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

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, and , encode putative cardiolipin synthases that have been linked to bile acid tolerance, but their physiological roles remain undefined. Phylogenetic analysis indicates that 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 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 , deletion of or 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 membranes and that ClsA and ClsB produce distinct cardiolipin products with unique acyl chain lengths and levels of unsaturation. Deletion of either gene led to Cls-specific remodeling of 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 membrane composition, metabolism, and stress resilience, and highlight cardiolipin as a key determinant of fitness under bile acid stress.