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

Abstract

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 ΔlapB 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 ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.

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

“…LapB is essential for bacterial viability due to its critical role in the regulated proteolysis of LpxC [ 9 , 12 ]. LpxC quantities are regulated by the LapB/FtsH/LapC complex, and at elevated temperatures by HslUV protease [ 9 , 12 , 13 ].…”

“…LapB is essential for bacterial viability due to its critical role in the regulated proteolysis of LpxC [ 9 , 12 ]. LpxC quantities are regulated by the LapB/FtsH/LapC complex, and at elevated temperatures by HslUV protease [ 9 , 12 , 13 ]. However, in E. coli , overexpression of SlrA sRNA, or the absence of Lpp lipoprotein or genetic backgrounds that reduce the total LPS content, can allow the growth of strains with deletion of lapAB genes [ 9 ].…”

“…Lipid A biosynthesis begins with the LpxA-catalyzed acylation of UDP-GlcNAc [ 2 , 3 , 4 , 6 , 7 ]. This product is further deacylated by the Zn ++ -dependent deacylase LpxC, whose quantities are tightly regulated by LapB, LapC, FtsH, and HslUV, constituting the first committed step in LPS biosynthesis, as the first reaction catalyzed by LpxA is energetically unfavorable [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Following the deacylation step, four additional enzymes (LpxD, LpxH, LpxB, and LpxK) act sequentially to generate the lipid IV A precursor [ 2 , 4 , 14 , 15 , 16 ].…”

“…This is achieved through the regulation of LpxC quantity and FabZ activity [ 19 , 27 ]. LpxC quantity is controlled by the proteolytic activity of the FtsH/LapB/LapC complex and HslUV proteases [ 12 , 13 ]. FtsH proteolytic activity for LpxC requires the heat shock protein LapB, whereas LapC acts antagonistically to prevent excessive degradation of LpxC, and thus together maintain a balanced biosynthesis of LPS [ 9 , 12 , 13 ].…”

“…LpxC quantity is controlled by the proteolytic activity of the FtsH/LapB/LapC complex and HslUV proteases [ 12 , 13 ]. FtsH proteolytic activity for LpxC requires the heat shock protein LapB, whereas LapC acts antagonistically to prevent excessive degradation of LpxC, and thus together maintain a balanced biosynthesis of LPS [ 9 , 12 , 13 ]. Similarly, UDP-GlcNAc serves as a precursor for LPS as well as peptidoglycan synthesis [ 3 ].…”