Cell Wall Hydrolases Research Articles

Effects of 1-Methylcyclopropene Treatment on Fruit Quality during Cold Storage in Apple Cultivars Grown in Korea

The effect of 1-methylcyclopropene (1-MCP) treatment on improving the storability of four apple cultivars (‘Hwangok’, ‘Picnic’, ‘Gamhong’, and ‘Fuji’) was investigated by analyzing the physiological and biochemical factors associated with their postharvest quality attributes. The flesh firmness, titratable acidity, and soluble solids content of the cultivars were higher in treated fruits than untreated fruits, while the opposite results were observed for ethylene production. In the treated fruits, the traits affected by 1-MCP varied depending on the cultivars used. Higher firmness and lower ethylene production were observed in the ‘Hwangok’ and ‘Picnic’ than ‘Gamhong’ and ‘Fuji’ cultivars. However, 1-MCP only affected weight loss in the ‘Gamhong’ cultivar, while the sugar content was affected in all of the cultivars except ‘Hwangok’. When analyzing cell wall hydrolase activities, 1-MCP differently affected the activities (β-galactosidase, α-galactosidase, β-glucosidase, α-mannosidase, β-xylosidase, and β-arabinosidase), with greater effects in the ‘Fuji’ and ‘Picnic’ cultivars and moderate effects in the ‘Gamhong’ and ‘Hwangok’ cultivars. In this study, the suppression of ethylene production by 1-MCP was positively associated with a transcriptional decrease in the ethylene biosynthesis genes MdACS1 and MdACO1. Overall, this study suggests that 1-MCP distinctly enhanced the storability of all apple cultivars, with a greater effect on ‘Hwangok’.

Diversification of LytM Protein Functions in Polar Elongation and Cell Division of Agrobacterium tumefaciens.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

Exebacase in Addition to Daptomycin against MRSA.

Exebacase is a lysin (cell wall hydrolase) with direct lytic activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). Time-kill analysis experiments illustrated bactericidal activity of exebacase-daptomycin against MRSA strains MW2 and 494. Furthermore, exebacase in addition to daptomycin (10, 6, and 4 mg/kg/day) in a two-compartment ex vivo pharmacokinetic/pharmacodynamic simulated endocardial vegetation model with humanized doses resulted in reductions of 6.01, 4.99, and 2.81 log10 CFU/g (from initial inoculum) against MRSA strain MW2.

TipB, a novel cell wall hydrolase, is required for efficient conjugative transfer of pXO16 from Bacillus thuringiensis sv. israelensis

pXO16, a large plasmid from Bacillus thuringiensis serovar israelensis, exhibits unique features. Not only is pXO16 able to transfer at high frequencies within few minutes, but it is also able to transfer among virtually all members of the Bacillus cereus group. Among the proteins encoded in the tip transfer locus of pXO16, TipB displays an atypical organization compared to known conjugative cell wall hydrolases with the large central soluble lytic transglycosylase (SLT) domain missing from the protein. We constructed a tipB deletion mutant which led to significant reduction in transfer efficiencies in both liquid and filter mating. The initial transfer frequencies could be restored when complementing tipB in trans thus showing the TipB implication in pXO16 conjugative transfer. Additionally, truncated versions of TipB were expressed in Escherichia coli to assess the protein lytic activity. When applied exogenously, TipB-2TM, in which the two N-terminal TM domains were removed, yielded a decrease of ca. 40% in optical density of B. thuringiensis sv. israelensis, a lytic activity that could partially be explained by the C-terminal CHAP-like domain. These results confirm TipB conjugative hydrolase function and provide new insights into pXO16 unique conjugative apparatus.

