Disruptions in outer membrane-peptidoglycan interactions enhance bile salt resistance in O-antigen-producing <i>E. coli</i>

Bile salts (BS) are antimicrobials that disrupt bacterial cell membranes and induce oxidative stress. The gut bacterium <i>Escherichia coli</i> is naturally resistant to BS, including the model strain K12 MG1655 that produces a lipopolysaccharide (LPS) without O-antigen (OAg) on the cell surface. Paradoxically, we have previously shown that restoring a wild-type like LPS with attached OAg (MG1655-S) sensitizes <i>E. coli</i> K12 to exogenous BS. In this study, we investigate this phenomenon. We show that mutations causing truncation of the LPS core oligosaccharide render MG1655-S strains even more susceptible to BS compared to MG1655. These mutants phenocopy a K-12 MG1655-S Δ<i>waaL</i> mutant, which is defective in OAg ligase, primarily due to periplasmic accumulation of the unligated lipid-linked UndPP-OAg. Through the characterization of BS-resistant suppressor mutants of MG1655-S Δ<i>waaL</i>, we identify key genetic disruptions involved in resistance. Notably, we observed the highest BS resistance in strains with a weaker connection between the outer membrane (OM) and peptidoglycan (PG), including strains lacking the major OM-anchored, PG-binding proteins OmpA or Lpp. Expressing versions of OmpA and Lpp that lack PG-binding capacity also enhanced the BS resistance. Our data suggest that BS-induced stress in OAg-producing <i>E. coli</i> is due to the spatial constraints between OM and PG and that mutations disrupting OM-PG interactions alleviate this stress, thereby enhancing BS resistance. These findings provide new insights into a major challenge <i>E. coli</i> faces in the gut environment, where it needs to produce OAg for stable colonization and immune evasion while resisting the antimicrobial activity of BS.IMPORTANCEEnteric bacteria residing in the human gut must withstand host-derived antimicrobial bile salts, but resistance mechanisms are not fully elucidated. In this study, we investigate bile salt resistance mechanisms in O-antigen (OAg)-producing <i>Escherichia coli</i> K-12. We show that the accumulation of carrier lipid-linked OAg in the periplasm of strains with truncated lipopolysaccharide (LPS) core oligosaccharide or defects in OAg ligase can sensitize <i>E. coli</i> more to bile salt, unless the physical links between outer membrane and peptidoglycan are disrupted, highlighting that bile salt-induced stress is attributed to spatial constraints between the outer membrane and peptidoglycan layer. Our work uncovers a previously unappreciated envelope stress response mechanism in <i>E. coli</i>, where reducing outer membrane-peptidoglycan connectivity mitigates bile salt-induced damage arising from OAg production. These findings reshape our understanding of how physical architecture and biosynthetic intermediates intersect to influence bacterial survival in hostile environments.