# 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
The high _tisB_ mRNA levels out-titrate the lstR-I (for inhibitor of SOS-induced toxicity by RNA) RNA antitoxin pool, despite its constitutive expression, and promotes cell growth arrest [39]. It was proposed that the lstR-1 antitoxin prevents inadvertent toxicity that results from leaky _tisB_ transcription. lstR-1 RNA base-pairs with the standby site of the active +42 _tisB_ mRNA and promotes RNA duplex degradation by RNase III to an inactive +106 _tisB_ mRNA, thus preventing ribosome access and toxin translation [41]. The effects of TisB on the membrane integrity, decreasing intracellular ATP levels and all essential cellular processes [30], were associated to _E.coli_ persistence in 2010. The [PERSON] group showed that ciprofloxacin, a DNA-damaging antibiotic targeting DNA gyrase and topoisomerase IV, increases persister levels via SOS-dependent induction of TisB [42]. A strain deleted for the _tisB_ locus showed a 10-to-100-fold decrease of ciprofloxacin-tolerant persisters, whereas deletion of the _istR-1_ locus resulted in a 10-to-100-fold increase [42]. Moreover, ectopic overexpression of TisB confers multidrug tolerance. The authors hypothesized that, in the presence of DNA-damaging antibiotics, the optimal strategy to survive is dual encompassing DNA repair activation and switching to a dormant state via the SOS-induced TisB expression [42]. The [PERSON] group demonstrated that the deletion of two regulatory RNA elements (i.e., lst-R1... ) and the inhibitory 5' UTR structure in the _tisB_  
Figure 2: Overview of the membrane-associated type I toxins inducing membrane perturbations as primary detected effect. Only the effects of membrane-associated type I toxins after induction of their overexpression in bacteria have been shown in the figure. mRNA [41] triggers stochastic TsB translation, leading to pore formations and superoxide production [35; 43]. This results in a depolarized sub-population that turns into persister cells even in the absence of strong SOS induction by exposure to ciprofloxacin [43; 44]. The deletion of the superoxide dismutases SodA and SodB impairs the stochastic TsB-dependent persisters' formation and the recovery of persister cells after ciprofloxacin exposure [35]. In 2012, two studies investigated the structure of TsB and its interaction with model of bacterial membranes. The first study showed by in vitro conductance measurements and polymer-exclusion experiments that TsB forms narrow anion-selective pores in planar lipids bilayers as consequence of the net-positive charge of TsB [45]. The other study confirmed the amphiphilic \(\alpha\)-helical conformation of TsB by circular dichroism, oriented circular dichroism, and molecular dynamic (MD) simulations. The authors showed a spontaneous insertion of TsB in lipid bilayers with a stable transmembrane alignment where the four charged side-chains of D5, K12, D22, and K26 lie on a narrow strip along with the polar face of the helix, with an alternating pattern of positive and negative charges. They also postulated formation of a spontaneous assembly as antiparallel dimers stabilized by four intermolecular salt bridges and an intermolecular hydrogen-bond interaction between Q19 residues (Table 1) [19]. The polar interface of the TsB helix could mediate the passage of protons across the hydrophilic lipid bilayer. Fluorescence dequenching experiments confirmed that TsB can form narrow pores of an estimated diameter of 0.15 nm that are impermeable to intracellular water-soluble components, thus allowing cell survival [19; 45]. The flows of protons and anions across TsB pores can dissipate the proton motive force (PMF), reduce the ATP production, and hijack the metabolism to a dormant state (Figure 2). Consequently, TsB leads to the shutdown of the major antibiotic targets and induces multidrug tolerance.