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

Abstract

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

Checkpoints That Regulate Balanced Biosynthesis of Lipopolysaccharide and Its Essentiality in Escherichia coli

“…Among these genes, transcription of the clsA gene is induced under a variety of stress conditions, and its product ClsA is the major cardiolipin synthase [ 104 , 105 , 106 ]. Recent studies showed that ClsA plays an essential role in strains synthesizing underacylated lipid A and is also required for the DksA-mediated multicopy suppression of strains that lack the six major cytoplasmic peptidyl-prolyl cis / trans isomerases ( Figure 4 c) [ 21 , 22 , 107 ]. A role for CL in Δ waaA strains synthesizing glycosylation free lipid IV A was hinted at because CL was found to prominently accumulate in such backgrounds, as shown by the mass spectrometric analysis of a mixture of lipid A and glycerophospholipids [ 20 , 21 ].…”

“…All amino acid exchanges in MsbA that suppress Δ( clsA lpxM ) map to positions shown to be contact regions with LPS or with drug molecules that highjack substrate binding of MsbA based on recently available structures [ 98 , 99 , 100 , 102 ]. Another independent study also showed a critical requirement for CL in the Δ lpxM background, and depletion experiments revealed that a substantial amount of LPS is retained in the IM, causing the lethality [ 22 ]. Taken together, these studies suggest coordination between CL and MsbA in lipid A trafficking, which is critical when the lipid A is underacylated and also highlights the integration of phospholipid and LPS biosynthetic pathways in balancing the OM composition.…”

“…To ensure that primarily LPS with hexa-acylated lipid A is translocated, MsbA provides an essential checkpoint as it selects species that are hexa-acylated by several orders of magnitude [ 19 , 20 ]. At this point, the viability of bacteria with either tetra- or penta-acylated lipid A requires the presence of cardiolipin (CL), which presumably aids in the transport of such LPS, constituting another checkpoint [ 21 , 22 ]. Once LPS is flipped by MsbA, it is delivered to the Lpt machinery for its final assembly in the OM.…”

Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis

“…Transposon (Tn) mutant libraries were generated in strains that contained or lacked LOS in the OM. Flanking regions of Tn insertions were sequenced (transposon insertion sequencing [Tn-seq]) ( 33 , 34 ) to find genes that impacted fitness during LOS deficiency. Tn-seq identified that every member of the elongasome and ElsL were likely essential during LOS deficiency.…”

Molecular Basis of Essentiality of Early Critical Steps in the Lipopolysaccharide Biogenesis in Escherichia coli K-12: Requirement of MsbA, Cardiolipin, LpxL, LpxM and GcvB

“…At the time of submission of this manuscript, an independent study has also shown synthetic lethality of Δ( clsA lpxM ) bacteria. However, in that work, the function of ClsA in deletion derivatives of waaA , lpxL , lpxP , ( lpxLMP ), and gmhD - waaA —as well as the viability of Δ( clsA lpxM ) at 30 °C—has not been addressed [ 72 ].…”