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

ABSTRACT 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. IMPORTANCE The gut bacterium Bacteroides fragilis is a normal member of the human intestinal microbiota that can also bloom to high abundance in inflamed guts. To survive within the gut, B. fragilis must maintain the integrity and function of its cell membrane. In this study, we report that the two cardiolipin synthase family genes in the B. fragilis genome impact the cell’s lipid profile in distinct ways and contribute differently to how B. fragilis responds to membrane stress. Our work provides insight into how a common gut bacterium adapts to conditions encountered in the intestine and improves understanding of membrane biology in this important group of gut microbes.