Description and functional analysis of the transcriptome from malting barley

The present study aimed to establish an early model of the malting barley transcriptome, which describes the expression of genes and their ontologies, identify the period during malting with the largest dynamic shift in gene expression for future investigation, and to determine the expression patterns of all starch degrading enzyme genes relevant to the malting and brewing industry. Large dynamic increases in gene expression occurred early in malting with differential expressed genes enriched for cell wall and starch hydrolases amongst many malting related categories. Twenty-five of forty starch degrading enzyme genes were differentially expressed in the malting barley transcriptome including eleven α-amylase genes, six β-amylase genes, three α-glucosidase genes, and all five starch debranching enzyme genes. Four new or novel α-amylase genes, one β-amylase gene (Bmy3), three α-glucosidase genes, and two isoamylase genes had appreciable expression that requires further exploration into their potential relevance to the malting and brewing industry.

Exebacase Is Active In Vitro in Pulmonary Surfactant and Is Efficacious Alone and Synergistic with Daptomycin in a Mouse Model of Lethal Staphylococcus aureus Lung Infection.

ABSTRACTExebacase (CF-301) is a novel antistaphylococcal lysin (cell wall hydrolase) in phase 3 of clinical development for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis, used in addition to standard-of-care antibiotics. In the current study, the potential for exebacase to treat S. aureus pneumonia was explored in vitro using bovine pulmonary surfactant (Survanta) and in vivo using a lethal murine pneumonia model. Exebacase was active against a set of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains, with an MIC90 of 2 μg/ml (n = 18 strains), in the presence of a surfactant concentration (7.5%) inhibitory to the antistaphylococcal antibiotic daptomycin, which is inactive in pulmonary environments due to specific inhibition by surfactant. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, exebacase synergized with daptomycin in the presence of surfactant in vitro, resulting in daptomycin MIC reductions of up to 64-fold against 9 MRSA and 9 MSSA strains. Exebacase was also observed to facilitate the binding of daptomycin to S. aureus and the elimination of biofilm-like structures formed in the presence of surfactant. Exebacase (5 mg/kg of body weight 1 time every 24 h [q24h], administered intravenously for 3 days) was efficacious in a murine model of staphylococcal pneumonia, resulting in 50% survival, compared to 0% survival with the vehicle control; exebacase in addition to daptomycin (50 mg/kg q24h for 3 days) resulted in 70% survival, compared to 0% survival in the daptomycin-alone control group. Overall, exebacase is active in pulmonary environments and may be appropriate for development as a treatment for staphylococcal pneumonia.

The potential of Trichoderma brevicompactum for controlling root rot on Atractylodes macrocephala

Root rot is an important disease of the medicinal plant Atractylodes macrocephala (bai zhu) in China. This study evaluates an antagonistic fungal agent, Trichoderma brevicompactum, as an alternative to chemical pesticides for control of root rot caused by Fusarium oxysporum. The inhibitory effect of T. brevicompactum on F. oxysporum was examined in dual-culture experiments, with mycelial growth of the pathogen found to be reduced by 74.4%. The results of an enzyme-linked immunosorbent assayindicated that T. brevicompactum produced enzymes such as cellulase and chitinase, suggesting that the fungal agent may inhibit F. oxysporum growth by producing cell wall hydrolases. Under greenhouse conditions, the efficacy of T. brevicompactum was evaluated based on its effects on root rot severity, plant height, root dry weight, and root fresh weight. When used as a soil treatment, T. brevicompactum reduced root rot severity by 48% to 58% and increased plant growth and biomass significantly when compared with the control (healthy) group. The plant growth-promoting effect of T. brevicompactum seems related to its production of secondary metabolites that contain an auxin (indoleacetic acid), and to the disease resistance induced in the host by the fungal agent. The results of this study suggest that T. brevicompactum not only controls root rot in A. macrocephala but may also promote growth of the plant.

