Possible mechanisms of evolved isobutanol tolerance. Examining genotypic adaptations to isobutanol stress in the context of known modes of solvent toxicity suggests that remodeling the cell envelope and stress response attenuation might be important mechanisms of adaptation in our evolved lineages. (A) Postulated mechanisms of adaptation involving the cell envelope. Asterisked (*) items were inferred from genoptyic data without direct experimental validation. We observe adaptations that may lead to alterations in cell envelope protein composition, downregulation of fimbriae biogenesis and upregulation of flagellar biogenesis, and alterations in peptidoglycan, membrane lipid composition, and lipopolysaccharide (LPS) composition. Collectively, these adaptations suggest that evolution may be remodeling the cell envelope to counteract the detrimental effects of isobutanol on the cell membrane. (B) Our results suggest that attenuation of RpoS activity may be a convergent adaptive effect associated with hfq, acrAB-tolC, relA, and spoT mutations. Hfq is required for translation of rpoS mRNA, RelA and SpoT synthesize signalling molecule ppGpp which upregulates RpoS, and one study suggests that AcrAB-TolC exports an unidentified quorum sensing signal (QSS) that upregulates RpoS, possibly via a periplasmic receptor [33, 39, 74]. All depicted regulatory interactions involve upregulation. Signalling molecules (ppGpp and QSS) are boxed in yellow. Effects/gene targets that might be ultimately linked to isobutanol tolerance are red. AcrAB-TolC quorum sensing model adapted from , using similar notation.