Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability.

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

Regulation of peptidoglycan synthesis by outer-membrane proteins.

Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/LpoB and their PBP-docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.

Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases.

Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.

Pbp4 provides transpeptidase activity to the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in Enterococcus faecalis.

Enterococci exhibit intrinsic resistance to cephalosporins, mediated in part by the class B penicillin-binding protein (bPBP) Pbp4 that exhibits low reactivity toward cephalosporins and thus can continue crosslinking peptidoglycan despite exposure to cephalosporins. bPBPs partner with cognate SEDS (shape, elongation, division, and sporulation) glycosyltransferases to form the core catalytic complex of peptidoglycan synthases that synthesize peptidoglycan at discrete cellular locations, although the SEDS partner for Pbp4 is unknown. SEDS-bPBP peptidoglycan synthases of enterococci have not been studied, but some SEDS-bPBP pairs can be predicted based on sequence similarity. For example, FtsW (SEDS)-PbpB (bPBP) is predicted to form the catalytic core of the peptidoglycan synthase that functions at the division septum (the divisome). However, PbpB is readily inactivated by cephalosporins, raising the question-how could the FtsW-PbpB synthase continue functioning to enable growth in the presence of cephalosporins? In this work, we report that the FtsW-PbpB peptidoglycan synthase is required for cephalosporin resistance of Enterococcus faecalis, despite the fact that PbpB is inactivated by cephalosporins. Moreover, Pbp4 associates with the FtsW-PbpB synthase and the TPase activity of Pbp4 is required to enable growth in the presence of cephalosporins in an FtsW-PbpB-synthase-dependent manner. Overall, our results implicate a model in which Pbp4 directly interacts with the FtsW-PbpB peptidoglycan synthase to provide TPase activity during cephalosporin treatment, thereby maintaining the divisome SEDS-bPBP peptidoglycan synthase in a functional state competent to synthesize crosslinked peptidoglycan. These results suggest that two bPBPs coordinate within the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in E. faecalis.

Lytic transglycosylase repertoire diversity enables intrinsic antibiotic resistance and daughter cell separation in Escherichia coli under acidic stress.

Peptidoglycan (PG) is an important architectural element that imparts physical toughness and rigidity to the bacterial envelope. It is also a dynamic structure that undergoes continuous turnover or autolysis. Escherichia coli possesses redundant PG degradation enzymes responsible for PG turnover; however, the advantage afforded by the existence of numerous PG degradation enzymes remains incompletely understood. In this study, we elucidated the physiological roles of MltE and MltC, members of the lytic transglycosylase (LTG) family that catalyze the cleavage of glycosidic bonds between disaccharide subunits within PG strands. MltE and MltC are acidic LTGs that exhibit increased enzymatic activity and protein levels under acidic pH conditions, respectively, and deletion of these two LTGs results in a pronounced growth defect at acidic pH. Furthermore, inactivation of these two LTGs induces increased susceptibility at acidic pH against various antibiotics, particularly vancomycin, which seems to be partially caused by elevated membrane permeability. Intriguingly, inactivation of these LTGs induces a chaining morphology, indicative of daughter cell separation defects, only under acidic pH conditions. Simultaneous deletion of PG amidases, known contributors to daughter cell separation, exacerbates the chaining phenotype at acidic pH. This suggests that the two LTGs may participate in the cleavage of glycan strands between daughter cells under acidic pH conditions. Collectively, our findings highlight the role of LTG repertoire diversity in facilitating bacterial survival and antibiotic resistance under stressful conditions.

The LpoA activator is required to stimulate the peptidoglycan polymerase activity of its cognate cell wall synthase PBP1a.

A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from E. coli and Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place.

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW.

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

PBP1A Directly Interacts with the Divisome Complex to Promote Septal Peptidoglycan Synthesis in Acinetobacter baumannii.

The class A penicillin-binding proteins (aPBPs), PBP1A and PBP1B, are major peptidoglycan synthases that synthesize more than half of the peptidoglycan per generation in Escherichia coli. Whereas aPBPs have distinct roles in peptidoglycan biosynthesis during growth (i.e., elongation and division), they are semiredundant; disruption of either is rescued by the other to maintain envelope homeostasis and promote proper growth. Acinetobacter baumannii is a nosocomial pathogen that has a high propensity to overcome antimicrobial treatment. A. baumannii contains both PBP1A and PBP1B (encoded by mrcA and mrcB, respectively), but only mrcA deletion decreased fitness and contributed to colistin resistance through inactivation of lipooligosaccharide biosynthesis, indicating that PBP1B was not functionally redundant with the PBP1A activity. While previous studies suggested a distinct role for PBP1A in division, it was unknown whether its role in septal peptidoglycan biosynthesis was direct. Here, we show that A. baumannii PBP1A has a direct role in division through interactions with divisome components. PBP1A localizes to septal sites during growth, where it interacts with the transpeptidase PBP3, an essential division component that regulates daughter cell formation. PBP3 overexpression was sufficient to rescue the division defect in ΔmrcA A. baumannii; however, PBP1A overexpression was not sufficient to rescue the septal defect when PBP3 was inhibited, suggesting that their activity is not redundant. Overexpression of a major dd-carboxypeptidase, PBP5, also restored the canonical A. baumannii coccobacilli morphology in ΔmrcA cells. Together, these data support a direct role for PBP1A in A. baumannii division and highlights its role as a septal peptidoglycan synthase. IMPORTANCE Peptidoglycan biosynthesis is a validated target of ß-lactam antibiotics, and it is critical that we understand essential processes in multidrug-resistant pathogens such as Acinetobacter baumannii. While model systems such as Escherichia coli have shown that PBP1A is associated with side wall peptidoglycan synthesis, we show herein that A. baumannii PBP1A directly interacts with the divisome component PBP3 to promote division, suggesting a unique role for the enzyme in this highly drug-resistant nosocomial pathogen. A. baumannii demonstrated unanticipated resistance and tolerance to envelope-targeting antibiotics, which may be driven by rewired peptidoglycan machinery and may underlie therapeutic failure during antibiotic treatment.

MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli.

Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin-binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain poorly understood. A few years ago, the conserved lytic transglycosylase MltG was identified as a potential terminase for PG synthesis in Escherichia coli. However, characterization of the in vivo function of MltG was hampered by the lack of a growth or morphological phenotype in ΔmltG cells. Here, we report the isolation of MltG-defective mutants as suppressors of lethal deficits in either aPBP or SEDS/bPBP PG synthase activity. We used this phenotype to perform a domain-function analysis for MltG, which revealed that access to the inner membrane is important for its in vivo activity. Overall, our results support a model in which MltG functions as a terminase for both classes of PG synthases by cleaving PG glycans as they are being actively synthesized.

Class A PBPs: It is time to rethink traditional paradigms.

Until recently, class A penicillin-binding proteins (aPBPs) were the only enzymes known to catalyze glycan chain polymerization from lipid II in bacteria. Hence, the discovery of two novel lipid II polymerases, FtsW and RodA, raises new questions and has consequently received a lot of attention from the research community. FtsW and RodA are essential and highly conserved members of the divisome and elongasome, respectively, and work in conjunction with their cognate class B PBPs (bPBPs) to synthesize the division septum and insert new peptidoglycan into the lateral cell wall. The identification of FtsW and RodA as peptidoglycan glycosyltransferases has raised questions regarding the role of aPBPs in peptidoglycan synthesis and fundamentally changed our understanding of the process. Despite their dethronement, aPBPs are essential in most bacteria. So, what is their function? In this review, we discuss recent progress in answering this question and present our own views on the topic.

ActS activates peptidoglycan amidases during outer membrane stress in Escherichia coli.

The integrity of the cell envelope of E. coli relies on the concerted activity of multi-protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD-transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a. Consequently, cells with defective OM biogenesis lyse if they lack any of these PG enzymes. Here we report that the morphological defects, and lysis associated with a ldtF mutant with impaired LPS transport, are alleviated by the loss of the predicted OM-anchored lipoprotein ActS (formerly YgeR). We show that ActS is an inactive member of LytM-type peptidoglycan endopeptidases due to a degenerated catalytic domain. ActS is capable of activating all three main periplasmic peptidoglycan amidases, AmiA, AmiB, and AmiC, which were previously reported to be activated only by EnvC and/or NlpD. Our data also suggest that in vivo ActS preferentially activates AmiC and that its function is linked to cell envelope stress.

Unconventional Antibacterials and Adjuvants.

The need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-ß-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus, discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of ß-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a ß-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of ß-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.

Photoaffinity labeling of benzophenone-containing salicylanilide compounds to give an insight into the mechanism in disrupting peptidoglycan formation.

A series of salicylanilide compounds was previously identified as antibacterial agents that inhibit the peptidoglycan formation. To find the exact binding mode, we synthesized a benzophenone-containing salicylanilide compound (1) and used it as a photoaffinity probe to label Acinetobacter baumannii penicillin-binding protein (PBP1b). After incubation and photo-irradiation, the labeled protein was subjected to trypsin digestion, dialysis enrichment, LC-ESI-MS/MS analysis, and Mascot search to reveal an octadecapeptide sequence 364RQLRTEYQESDLTNQGLR381 that was labeled at E372. Our molecular docking experiments suggest a hydrophobic pocket surrounded by R367 and E372 is the binding site of salicylanilide 1. The pocket lies in between the transglycosylase and transpeptidase domains, thus binding of salicylanilide 1 can block the propagation pathway to disrupt the growth of peptidoglycan chain.

SEDS proteins are a widespread family of bacterial cell wall polymerases.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.

Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli.

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.

CwlQ Is Required for Swarming Motility but Not Flagellar Assembly in Bacillus subtilis.

Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown.IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75-Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation.

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by 'autolysins', enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low-salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

Simultaneously inhibiting undecaprenyl phosphate production and peptidoglycan synthases promotes rapid lysis in Escherichia coli.

Peptidoglycan (PG) is a highly cross-linked polysaccharide that encases bacteria, resists the effects of turgor and confers cell shape. PG precursors are translocated across the cytoplasmic membrane by the lipid carrier undecaprenyl phosphate (Und-P) where they are incorporated into the PG superstructure. Previously, we found that one of our Escherichia coli laboratory strains (CS109) harbors a missense mutation in uppS, which encodes an enzymatically defective Und-P(P) synthase. Here, we show that CS109 cells lacking the bifunctional aPBP PBP1B (penicillin binding protein 1B) lyse during exponential growth at elevated temperature. PBP1B lysis was reversed by: (i) reintroducing wild-type uppS, (ii) increasing the availability of PG precursors or (iii) overproducing PBP1A, a related bifunctional PG synthase. In addition, inhibiting the catalytic activity of PBP2 or PBP3, two monofunctional bPBPs, caused CS109 cells to lyse. Limiting the precursors required for Und-P synthesis in MG1655, which harbors a wild-type allele of uppS, also promoted lysis in mutants lacking PBP1B or bPBP activity. Thus, simultaneous inhibition of Und-P production and PG synthases provokes a synergistic response that leads to cell lysis. These findings suggest a biological connection that could be exploited in combination therapies.