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This study investigated the role of the mycolyltransferase Ag85A, a key enzyme in mycomembrane synthesis, in regulating plasma membrane domains and cell envelope organization in\n                      <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                      . Using \u2206\n                      <jats:italic toggle=\"yes\">ag85A<\/jats:italic>\n                      deletion mutants, we combined microscopy, subcellular fractionation, mass spectrometry, and lipid analysis to evaluate changes in membrane structure, intracellular chemical accumulation, and lipid composition. The deletion of\n                      <jats:italic toggle=\"yes\">ag85A<\/jats:italic>\n                      led to destabilization of the plasma membrane domain, increased chemical accumulation, and altered cell envelope lipid compositions. Consistent with prior studies, \u0394\n                      <jats:italic toggle=\"yes\">ag85A<\/jats:italic>\n                      accumulated mycolates, specifically in the free lipid pool rather than in the covalently linked cell wall fractions. In addition, the accumulation of an uncharacterized glycolipid species was absent or reduced in the wild-type and complemented strains; these findings indicate that Ag85A loss alters the distributions of mycolic acid-containing lipids and is associated with destabilization of the plasma membrane domain, highlighting a functional link between the processes of lipids and plasma membrane architecture.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        The unique cell envelope of mycobacteria plays a vital role in survival by protecting against immune responses and antibiotic treatments. In this study, we examined the consequences of deleting\n                        <jats:italic toggle=\"yes\">ag85A<\/jats:italic>\n                        , a well-characterized mycolyltransferase that contributes to the assembly of the mycomembrane lipid layer. In addition to known phenotypes, such as increased drug sensitivity and disrupted biofilm formation, our data reveal alterations in membrane-associated lipid distributions and suggest a possible link between membrane synthesis and plasma membrane organization. These findings provide new insight into the broader cellular impact of Ag85A loss and suggest avenues for further investigation into how lipid biosynthesis interfaces with membrane architecture. Understanding this relationship may inform new therapeutic strategies that destabilize the protective envelope of pathogenic mycobacteria.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jb.00240-26","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T16:42:48Z","timestamp":1784047368000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["<i>ag85A<\/i>\n                    loss promotes free mycolate accumulation and sensitizes plasma membrane domain to disruption"],"prefix":"10.1128","volume":"208","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5419-6123","authenticated-orcid":true,"given":"Takehiro","family":"Kado","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01d2sez20","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Missouri State University","place":["Springfield, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"family":"Shivangi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05vt9qd57","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers University","place":["Newark, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paige H.","family":"Bosworth","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0072zz521","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, University of Massachusetts","place":["Amherst, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jake","family":"Jordan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01d2sez20","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Missouri State University","place":["Springfield, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joel S.","family":"Freundlich","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05vt9qd57","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers University","place":["Newark, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8232-3246","authenticated-orcid":true,"given":"M. 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