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

Abstract

In <i>Escherichia coli</i>, cardiolipin (CL) is the least abundant of the three major glycerophospholipids in the gram-negative cell envelope. However, <i>E. coli</i> 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 <i>clsA</i> Here, we demonstrate that CL produced by ClsA enhances LPS transport. Suppressors of <i>clsA</i> and <i>lpxM</i> essentiality were identified in <i>msbA</i>, a gene that encodes the indispensable LPS ABC transporter. Depletion of ClsA in ∆<i>lpxM</i> 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<i>lpxM</i> 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<i>clsA</i> Δ<i>lpxM</i> 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.