An Essential Membrane Protein Modulates the Proteolysis of LpxC to Control Lipopolysaccharide Synthesis in Escherichia coli

Abstract

Gram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share an intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adapter protein LapB (YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM (PbgA) participates in this regulatory pathway. Suppressors of YejM essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of ΔyejM mutants. Furthermore, the stability of LpxC was shown to be reduced in cells lacking YejM, and genetic and physical interactions between LapB and YejM were detected. Taken together, our results are consistent with a model in which YejM directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis. IMPORTANCE The outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this study, we identified an essential protein of unknown function as a key new factor in modulating LPS synthesis in the model bacterium Escherichia coli. Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.

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

“…Another variant of LpxC R230C as the suppressor of lapC mutants was also isolated three times that also resulted in elevated levels of LpxC. During the completion of this work, two independent studies also reported the isolation of suppressors of lapC that caused alterations of either V37 or R230 amino acids, respectively [35,36]. It is of interest that we isolated additional variants of LpxC that suppress lapC190 and lapC377fs mutations, which were not reported in other complementary studies mentioned above, providing a broader model of LpxC regulation and help in understanding the function of LapC.…”

“…Thus, in the first approach, Tn10-linked point mutations that simultaneously exhibited the elevated rpoEP3 promoter activity, the sensitivity to the LpxC inhibitor CHIR090 to different extents and membrane permeability defects were analyzed leading to the identification of three point mutations: one having a frame-shift mutation resulting in truncation after 377 amino acid residue isolated three times, the second mutation introduces a stop codon after amino acid residue 190, another resulting into a single amino acid F349S substitution, in the essential yejM gene. During the completion of this work, YejM has been speculated to either maintain lipid homeostasis or be involved in the transport of cardiolipin or exhibit a metal-dependent phosphatase activity [31][32][33][34][35][36][37]. We designated this gene lapC based on identified genetic and biochemical interactions of LapC with LapB.…”

Restoring Balance to the Outer Membrane: YejM’s Role in LPS Regulation

“…Three consecutive papers from the Silhavy ( 10 ), Bernhardt ( 12 ), and Misra ( 13 ) laboratories revealed the essential role of YejM in E. coli through suppressor analysis. The Misra group began by generating suppressors to large antibiotics (erythromycin and vancomycin) in a hypersensitive E. coli mutant that produces a periplasmic-truncated YejM ( 13 ).…”

“…Furthermore, Bernhardt and colleagues isolated viable suppressors in E. coli lacking the complete YejM protein, and these suppressors were mapped to lpxC and lapB ( 12 ). Like the Misra laboratory, they also revealed that stabilizing LpxC levels rescued yejM mutants ( 12 ).…”

“Asymmetry Is the Rhythmic Expression of Functional Design,” a Quotation from Jan Tschichold

“…1A). Recent papers, one of which is in this issue of the journal (3), find a regulatory role for one of the last essential genes (yejM) in Escherichia coli that lacks a clear function (4,5). The papers provide evidence that YejM senses an intermediate in these biosynthetic pathways to regulate the stability of LpxC, a key enzyme in the biosynthesis of LPS (6,7), to balance the synthesis of PL with LPS and maintain OM asymmetry.…”