## 2 Results
### OrtT is an Orphan Toxin  
To further characterize this new toxin, we explored if it forms a toxin-antitoxin pair by interacting with other protein/RNA. Although _ortT-ydcY_ does not form an operon (Figure 3A), we tested whether YdcY can act as an antitoxin by constructing pCA24N-_ortT-ydcY_ (Figure S2A). Using this plasmid we found that YdcY does not act as an antitoxin as OrtT-mediated toxicity was not reduced when YdcY was co-produced with OrtT (Figure 3B). Note that YdcY was confirmed to be a protein by running an SDS-PAGE (Figure S3). Figure 2: OrtT causes cell lysis via membrane damage and reduces cellular ATP. (**A**) Superimposed protein model of GhoT (green) and OrtT (cyan). Phe38 is blue for GhoT and yellow for OrtT, and Ile21 is red for GhoT and magenta for OrtT; (**B**) Growth of BW25113 wild-type cells carrying different plasmids in LB medium with chloramphenicol (30 \(\upmu\)g/mL) with 0.1 mM IPTG introduced at 1h. Two independent cultures were evaluated. Error bar represents standard deviation; (**C**) Cell lysis assay with BW25113 wild-type cells producing OrtT or with empty vector. LB medium was used with chloramphenicol (30 \(\upmu\)g/mL) and 2 mM IPTG (added at OD\({}_{600}\) 0.5). Two independent cultures were evaluated. Error bar represents standard deviation; (**D**) TEM images of BW25113 wild-type cells producing OrtT (top two rows) or with empty vector (bottom row). Left column shows longitudinal sections, and the right column shows horizontal sections. The blue arrow shows the enlargement of the periplasmic space, the red arrow shows nucleoid condensation, and the green arrow shows the empty cytoplasmic space. Scale bar indicates 0.5 \(\upmu\)m; (**E**) Change in relative intracellular ATP level due to OrtT production in BW25113 wild-type cells. LB medium was used with chloramphenicol (30 \(\upmu\)g/mL) and cells were treated with 1 mM IPTG (added at OD\({}_{600}\) 0.5) for 3 h. The ATP levels were calculated as RLU/OD\({}_{600}\) (relative light units/optical cell density at 600 nm) and normalized to that of the strain with the empty vector pCA24N. Plasmid abbreviations: Empty, pCA24N; OrtT, pCA24N-_ortT_; OrtTF38R, pCA24N-_ortT_F38R having Phe38 substituted by Arg; GhoT, pCA24N-_ghoT_; GhoTF38R, pCA24N-_ghoT_F38R having similar F38R substitution. GhoS is the antitoxin for GhoT, and it functions by cleaving the _ghoT_ transcript [18]. Since OrtT is similar to GhoT, we tested whether _ortT_ mRNA is a substrate for GhoS by constructing pCA24N-_ortT-ghoS_ (Figure S2B). We found that GhoS does not attenuate OrtT toxicity (Figure 3B). These plasmids (_i.e._, pCA24N-_ortT-ydcY_ and pCA24N-_ortT-ghoS_) were constructed in a way that the toxin and putative antitoxin genes are under the control of the same promoter. Note that such co-expression of toxins and antiotoxins from the same promoter has been used for different TA systems such as MqsRA (Figure S4) [43] and VapBC [44]. We also considered the possibility of OrtT to be regulated by an RNA antitoxin. Type I toxin genes are generally secluded by large intergenic distances ranging from 492 to 1181 bp upstream and 282-708 bp downstream [14]. Hence, it was unlikely for OrtT to be a member of type I TA family considering the relatively small intergenic distances (_i.e._, 194 bp upstream and 85 bp downstream). Nonetheless, we tested for RNA interactions by cloning the upstream and downstream intergenic region between _ortT_ and neighboring genes _yncL_ and _ydcY_, respectively, to construct pCA24N-_ortT_-UTR  
Figure 3: OrtT is an orphan toxin. (**A**) The chromosomal region around _ortT_ gene in _E. coli.__ortT_ is represented by a red arrow and _ydcY_ is shown by the grey arrow.