Non-redundant cardiolipin synthase family proteins shape lipid composition and stress resilience in Bacteroides...

Gut-resident bacteria must withstand membrane-disrupting stresses, such as bile acids, to persist in the intestinal environment. The Bacteroidota are a dominant phylum of the human gut microbiota. However, the genes responsible for the synthesis of membrane lipids in this phylum remain relatively uncharacterized. Bacteroides fragilis is a prevalent member of this phylum and encodes two predicted cardiolipin synthase family proteins, ClsA and ClsB. We previously identified both cls genes as bile-acid fitness factors in B. fragilis P207, but the contributions of clsA and clsB to cell physiology remained unclear. Here, we combine targeted gene deletion with high-resolution lipidomics, metabolomics, and elemental mass spectrometry to show that ClsA and ClsB have distinct and non-redundant functions in the cell. Each cls gene differs in its growth-phase expression pattern, and deletion of each gene produces distinct effects on cell morphology, fitness under membrane-perturbing stress, and the broader metabolome. clsA and clsB strongly influence the levels of different glycyl-seryl peptidolipids, a Bacteroides lipid family that includes immune-active lipids, and whose loss impairs bile acid resistance and gut colonization. In contrast to the acute ion-gradient disruption caused by the secondary bile acid deoxycholate, deletion of clsA and clsB did not measurably alter steady-state intracellular ion levels under standard growth conditions. Together, these results define related but non-redundant roles for two cardiolipin synthase family proteins in a common member of the human gut microbiota.