A surface-exposed cardiolipin synthase provides an unexpected paradigm for maintaining the Gram-negative outer membrane

Gram-negative bacteria have long been thought to confine primary glycerophospholipid synthesis to the inner membrane (IM). Here, we challenge this paradigm by identifying ClsO, an outer membrane (OM) lipoprotein from <i>Acinetobacter baumannii</i> that functions as a cardiolipin synthase. In contrast to canonical cardiolipin synthases, which are restricted to the IM, ClsO is surface-exposed and deposits cardiolipin into the outer leaflet of the OM, colocalizing with <i>Acinetobacter</i> lipooligosaccharide. Loss of ClsO sensitizes cells to membrane disrupting agents, such as bile salts, and key antibiotics, implicating ClsO in OM barrier integrity. ClsO activity increases when mislocalized glycerophospholipids accumulate at the cell surface, such as in cells lacking Mla (maintenance of lipid asymmetry) system, consistent with a surface-active site mechanism. Engineering mutations in ClsO that prevent its trafficking to the OM was found to abolish nearly all enzymatic activity. Remarkably, we found that ClsO homologs are widespread across Proteobacteria and confirmed several to be functional cardiolipin synthases, indicating a conserved strategy of surface lipid remodeling. These findings redefine the spatial organization of lipid metabolism in Gram-negative bacteria and reveal an adaptive mechanism to maintain OM integrity when membrane asymmetry is compromised.