Charge Requirements of Lipid II Flippase Activity in Escherichia coli

Abstract

Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport. Most bacteria produce a rigid peptidoglycan (PG) layer that is essential for defining cell shape and providing protection against osmotic lysis (1, 2). This mesh-like PG macromolecule consists of glycan strands of repeating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units that are bridged by the cross-linking of peptide stems extending from the MurNAc moiety. The PG layer is localized outside the cytoplasmic membrane, and as the cell grows, newly synthesized PG material must be incorporated into the preexisting polymer. Since the disaccharide-pentapeptide building blocks are made in the cytoplasm, the cytoplasmic membrane separates PG precursor synthesis from its utilization. Therefore, transport of the disaccharide-pentapeptide across the membrane is an essential step in PG biogenesis.In Escherichia coli, PG is present in the periplasm, the aqueous compartment between the inner and outer membranes (3). Like in all PG-producing bacteria, the disaccharide-pentapeptide PG precursor is synthesized onto the lipid carrier undecaprenol at the inner leaflet of the inner membrane (IM) (reviewed in references 4-7). This lipid-linked precursor, known as lipid II (undecaprenyl-pyrophosphoryl-MurNAc-[pentapeptide]-GlcNAc), must be flipped across the IM and delivered to the outer leaflet of the IM. Once lipid II is exposed to the periplasm, t...

A viral protein antibiotic inhibits lipid II flippase activity

“…The SCAM reactivity patterns for five positions in aqueous domains were unaffected by lys M induction, but five TMD positions exhibited altered MTSES sensitivity, with four being converted from partial to complete reactivity. These results suggest that Lys M binds to MurJ and causes a conformational change that locks MurJ into one of the two conformations proposed to constitute the lipid II flipping cycle, with the solvent-filled cavity facing either the periplasm or cytoplasm 10–12 (Supplementary Fig. 7).…”

“…7). Accordingly, induction of lys M in cells overexpressing non-functional alleles of murJ that produce normal levels of protein 12 did not protect against Lys M -dependent lysis, whereas the wild-type allele does (Supplementary Fig. 4d).…”

Filling holes in peptidoglycan biogenesis of Escherichia coli

“…The predicted structure includes a V-shaped solvent-exposed cavity that contains several charged residues required for function [25]. Furthermore, modification of some engineered cysteine substitutions in this cavity with sulfhydryl-reacting probes inactivates MurJ in E. coli [26].…”

“…First, MurJ belongs to the MOP exporter superfamily, which includes members such as Wzx flippases of Und-PP-linked oligosaccharides similar to lipid II [18,27]. Second, like Wzx, MurJ adopts a structure with a central hydrophilic cavity located within the plane of the membrane that contains residues essential for function [23,25,28]. Thus, MurJ is evolutionarily related to flippases of Und-P-linked oligosaccharides and shares functionally relevant, structural features with them.…”

“…In this model, the essential charged residues in the cavity of MurJ could interact with either the hydrophilic moiety of lipid II or the counter ion [21,24,25]. Alternatively, since those essential residues are localized in the periplasmic half of the solvent-exposed cavity, MurJ could first engage with lipid II in an inward-facing conformation through a low-affinity site; this interaction could result in a conformational change that could open the periplasmic side of the cavity, allowing lipid II to interact with higher affinity with the aforementioned charged residues, thereby driving transport directionally to the periplasmic side of the membrane [21].…”