to incubate at 37 \({}^{\circ}\)C overnight. Then, 100 \(\upmu\)L of the planktonic culture was transferred to another 96-well plate without disturbing the underlying biofilm, and the cell density of the planktonic culture was measured (OD\({}_{600}\)) using a Multiscale Go plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Biofilm volume was measured by adding 100 \(\upmu\)L fresh LB and 30 \(\upmu\)L Gram crystal violet (CV) (Remel, San Diego, CA, USA); 3 g crystal violet, 50 mL isopropanol, 50 mL ethanol, 900 mL purified water) to the original plate and allowing it to incubate for 1 h for staining. Plates were washed with water and air dried, and CV was solubilized with an ethanol:acetone (4:1) solution. The OD\({}_{570}\) was determined from this solution, and the biofilm volume was calculated as the ratio of OD\({}_{570}\) to OD\({}_{600}\)[49; 50]. Biofilm inhibition assays: For biofilm formation on polystyrene surfaces, flat-bottom 96-well microtiter plates (Corning Inc.) were used.