Orphan Toxin OrtT (YdcX) of Escherichia coli Reduces Growth during the Stringent Response

Abstract

Toxin/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin) is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.

Combatting bacterial persister cells

“…It is now clear that the fitness of bacteria is enhanced if they are able to reduce their metabolism during stress. For example, membrane toxin GhoT (Wang et al, ) reduces cell growth by decreasing ATP production and the proton motive force during antibiotic stress to make the cell more fit (Cheng et al, ), and membrane toxin OrtT reduces cell growth by reducing ATP during the stringent response (e.g., nutrient stress) to make the cell more fit (Islam et al, ).…”

Identification of novel genes including rpmF and yjjQ critical for Type II persister formation in Escherichia coli

“…Of these genes, phage shock protein ( pspC ) has been reported to be involved in indole-mediated persister formation (Vega et al, 2012b). And ydcX also participates in persister formation as an orphan toxin (Islam et al, 2015), and ssrA involved in trans-translation has previously been shown to be involved in persister formation (Li et al, 2013).…”

“…The 13 overlapping genes of S2/S1 and S3/S1 were supposed to be of significance for both “boot-up” and “shoot-up” of persister formation and required a thorough investigation. Apart from two genes ( ssrA and ydcX ) which have been known to participate in the persister formation (Li et al, 2013; Islam et al, 2015), the other 11 overlapping genes have not been reported to be related to persistence.…”

“…Because ssrA and ydcX have previously been shown to be involved in persister formation (Li et al, 2013; Islam et al 2015), we focused on assessing the impact of the knockout strains on persister levels of the remaining 11 of the 13 previously unreported overlapping genes under ampicillin treatment. However, only 7 knockout mutants ( ΔgnsA, ΔybfA, ΔyjjQ, ΔyhdU, ΔcsgD, ΔyohJ and ΔrpmF ) were successfully constructed presumably because some genes are either essential, or lack of sequence homolog (i.e.…”

“…Specially, 13 genes overlap in both comparison groups. Apart from 4 genes ( gnsB, ymdF, yncN, ybfA ) with uncharacteristic functions and 2 genes ( ssrA, ydcX ) with definite effect on persistence (Li et al, 2013; Islam et al, 2015), the remaining 7 overlapping genes were mapped to ribosomal protein ( rpmF ), regulator of phosphatidylethanolamine synthesis ( gnsA ), membrane protein ( yohJ, yhdU ), DNA-binding transcriptional activator ( csgD ), transcription factor ( yjjQ ) and cytoplasmic sugar-binding protein ( rbsD ). We confirm two genes ( rpmF and yjjQ ) play crucial roles in persister formation under multiple antibiotic treatment and stress conditions.…”