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The gut bacterium\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      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 \u0394\n                      <jats:italic toggle=\"yes\">waaL<\/jats:italic>\n                      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 \u0394\n                      <jats:italic toggle=\"yes\">waaL<\/jats:italic>\n                      , 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\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      is due to the spatial constraints between OM and PG and that mutations disrupting OM\u2013PG interactions alleviate this stress, thereby enhancing BS resistance. These findings provide new insights into a major challenge\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      faces in the gut environment, where it needs to produce OAg for stable colonization and immune evasion while resisting the antimicrobial activity of BS.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Enteric 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\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                        , where reducing outer membrane\u2013peptidoglycan 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.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.02184-25","type":"journal-article","created":{"date-parts":[[2025,8,28]],"date-time":"2025-08-28T12:00:17Z","timestamp":1756382417000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Disruptions in outer membrane-peptidoglycan interactions enhance bile salt resistance in O-antigen-producing\n                    <i>E. coli<\/i>"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5867-6332","authenticated-orcid":true,"given":"Jilong","family":"Qin","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03pnv4752","id-type":"ROR","asserted-by":"publisher"}],"name":"Centre for 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