Non-redundant cardiolipin synthases shape membrane composition and support stress resilience in Bacteroides fragilis.

Bacteroides fragilis is an anaerobic resident of the human gut known to tolerate the toxic effects of host-produced and microbially-modified bile acids. Two conserved genes, clsA and clsB, encode putative cardiolipin synthases that have been linked to bile acid tolerance, but their physiological roles remain undefined. Phylogenetic analysis indicates that Bacteroides spp. ClsA and ClsB diverge from the well-characterized cardiolipin synthases of Gammaproteobacteria and Firmicutes. Here, we show that these enzymes have distinct cardiolipin synthase activities and make non-redundant contributions to B. fragilis fitness under gut-relevant stress conditions, including osmotic stress, disruption of membrane potential, and exposure to the bile acid deoxycholate. Although deoxycholate treatment perturbed K⁺/Na⁺ homeostasis in B. fragilis, deletion of clsA or clsB did not significantly alter intracellular ion levels, suggesting that cardiolipin loss does not substantially impact ion balance under standard cultivation conditions. High-resolution lipidomic analyses showed that cardiolipin comprises less than 1% of B. fragilis membranes and that ClsA and ClsB produce distinct cardiolipin products with unique acyl chain lengths and levels of unsaturation. Deletion of either cls gene led to Cls-specific remodeling of B. fragilis envelope lipid content, which was also associated with shifts in non-lipid metabolites indicative of stress-induced metabolic changes. These results define distinct roles for ClsA and ClsB in shaping B. fragilis membrane composition, metabolism, and stress resilience, and highlight cardiolipin as a key determinant of fitness under bile acid stress.