Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

Butler, Emily K.; Davis, Rebecca; Bari, Vase; Nicholson, Paul A.; Ruiz, Natividad

doi:10.1128/jb.00731-13

Cited by 63 publications

(150 citation statements)

References 72 publications

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“…Additional support for the idea that MurJ homologs are flippases comes from their membership in the MOP exporter superfamily and mutagenesis studies in E. coli suggesting that MurJ Ec contains a solvent-exposed transmembrane channel (26). Collectively, our data lead us to propose that Amj is the founding member of a new family of lipid II flippases.…”

Section: Discussionmentioning

confidence: 64%

“…Thus, we anticipate that Amj oligomerizes to assemble a conduit for lipid II transport. It has been proposed that MurJ forms a C-shaped portal that allows transport of the hydrophilic diphosphate-disaccharide-pentapeptide portion of lipid II while accommodating its hydrophobic polyisoprenoid moiety within the lipid bilayer (26). Whether MurJ uses energycoupled transport or facilitated diffusion in translocating lipid II remains an open question.…”

Section: Discussionmentioning

confidence: 99%

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MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

Meeske

Sham

Kimsey

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

172

176

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Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJ Bs ; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJ Bs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJ Bs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ M and cells lacking MurJ Bs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.T he bacterial cell wall or peptidoglycan (PG) is composed of glycan strands cross-linked together by short peptides. This 3D meshwork protects the cell from osmotic lysis and determines shape, and its assembly is the target of some of our most successful antibiotics. Cell wall synthesis begins in the cytoplasm, where a set of highly conserved enzymes catalyze the formation of the lipid-linked precursor lipid II, which is composed of undecaprenyl-pyrophosphate (UndPP) linked to N-acetylglucosamine-N-acetylmuramic acid pentapeptide. Lipid II is synthesized on the inner face of the cytoplasmic membrane (1). The molecule is then translocated to the outer face of the membrane, where the disaccharide-peptide monomer is incorporated into the existing PG by cell wall synthetic machineries composed of penicillin-binding proteins and additional factors (2). The enzymes that transport lipid II across the membrane have been the subject of extensive research and speculation (3-6). Recent work in Escherichia coli has provided strong evidence that the polytopic membrane protein MurJ is required for lipid II transport across the membrane, and is likely to be a lipid II flippase (6). MurJ is a member of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (7). It is broadly conserved among Eubacteria and essential for viability in many organisms. Intriguingly, work in the model gram-positive bacterium Bacillus subtilis indicates that cells lacking four MOP superfamily members similar to MurJ are viable and have...

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Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

Meeske

Sham

Kimsey

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

172

176

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“…The IM protein MurJ is essential for PG biogenesis and functions as the lipid II flippase (9)(10)(11). In agreement with this function, (i) MurJ depletion leads to the accumulation of PG precursors and cell lysis (10)(11)(12); (ii) MurJ belongs to the MVF family of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily, which includes other flippases (13); (iii) MurJ contains a central hydrophilic cavity within the hydrophobic core of the membrane (14); (iv) residues within this hydrophilic cavity are essential for MurJ function (14); and (v) chemical inactivation of MurJ function rapidly stops lipid II flipping and leads to the accumulation of lipid II at the inner leaflet of the IM (9). Although we do not understand how MurJ functions in lipid II translocation, it likely shares functional features with members of the MOP exporter superfamily.…”

mentioning

confidence: 85%

“…Furthermore, the two proteins are functionally interchangeable and share 54% identity (determined by Clustal Omega alignment [21]). The essentiality of 5 charged residues that are homologous to essential residues located within the cavity of E. coli MurJ (14) has been investigated in MurJ BC ; of these residues, only 3 are essential for MurJ BC function in B. cenocepacia (12). Thus, evidence exists for both conserved and organism-specific requirements for MurJ activity.…”

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confidence: 99%

Charge Requirements of Lipid II Flippase Activity in Escherichia coli

et al. 2014

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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...

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“…Next, the FemXAB transpeptidases catalyse the coupling of a pentaglycin-moiety to the D-lysine of the pentapeptide u it a d fu the o e, the a idatio of the -D-glutamate of the pentapeptide unit by the MurT/GatD two-enzyme complex leading to the corresponding D-isoglutamin (Münch et al 2012;Zapun et al 2013). This modified lipid II now contains the complete peptidoglycan monomer linked via a pyrophosphate to the bactoprenol membrane anchor, which is translocated through the phospholipid membrane by the MurJ flippase and presented on the extracellular membrane surface (Mohammadi et al 2011;Butler et al 2013a;Sham et al 2014;Meeske et al 2015). On the extracellular membrane surface the peptidoglycan precursor is released, incorporated into the growing peptidoglycan layer and crosslinked (Zapun et al 2013) with each other catalyzed by the PBPs (Sauvage et al 2008).…”

Section: Lessons Learned From Druggable Targets In Bacteriamentioning

confidence: 99%

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

Klahn

Brönstrup

2016

Current Topics in Microbiology and Immunology

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Abstract:The development of bacterial resistance against current antibiotic drugs necessitates a continuous renewal of the arsenal of efficacious drugs. This imperative has not been met by the output of antibiotic research and development of the past decades for various reasons, including the declining efforts of large pharma companies in this area. Moreover, the majority of novel antibiotics are chemical derivatives of existing structures that represent mostly step innovations, implying that the available chemical space may be exhausted. This review negates this impression by showcasing recent achievements in lead finding and optimization of antibiotics that have novel or unexplored chemical structures. Not surprisingly, many of the novel structural templates like teixobactins, lysocin, griselimycin, or the albicidin/cystobactamid pair were discovered from natural sources. Additional compounds were obtained from the screening of synthetic libraries and chemical synthesis, including the gyrase-inhibiting NTBI s a d spiropyrimidinetrione, the tarocin and targocil inhibitors of wall teichoic acid synthesis, or the boronates and diazabicyclo[3.2

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Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

Cited by 63 publications

References 72 publications

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

Charge Requirements of Lipid II Flippase Activity in Escherichia coli

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

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