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.

Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease.

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of Δ revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of Δ by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated , suggesting LapB and LapC act antagonistically.

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl Isomerases Identifies Three New PPIase Activities in That Includes the DksA Transcription Factor.

Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl isomerases (PPIs) in confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of Δ6 strains. Interestingly, viability of Δ6 bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda.

Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Isomerases and Their Collective Essentiality in .

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl isomerases (PPIs). The cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed.