Cardiolipin aids in lipopolysaccharide transport to the gram-negative outer membrane.

In , cardiolipin (CL) is the least abundant of the three major glycerophospholipids in the gram-negative cell envelope. However, harbors three distinct enzymes that synthesize CL: ClsA, ClsB, and ClsC. This redundancy suggests that CL is essential for bacterial fitness, yet CL-deficient bacteria are viable. Although multiple CL-protein interactions have been identified, the role of CL still remains unclear. To identify genes that impact fitness in the absence of CL, we analyzed high-density transposon (Tn) mutant libraries in combinatorial CL synthase mutant backgrounds. We found LpxM, which is the last enzyme in lipid A biosynthesis, the membrane anchor of lipopolysaccharide (LPS), to be critical for viability in the absence of Here, we demonstrate that CL produced by ClsA enhances LPS transport. Suppressors of and essentiality were identified in , a gene that encodes the indispensable LPS ABC transporter. Depletion of ClsA in ∆ mutants increased accumulation of LPS in the inner membrane, demonstrating that the synthetic lethal phenotype arises from improper LPS transport. Additionally, overexpression of ClsA alleviated Δ defects associated with impaired outer membrane asymmetry. Mutations that lower LPS levels, such as a YejM truncation or alteration in the fatty acid pool, were sufficient in overcoming the synthetically lethal Δ Δ phenotype. Our results support a model in which CL aids in the transportation of LPS, a unique glycolipid, and adds to the growing repertoire of CL-protein interactions important for bacterial transport systems.