## 2 Results
### Physiological Relevance of Toxin OrtT  
Because both OrtT and GhoT are toxins that corrupt the membrane, we investigated if these two toxins serve a similar role in cell physiology. GhoT decreases cellular metabolism in the presence of carbenicillin and cefoxitin helping cells withstand this kind of membrane stress [42]. Hence, we first investigated whether OrtT was involved in the same pathway to slow metabolism in presence of carbenicillin. Surprisingly, OrtT was not involved in the stress response against carbenicillin (Figure 4A) since cells which lack OrtT (_i.e._, \(\Delta\)_ortT_\(\Delta\)_Kan_) had low metabolism like wild-type cells whereas cells lacking GhoT (_i.e._, \(\Delta\)_ghoT_\(\Delta\)_Kan_) showed high metabolism under this stressful condition. Note that only strains which lack the antibiotic resistance marker were used so that the only difference between the strains is deletion of the toxin gene. Next, we investigated whether OrtT participates in a similar adaptive stress response with other antimicrobials. We took advantage of the PortEco database that indicates the results for large-scale phenotypic screening for all of the Keio mutants and that indicates \(\Delta\)_ortT_ has a negative fitness score for trimethoprim (TMP) [45]. TMP is an antifolate drug that inhibits the synthesis of THF by binding to dihydrofolate reductase (DHFR) [46]. THF plays vital role in synthesis of amino acid serine and methionine (Figure S5), and low THF induces the stringent response and causes bacteria to enter into a non-proliferating state [5]. In addition, THF is an essential precursor for purine and thymidine triphosphate synthesis (Figure S5) and interference with THF metabolism inhibits bacterial DNA synthesis [47]. We found that the \(\Delta\)_ortT_\(\Delta\)_Kan_ mutant had dramatically higher metabolism compared to wild-type strain in response to 1 \(\mu\)g/mL TMP (Figure 4B), indicating OrtT acts as a metabolic brake to slow cellular metabolism under conditions that are unfavorable for amino acid and DNA synthesis. Critically, while the \(\Delta\)_ortT_\(\Delta\)_Kan_ strain was metabolically active in the presence of TMP, the \(\Delta\)_ghoT_\(\Delta\)_Kan_ strain was as sensitive to TMP as the wild-type cells (Figure 4B). These results indicate that toxins OrtT and GhoT have different physiological roles in cell fitness even though they share 63% protein identity. Furthermore, to corroborate that OrtT acts specifically in the THF synthesis pathway, we performed a similar metabolic assay using sulfamethoxazole (SMZ). SMZ is often used along with TMP for their synergistic effect against bacteria since SMZ inhibits dihydroprotease synthetase, whereas TMP inhibits DHFR [48] (Figure S6). As expected, the \(\Delta\)_ortT_\(\Delta\)_Kan_ mutant showed higher metabolism compared to the wild-type host in response to 60 \(\upmu\)g/mL SMZ (Figure 4C) which corroborates the previous result with TMP. Figure 4: OrtT reduces cellular metabolism under THF-related antimicrobial stress. (**A**) Metabolic activity of the wild-type, \(\Delta\)_ortT_\(\Delta\)_Kan_, and \(\Delta\)_ghoT_\(\Delta\)_Kan_ strains in response to 3 \(\upmu\)g/mL carbenicillin or water (negative control); (**B**) Metabolic activity of the wild-type, \(\Delta\)_ortT_\(\Delta\)_Kan_, and \(\Delta\)_ghoT_\(\Delta\)_Kan_ strains in response to 1 \(\upmu\)g/mL TMP or DMSO (negative control); (**C**) Metabolic activity of the wild-type strain and \(\Delta\)_ortT_\(\Delta\)_Kan_ in response to 60 \(\upmu\)g/mL SMZ or DMSO (negative control). Two independent cultures were evaluated for each experiment. Error bar represents standard deviation. Since metabolic activity is measured at 590 nm and cell growth is measured at 600 nm, we suspected OrtT might also influence cell growth when challenged with TMP. To investigate this, we performed the growth assay with wild-type and \(\Delta\)_ortT_\(\Delta\)_Kan_ cells with or without TMP treatment.