Coordinated Biosynthesis of Essential Cell Envelope Components: Lipopolysaccharide and Fatty Acids Requires LapD,...

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of Gram-negative bacteria, and its levels are tightly co-regulated with phospholipid (PL) amounts. This homeostatic regulation necessitates the involvement of numerous genes, including lapD in a poorly defined manner. To understand the function of LapD, we took advantage of the synthetic lethal phenotype conferred by the concomitant absence of LapD and myristoyltransferase LpxM or heptosyltransferase WaaC and isolated extragenic suppressors that could bypass this lethality. Suppressor analyses of Δ(lapD lpxM) bacteria identified five single amino acid exchanges in AccA and two in each of AccC and AccD. These proteins comprise different subunits of the acetyl-CoA carboxylase complex, which catalyzes the rate-limiting step in the initiation of fatty acid synthesis, mediating the conversion of acetyl-CoA to malonyl-CoA. Fatty acid analysis revealed that these mutations restored the ratio of saturated to unsaturated fatty acids and repressed elevated PL levels. Suppressor analyses of Δ(lapD waaC) identified a single amino acid substitution in LptD, which is required for LPS assembly in the OM, and in NlpI, which regulates the amount of peptidoglycan hydrolase MepS. These results posit LapD as the point of critical regulation of homeostatic control of three essential cell envelope components.