Phage vB_PaeS-PAJD-1 Rescues Murine Mastitis Infected With Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative pathogen that causes a variety of infections in humans and animals. Due to the inappropriate use of antibiotics, multi-drug resistant (MDR) P. aeruginosa strains have emerged and are prevailing. In recent years, cow mastitis caused by MDR P. aeruginosa has attracted attention. In this study, a microbial community analysis revealed that P. aeruginosa could be a cause of pathogen-induced cow mastitis. Five MDR P. aeruginosa strains were isolated from milk diagnosed as mastitis positive. To seek an alternative antibacterial agent against MDR, P. aeruginosa, a lytic phage, designated vB_PaeS_PAJD-1 (PAJD-1), was isolated from dairy farm sewage. PAJD-1 was morphologically classified as Siphoviridae and was estimated to be about 57.9 kb. Phage PAJD-1 showed broad host ranges and a strong lytic ability. A one-step growth curve analysis showed a relatively short latency period (20 min) and a relatively high burst size (223 PFU per infected cell). Phage PAJD-1 remained stable over wide temperature and pH ranges. Intramammary-administered PAJD-1 reduced bacterial concentrations and repaired mammary glands in mice with mastitis induced by MDR P. aeruginosa. Furthermore, the cell wall hydrolase (termed endolysin) from phage PAJD-1 exhibited a strong bacteriolytic and a wide antibacterial spectrum against MDR P. aeruginosa. These findings present phage PAJD-1 as a candidate for phagotherapy against MDR P. aeruginosa infection.

Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth.

Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise as a novel class of antibacterial agents. In this study, we recombinantly expressed and characterized a cell wall hydrolase (CWH) lysin from C. difficile phage, phiMMP01. The full-length CWH displayed lytic activity against selected C. difficile strains. However, removing the N-terminal cell wall binding domain, creating CWH351—656, resulted in increased and/or an expanded lytic spectrum of activity. C. difficile specificity was retained versus commensal clostridia and other bacterial species. As expected, the putative cell wall binding domain, CWH1—350, was completely inactive. We also observe the effect of CWH351—656 on preventing C. difficile spore outgrowth. Our results suggest that CWH351—656 has therapeutic potential as an antimicrobial agent against C. difficile infection.

Characteristics for phage-encoded cell wall hydrolase of LysSAP27 to reduce staphylococcal food poisoning.

The online version contains supplementary material available at 10.1007/s10068-021-00910-2.

Determination of MIC Quality Control Parameters for Exebacase, a Novel Lysin with Antistaphylococcal Activity.

ABSTRACTExebacase (CF-301), a novel, antistaphylococcal lysin (cell wall hydrolase) is the first agent of this class to enter late-stage clinical development (phase 3, NCT04160468) for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis. A multilaboratory Clinical and Laboratory Standards Institute (CLSI) M23-defined tier 2 quality control (QC) study was conducted to establish exebacase QC ranges for a new reference broth microdilution method. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were selected as reference QC strains. Broth microdilution MIC QC ranges for exebacase spanned 4 log2 dilutions and contained 99.2% of the MIC results generated for the two reference strains. The QC ranges for exebacase were defined as 0.25 to 2 μg/ml and 8 to 64 μg/ml against S. aureus ATCC 29213 and E. faecalis ATCC 29212, respectively, and were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. These QC ranges established for use with the reference broth microdilution method developed for exebacase susceptibility testing will ensure the test performance and accuracy of results generated during clinical development.

Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.

Building blocks and blueprints for bacterial autolysins.

Bacteria utilize a wide variety of endogenous cell wall hydrolases, or autolysins, to remodel their cell walls during processes including cell division, biofilm formation, and programmed death. We here systematically investigate the composition of these enzymes in order to gain insights into their associated biological processes, potential ways to disrupt them via chemotherapeutics, and strategies by which they might be leveraged as recombinant antibacterial biotherapies. To do so, we developed LEDGOs (lytic enzyme domains grouped by organism), a pipeline to create and analyze databases of autolytic enzyme sequences, constituent domain annotations, and architectural patterns of multi-domain enzymes that integrate peptidoglycan binding and degrading functions. We applied LEDGOs to eight pathogenic bacteria, gram negatives Acinetobacter baumannii, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aeruginosa; and gram positives Clostridioides difficile, Enterococcus faecium, Staphylococcus aureus, and Streptococcus pneumoniae. Our analysis of the autolytic enzyme repertoires of these pathogens reveals commonalities and differences in their key domain building blocks and architectures, including correlations and preferred orders among domains in multi-domain enzymes, repetitions of homologous binding domains with potentially complementarity recognition modalities, and sequence similarity patterns indicative of potential divergence of functional specificity among related domains. We have further identified a variety of unannotated sequence regions within the lytic enzymes that may themselves contain new domains with important functions.

