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

To identify the physiological factors that limit the growth of K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IV derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ() identified two independent single-amino-acid substitutions-P50S and R310S-in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene was found to be essential for the growth of Δ, Δ, Δ, and Δ() bacteria, with a conditional lethal phenotype of Δ(), which could be overcome by suppressor mutations in MsbA. Suppressor mutations A20D or G53V, causing a constitutive incorporation of phosphoethanolamine () in the lipid A, could abolish the Ca sensitivity of Δ(), thereby compensating for absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ() bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis.