# Bacterial Type I Toxins: Folding and Membrane Interactions
## 3 Membrane-Associated Type I Toxins Inducing Membrane Perturbations as a Primary Detected Effect
### Membrane-Associated Type I Toxins Inducing Pore Formation
Changes in conductance using specific PEGylation demonstrated that HokB crosses the lipid bilayer with its positively charged N-terminal domain extending in the cytoplasm, whereas the negatively charged C-terminal domain extends in the periplasm [34]. Thanks to a m-Cherry tag at the N-terminal domain, the authors showed by microscopy cluster formation of HokB peptides at the _E. coli_ membrane, consequently to an in vivo pore formation. By blocking these HokB pores with PEG 1000 in vivo, the group elegantly proved that pore formation by HokB is directly linked to _E. coli_ persistence and results in a decrease of the cellular energy ratio and the ATP efflux within 4 h upon induction. Moreover, the authors demonstrated in vitro that the membrane potential controls the size of HokB pores since, when the potential applied to the membrane is high, mature pores are formed and provoke both a membrane depolarization and an ATP efflux, leading to persistence in metabolically active cells (Figure 2). When the applied potential is low, intermediate pores can be formed. They induce ATP/ADP ratio drop, but no persister cells formation [34]. HokB overexpression triggers membrane depolarization and reactive oxygen species (ROS) formation within 1 h upon induction, leading to growth inhibition which could contribute to persister cells formation [35] (Figure 2). Recently, mechanisms that control the formation and the awakening of HokB-induced persister cells have been identified [36]. HokB contains three cysteine residues of which cysteines C9 and C14 are predicted to be part of a transmembrane \(\alpha\)-helix anchored to the membrane and cysteine C46 to be present in the periplasmic C-terminal domain (Table 1). Construction of cysteine-to-serine substitution mutants demonstrated that the periplasmic cysteine C46 is responsible for HokB dimerization by disulfide bridge formation as well as for HokB-induced membrane depolarization and persister cells formation [36]. The inter-peptide disulfide bridge formation between two periplasmic C46 residues is mediated by the periplasmic oxidoreductase DsbA. This enzyme is essential for HokB dimerization and stability and, consequently, for pore formation, membrane depolarization, ATP efflux, and persister cells formation [36]. A positive correlation between the concentration of HokB peptides and the dormancy duration in persister cells was evidenced by microfluidics and single-cell approaches. It suggests that pore disassembly may be involved in the awakening of HokB-induced persister cells. Upon awakening, the periplasmic oxidoreductase DsbC was shown to reduce the disulfide bridges and induce HokB monomerization, thus promoting DegQ-mediated degradation of HokB peptides. This pore destabilization leads to membrane repolarization and ATP production by the NADH-dehydrogenase complex I and to resume the growth of HokB-induced persister cells [36]. The TisB type I toxin is another pore-forming peptide expressed in _E. coli_ from the _tisB/lst-I_ locus that was discovered by the first genome-wide searches for bacterial small RNAs [37; 38]. The [PERSON] group demonstrated that _tisB_/lstR-1 is the first type I TA system involved in the SOS response [39]. The SOS response is initiated when the recombinase RecA senses DNA damage and activates cleavage of the global repressor LexA [40]. The _tisB_ (for toxicity induced by SOS) gene encodes a 29-amino-acid peptide located in the inner membrane of _E. coli_[30]. Its ectopic overexpression provokes membrane damages and a rapid drop in ATP levels that results to a drastic decrease in transcription, translation, and replication rates, leading to cell death within 1 h upon induction (Figure 2) [30; 39]. Under normal growth conditions, the transcription of _tisB_ is repressed by LexA. When SOS conditions are encountered, RecA-induced cleavage of LexA derepresses _tisB_[39].