Vaccination with LytA, LytC, or Pce of Streptococcus pneumoniae Protects against Sepsis by Inducing IgGs That Activate the Complement System.

The emergence of non-vaccine serotypes of Streptococcus pneumoniae after the use of vaccines based in capsular polysaccharides demonstrates the need of a broader protection vaccine based in protein antigens and widely conserved. In this study, we characterized three important virulence factors of S. pneumoniae namely LytA, LytC, and Pce as vaccine candidates. These proteins are choline-binding proteins that belong to the cell wall hydrolases’ family. Immunization of mice with LytA, LytC, or Pce induced high titers of immunoglobulins G (IgGs) of different subclasses, with IgG1, IgG2a, and IgG2b as the predominant immunoglobulins raised. These antibodies activated the classical pathway of the complement system by increasing the recognition of C1q on the surface of pneumococcal strains of different serotypes. Consequently, the key complement component C3 recognized more efficiently these strains in the presence of specific antibodies elicited by these proteins, activating, therefore, the phagocytosis. Finally, a mouse sepsis model of infection was established, confirming that vaccination with these proteins controlled bacterial replication in the bloodstream, increasing the survival rate. Overall, these results demonstrate that LytA, LytC, and Pce can be protein antigens to be contained in a future universal vaccine against S. pneumoniae.

Changes of DNA Methylation Patterns Reveal Epigenetic Modification of Dormancy Release-Related Genes Is Induced by Chilling in Tree Peony.

DNA methylation is an important epigenetic regulator of gene expression. Application of 5-azacytidine (a methylation inhibitor) significantly promoted bud sprouting rate and the elongation of branches and leaves in "Luhehong" and "Fengdanbai." In total, 11,166 and 11,443 fragments were obtained by methylation-sensitive amplified polymorphism (MSAP) analysis during chilling-induced dormancy release in the two varieties, respectively. Total methylation levels were high in dormant buds, mainly for hemimethylation, which were slowly increased by short-term chilling (7 days) and decreased by long-term chilling. Compared with 0 day, the ratio of the methylation downregulated group increased during dormancy release, whereas that of the upregulated group declined gradually. These variations were consistent with the dynamic expressions of DNA methyltransferase/demethylase genes and their enzyme activity changes. In total, 13 polymorphic MSAP fragments were similar to known proteins (E-value <1e-5), and their methylation statuses were consistent with their expression patterns. The expression change of PsCWH, encoding cell wall hydrolase, might be due to DNA methylation ratios of CpG sites identified by bisulfite sequencing. These results indicated that chilling accumulation promoted bud dormancy release and sprouting through DNA methylation modification of specific genes. This study would provide new insights into the molecular mechanism underlying dormancy release in tree peony.

