# 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 Membrane Depolarization and/or Permeability
IbsC and ShoB overexpressions induce membrane depolarization within 5 min upon induction followed by 20 min of incubation with dye (50% and 98% of depolarized cells for IbsC and ShoB, respectively), leading to a massive decrease of cell viability within 30 min upon induction (Figure 2) [56]. IbsC and ShoB are predicted to display a transmembrane domain. The formation of an \(\alpha\)-helix has been confirmed by CD experiment for IbsC (Table 1) [20]. A mutagenesis approach revealed that the IbsC sequence can be reduced to 15 amino acids with a minimum of 10 hydrophobic residues to retain toxicity and probably keep the helix translocation across the inner membrane. This minimal sequence requires the C-terminal residues and the hydrophobic residues near the center of IbsC to keep toxicity (residues 6 to 19), suggesting that these residues are probably involved in the promotion of a proper transmembrane 3D conformation and/or protein-protein interactions. On the contrary, the N-terminal residues deletion did not impact the toxicity of IbsC, except for V5 [20]. Whole genome expression analyses carried 20 min after IbsC, ShoB, LdrD, or TisB overexpressions showed the induction of a common set of genes encoding membrane proteins and/or proteins involved in sugar transport and in stress response, as the _soxS_ mRNA encoding a regulator of the superoxide stress response [56]. These observations are not surprising as these type I toxins are localized on membrane and induce cell toxicity. The individual overexpression of each toxin also induces the expression of specific genes. Interestingly, IbsC overexpression specifically increases the expression of the _pspABCDE_ operon where PspA protein, known to be induced by pore-forming proteins [58], can interact with phospholipids to block the passage of protons across the damaged membrane [56]. Moreover, ShoB overexpression up-regulates the expression of genes involved in sugar transport. These gene regulations could be linked to a direct effect of the toxins on membrane or metabolism or to a secondary consequence of membrane depolarization [56; 59]. The 35-amino acid LdrA toxin, which belongs to the Ldr (for Long direct repeat) family of type I TA systems, rapidly induces intracellular ATP drop within 2 min upon induction. This results in a simultaneous inhibition of DNA replication, transcription, and translation within 10 min upon induction and, finally, to cell growth arrest within 30 min upon induction (Figure 2) [60]. This timing strongly suggests a direct effect of LdrA on the integrity of the membrane. LdrA forms an \(\alpha\)-helix confirmed by circular dichroism and is predicted by MD simulations to cross the inner membrane of _E. coli_ with positively charged C-terminal domain located in the cytosol (Table 1) [60]. The toxicity of LdrA is abolished when a His-tag is added at the N-terminal domain, suggesting the importance of this domain for the anchoring in the inner membrane, leading to the inhibition of ATP synthesis [60]. It can be noted that LdrA is the one of few type I toxins for which the extracellular N-terminal part has been demonstrated to be essential for its toxicity. As for Lpt, the molecular mechanisms involved in the regulation of LdrA expression by its antitoxin remain to be deciphered. The DinQ type I toxin from the _dinQ_/AgrB TA system is a 27-amino-acid peptide identified in _E. coli_ as a new gene of the LexA regulon involved in the SOS response to DNA damaging agents like UV [37; 57; 61]. DinQ is located within the inner membrane of _E. coli_ and its ectopic overexpression induces membrane depolarization within 5 min upon induction followed by 20 min of incubation with the dye, leading to cell death and a high UV sensitivity counteracted by the AgrB RNA antitoxin [62]. AgrB base pairs to the _dinQ_ translationally active +44 transcript, resulting in the sequestration of the SD sequence, the cleavage of the duplex by RNase III, and, consequently, preventing ribosome access and toxin translation [63].