## 3 Discussion  
In our previous study, the physiological role of type V toxin GhoT was ascertained; GhoT causes membrane damage by reducing cellular ATP level and reduces metabolism in the presence of various antimicrobials related to cell-wall synthesis (_i.e._, carbenicillin and cefoxitin) [42]. In the present study, we have characterized a new GhoT-like toxin, OrtT, in _E. coli_. OrtT is a small, hydrophobic peptide that shares 63% protein identity with GhoT, and we have demonstrated that: (i) OrtT strongly inhibits growth and causes cell lysis; (ii) removal of the start codon encoding _ortT_ restores growth proving OrtT is active as a protein; (iii) OrtT damages the cell membrane and depletes cellular ATP, (iv) the F38R substitution reduces OrtT toxicity; (v) unlike GhoT, OrtT is most likely an orphan toxin as it is neither encoded by a bicistronic operon nor under _in trans_ regulation by an antitoxin encoded elsewhere in the genome; (vi) OrtT reduces cellular metabolism in the presence of antimicrobials like TMP and SMZ; and (vii) _ortT_ is activated under stringent conditions; so, its role in cell physiology appears to be to reduce growth during nutritional stress. Based on the structural homology between GhoT and OrtT, we introduced the same mutation (_i.e._, F38R) in OrtT that allowed us previously to create a non-toxic version of GhoT [42] in order to characterize OrtT. Despite the absence of a hydrophobic interaction between Phe38 and Ile21 (that presumably stabilized GhoT) in OrtT (Figure 2A), toxicity was reduced upon F38R substitution indicating OrtT might be functionally similar to GhoT. Moreover, similarities in the morphological changes in the membrane upon OrtT and GhoT production (visualized via TEM) indicates that these toxins might be utilized in a similar fashion by the cells to escape unfavorable environmental stimuli. Surprisingly, deletion of _ortT_ did not have the same impact on metabolism in presence of carbenicillin as _ghoT_. In addition, _ortT_ was advantageous for the cells in response to a completely different set of antibiotics, TMP and SMZ, related to THF synthesis whereas the _ghoT_ mutation was unrecognizable under the same conditions. Hence, OrtT serves a different physiological role than GhoT. Furthermore, _ortT_ mRNA contains two MqsR-preferred 5'-GCU cleavage sites [56] which are absent in _ghoT_ mRNA indicating OrtT is likely not active under conditions that activate MqsR whereas \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Conditions**} & \multirow{2}{*}{**Strains**} & **Fold Change of** & \multirow{2}{*}{**Treatment Description**} \\  & & _ortT_ **Induction** & \\ \hline \multirow{2}{*}{TMP treatment} & \multirow{2}{*}{BW25113} & \multirow{2}{*}{3.1 \(\pm\) 1.5} & Exponentially-growing cells were treated with \\  & & & TMP (75 \(\upmu\)g/mL) for 2 h. \\ \hline \multirow{2}{*}{RelA production} & \multirow{2}{*}{BW25113/pCA24N-_relA_} & \multirow{2}{*}{2.3 \(\pm\) 0.6} & Exponentially-growing cells were treated with \\  & & & IPTG (1 mM) to induce RelA production for 2 h. \\ \hline \multirow{2}{*}{MUP treatment} & \multirow{2}{*}{BW25113} & \multirow{2}{*}{4.61 \(\pm\) 0.29} & Exponentially-growing cells were treated with \\  & & & MUP (100 \(\upmu\)g/mL) for 2 h. \\ \hline \multirow{2}{*}{SHX treatment} & \multirow{2}{*}{BW25113} & \multirow{2}{*}{89 \(\pm\) 80} & Exponentially-growing cells were treated with \\  & & & SHX (1 mg/mL) for 2 h. \\ \hline \multirow{2}{*}{Heat shock} & \multirow{2}{*}{BW25113} & \multirow{2}{*}{2.0 \(\pm\) 0.2} & Exponentially-growing cells were shifted from \\  & & & 30 \(\upcirc\) to 43 \(\upcirc\) for 30 min. \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Summary of qRT-PCR results to investigate whether _ortT_ expression is induced under stringent conditions. All qRT-PCR experiments were performed with two biological replicates each with two technical replicates. MqsR activates GhoT [32]. Unlike GhoT, OrtT also increases swarming motility [57].