Penicillin and related beta-lactams comprise one of our oldest and most widely used antibiotic therapies. These drugs have long been known to target enzymes called penicillin-binding proteins (PBPs) ...that build the bacterial cell wall. Investigating the downstream consequences of target inhibition and how they contribute to the lethal action of these important drugs, we demonstrate that beta-lactams do more than just inhibit the PBPs as is commonly believed. Rather, they induce a toxic malfunctioning of their target biosynthetic machinery involving a futile cycle of cell wall synthesis and degradation, thereby depleting cellular resources and bolstering their killing activity. Characterization of this mode of action additionally revealed a quality control function for enzymes that cleave bonds in the cell wall matrix. The results thus provide insight into the mechanism of cell wall assembly and suggest how best to interfere with the process for future antibiotic development.
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•Beta-lactams cause the activity of their target pathway to become toxic•Toxicity stems from a futile cycle of cell wall synthesis and degradation•Degradation is catalyzed by the lytic transglycosylase enzyme Slt•Slt contributes to quality control in cell wall assembly
Penicillin and related beta-lactam antibiotics induce a toxic, futile cycle of peptidoglycan (PG) synthesis and degradation that depletes cellular stores of the PG building blocks, explaining the effectiveness of these workhorse drugs.
Tol-Pal is a multiprotein system present in the envelope of Gram-negative bacteria. Inactivation of this widely conserved machinery compromises the outer membrane (OM) layer of these organisms, ...resulting in hypersensitivity to many antibiotics. Mutants in the tol-pal locus fail to complete division and form cell chains. This phenotype along with the localization of Tol-Pal components to the cytokinetic ring in Escherichia coli has led to the proposal that the primary function of the system is to promote OM constriction during division. Accordingly, a poorly constricted OM is believed to link the cell chains formed upon Tol-Pal inactivation. However, we show here that cell chains of E. coli tol-pal mutants are connected by an incompletely processed peptidoglycan (PG) layer. Genetic suppressors of this defect were isolated and found to overproduce OM lipoproteins capable of cleaving the glycan strands of PG. Among the factors promoting cell separation in mutant cells was a protein of previously unknown function (YddW), which we have identified as a divisome-localized glycosyl hydrolase that cleaves peptide-free PG glycans. Overall, our results indicate that the cell chaining defect of Tol-Pal mutants cannot simply be interpreted as a defect in OM constriction. Rather, the complex also appears to be required for the activity of several OM-localized enzymes with cell wall remodeling activity. Thus, the Tol-Pal system may play a more general role in coordinating OM invagination with PG remodeling at the division site than previously appreciated.
Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin ...and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.
Summary
Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin‐binding proteins (PBPs), the targets of penicillin and related ...antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG‐dependent nascent PG processing in vivo, and bacterial two‐hybrid analysis identified an MltG‐PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild‐type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.
The cell wall layer of bacteria is composed of long glycan chains crosslinked to one another by attached peptide moieties. It has been known for many years that glycan polymerization and crosslinking are catalyzed by the penicillin‐binding proteins (PBPs). However, it has remained unclear how glycan chain length is controlled. Here, we identify MltG as a candidate terminase enzyme that cleaves nascent glycans produced by the PBPs to help determine chain length.
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
, 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
and
, and LpxC overproduction was shown to be sufficient to allow survival of Δ
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.
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
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.
Summary
Most bacteria are surrounded by a complex cell envelope. As with many biological processes, studies of envelope assembly have benefited from cell‐based assays for detecting protein–protein ...interactions. These assays use simple readouts and lack a protein purification requirement, making them ideal for early stage investigations. The most widely used two‐hybrid interaction assay for proteins involved in envelope biogenesis is based on the reconstitution of adenylate cyclase activity from a split enzyme. Because adenylate cyclase is only functional in the cytoplasm, both protein fusions used in the assay must have a terminus located in this compartment. However, many envelope assembly factors are wholly extracytoplasmic. Detecting interactions involving such proteins using two‐hybrid systems has therefore been problematic. To address this issue, we developed a cytological assay in Escherichia coli based on PopZ from Caulobacter crescentus. Here, we demonstrate the utility of this PopZ‐Linked Apical Recruitment (POLAR) method for detecting interactions between proteins located in different cellular compartments. Additionally, we report that recruitment of an active peptidoglycan synthase to the cell pole is detrimental for E. coli and that interactions between proteins in the inner and outer membranes of the Gram‐negative envelope may provide a mechanism for recruiting protein complexes to subpolar sites.
The POLAR system detects interactions between proteins with different subcellular localizations within the cell envelope.
Cytokinesis in gram-negative bacteria requires the constriction of all three cell envelope layers: the inner membrane (IM), the peptidoglycan (PG) cell wall and the outer membrane (OM). In order to ...avoid potentially lethal breaches in cell integrity, this dramatic reshaping of the cell surface requires tight coordination of the different envelope remodeling activities of the cytokinetic ring. However, the mechanisms responsible for this coordination remain poorly defined. One of the few characterized regulatory points in the envelope remodeling process is the activation of cell wall hydrolytic enzymes called amidases. These enzymes split cell wall material shared by developing daughter cells to facilitate their eventual separation. In Escherichia coli, amidase activity requires stimulation by one of two partially redundant activators: EnvC, which is associated with the IM, and NlpD, a lipoprotein anchored in the OM. Here, we investigate the regulation of amidase activation by NlpD. Structure-function analysis revealed that the OM localization of NlpD is critical for regulating its amidase activation activity. To identify additional factors involved in the NlpD cell separation pathway, we also developed a genetic screen using a flow cytometry-based enrichment procedure. This strategy allowed us to isolate mutants that form long chains of unseparated cells specifically when the redundant EnvC pathway is inactivated. The screen implicated the Tol-Pal system and YraP in NlpD activation. The Tol-Pal system is thought to promote OM invagination at the division site. YraP is a conserved protein of unknown function that we have identified as a new OM-localized component of the cytokinetic ring. Overall, our results support a model in which OM and PG remodeling events at the division site are coordinated in part through the coupling of NlpD activation with OM invagination.
Cell division in bacteria is mediated by a multiprotein assembly called the divisome. A major function of this machinery is the synthesis of the peptidoglycan (PG) cell wall that caps the daughter ...poles and prevents osmotic lysis of the newborn cells. Recent studies have implicated a complex of FtsW and FtsI (FtsWI) as the essential PG synthase within the divisome; however, how PG polymerization by this synthase is regulated and coordinated with other activities within the machinery is not well understood. Previous results have implicated a conserved subcomplex of division proteins composed of FtsQ, FtsL, and FtsB (FtsQLB) in the regulation of FtsWI, but whether these proteins act directly as positive or negative regulators of the synthase has been unclear. To address this question, we purified a five-member Pseudomonas aeruginosa division complex consisting of FtsQLB-FtsWI. The PG polymerase activity of this complex was found to be greatly stimulated relative to FtsWI alone. Purification of complexes lacking individual components indicated that FtsL and FtsB are sufficient for FtsW activation. Furthermore, support for this activity being important for the cellular function of FtsQLB was provided by the identification of two division-defective variants of FtsL that still form normal FtsQLB-FtsWI complexes but fail to activate PG synthesis. Thus, our results indicate that the conserved FtsQLB complex is a direct activator of PG polymerization by the FtsWI synthase and thereby define an essential regulatory step in the process of bacterial cell division.
During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to ...prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC− defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.
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