# Bacterial Type I Toxins: Folding and Membrane Interactions
## 4 Membrane-Associated Type I Toxins Inducing Cell Morphology Changes as a Primary Detected Effect
The BsrG type I toxin is a component of the temperature-dependent _bsrG_/SR4 TA system located on the SP\(\beta\) pphage region of the _Bacillus subtilis_ chromosome [73]. The _bsrG_ mRNA encodes a 38-amino-acid hydrophobic peptide whose ectopic overexpression causes cell lysis on agar plates. This toxicity is reversed by the bifunctional SR4 RNA antitoxin that interacts with _bsrG_ mRNA via its overlapping 3'-end, leading to the RNase III-mediated degradation of the _bsrG_/SR4 duplex and to the _bsrG_ mRNA translation inhibition by sequestering the Shine-Dalgarno (SD) sequence [73; 74]. To analyze the mechanism of action of BsrG toxin, the _bsrG_ gene is integrated into the _aprE_ locus of the _B. subtilis_ chromosome under the control of an IPTG-inducible promoter. In this condition, the BsrG expression causes a slight growth inhibition of _B. subtilis_ 3 h after IPTG induction (Figure 3) [75]. Although the BsrG toxin is associated with the cell membrane, its IPTG-inducible expression induces neither membrane permeabilization and depolarization, nor alteration of  
Figure 3: Overview of the membrane-associated type I toxins inducing cell morphology changes as a 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. membrane fluidity and intracellular ATP level [75]. However, _bsrG_ induction provokes morphological abnormalities visualized by 3D-structured illumination microscopy such as reduced cell size, irregular shape, distorted cell division planes, membrane invaginations, and irregular septa [75]. BsrG also induces nucleoid condensation without alterations in chromosome segregation and replication and promotes a global inhibition of transcription and translation as a secondary consequence of its toxicity. After a detailed analysis of the different effects of BsrG overexpression, the authors concluded that BsrG stimulates fatty acids biosynthesis, causing invaginations of the cytoplasmic membrane, leading to the delocalization of the cytoskeletal protein MreB and the accompanied cell wall synthesis machinery. This triggers cell lysis, which is dependent of LytC and LytD autolysins [75]. However, the direct cellular target of BsrG is not the cell wall synthesis machinery and remains to be identified [76]. From a structural point of view, addition of the monomeric superfolder GFP to the C-terminus of BsrG removes the toxicity, suggesting that the cationic and polar C-terminal domain predicted to be located inside the cell (Table 1) is essential for the interaction of BsrG with an intracellular target [75]. The _H. pylori_ AapA1 type I toxin is a 30-amino-acid peptide from the _aapA1_/IsoA1 TA system initially discovered by global transcriptome analysis in _H. pylori_ 26695 strain [77]. Structural rearrangements of the _aapA1_ mRNA leads to a duplex formation with the _cis_-encoded IsoA1 RNA antitoxin [78]. This interaction promotes the translation inhibition of the _aapA1_ mRNA and its degradation by RNase III, thus preventing toxin synthesis under normal growth conditions [78]. The ectopic overexpression of AapA1 leads to a growth arrest characterized by a rapid morphological transformation of _H. pylori_ from spiral-shaped bacteria to round coccoid cells [78; 79]. This transformation occurs as early as 2.5 h after toxin induction that corresponds to the division time of _H. pylori_. AapA1 is exclusively located to the inner membrane [24; 79]. The intracellular ATP level and the membrane potential are weakly affected 6 h and 8 h after AapA1 induction, whereas most cells are transformed into viable coccoids (Figure 3) [79]. Experiments using in vitro membrane models, Plasmon waveguide resonance, MD simulations, and Cryo-EM demonstrated that the interaction between AapA1 and cell membrane induces a lipid reorganization and a thinning of the bilayer lipids without severe membrane disruption [24].