Lytic characterization and application of listerial endolysins PlyP40 and PlyPSA in queso fresco

Queso fresco (QF) is a fresh Hispanic-style cheese that is commonly associated with the human foodborne pathogen Listeria monocytogenes and outbreaks of listeriosis in the United States. Endolysins, cell wall hydrolases derived from bacteriophages, are promising candidates for controlling bacterial pathogens in food systems. In this study, we characterized the lytic capabilities of 2 endolysins, PlyP40 and PlyPSA, under varying conditions (pH, temperature, salt concentration) and compared their activities with those of the previously described endolysin PlyP100. We showed that PlyP40 was effective, showing at least a 33% reduction in cellular debris, against a broader range of Listeria than PlyPSA, which showed little lytic activity toward Listeria strains not from serovar 4. Both endolysins were also capable of maintaining lytic activity to varying extents at refrigeration temperature. The effect of salt concentration and pH differed between PlyP40 and PlyPSA. Furthermore, we added the endolysins to QF and monitored their ability to control L. monocytogenes contamination over 28 d of cold storage. Both PlyP40 and PlyPSA were capable of lowering QF inoculum cell counts compared with the control; however, both were less effective than the previously characterized PlyP100. Further characterization of endolysins will continue to open opportunities to optimization and implementation in a variety of food matrices for controlling pathogen contamination.

Horizontally acquired antibacterial genes associated with adaptive radiation of ladybird beetles

BackgroundHorizontal gene transfer (HGT) has been documented in many herbivorous insects, conferring the ability to digest plant material and promoting their remarkable ecological diversification. Previous reports suggest HGT of antibacterial enzymes may have contributed to the insect immune response and limit bacterial growth. Carnivorous insects also display many evolutionary successful lineages, but in contrast to the plant feeders, the potential role of HGTs has been less well-studied.ResultsUsing genomic and transcriptomic data from 38 species of ladybird beetles, we identified a set of bacterial cell wall hydrolase (cwh) genes acquired by this group of beetles. Infection with Bacillus subtilis led to upregulated expression of these ladybird cwh genes, and their recombinantly produced proteins limited bacterial proliferation. Moreover, RNAi-mediated cwh knockdown led to downregulation of other antibacterial genes, indicating a role in antibacterial immune defense. cwh genes are rare in eukaryotes, but have been maintained in all tested Coccinellinae species, suggesting that this putative immune-related HGT event played a role in the evolution of this speciose subfamily of predominant predatory ladybirds.ConclusionOur work demonstrates that, in a manner analogous to HGT-facilitated plant feeding, enhanced immunity through HGT might have played a key role in the prey adaptation and niche expansion that promoted the diversification of carnivorous beetle lineages. We believe that this represents the first example of immune-related HGT in carnivorous insects with an association with a subsequent successful species radiation.

English

Bovine mastitis is an inflammatory response mainly caused by Staphylococcus aureus. Lysin is a cell wall hydrolase encoded and synthesized by a bacteriophage, which can kill specific Gram-positive bacteria. In this study, phage lysin “LysGH15” is used to treat the mice mastitis caused by S. aureus. The purified lysGH15 showed strong bactericidal activity in vitro. When treated with 25μg/mL of the LysGH15, the bacterial counts of S. aureus dropped approximately 5 log units within 10 min. In the in vivo experiments, the administration of LysGH15 significantly (P&lt;0.05) reduced the colonies of S. aureus and alleviated damage to the breast tissue. Also, the levels of IL-6 and TNF-α in breast tissue were significantly decreased. It indicates that the LysGH15 can effectively treat the murine mastitis caused by S. aureus. This study demonstrated the potential of LysGH15 as an alternative to antibiotics for treating bovine mastitis caused by S. aureus

Comparative transcriptome analysis reveals the regulatory effects of acetylcholine on salt tolerance of Nicotiana benthamiana

Salinity is a major cause of crop losses worldwide. Acetylcholine (ACh) can ameliorate the adverse effects of abiotic stresses on plant growth, including salinity stress; however, the underlying molecular mechanisms of this process are unclear. Here, seedlings of Nicotiana benthamiana grown under normal conditions or exposed to 150 mmol L−1 NaCl salinity stress were then treated with a root application of 10 μM ACh. Exogenous ACh application resulted in the downregulation of the activity of the antioxidant enzymes, ascorbate peroxidase, and catalase. ACh-treated plants had lower levels of reactive oxygen species, including the superoxide anion radical and hydrogen peroxide. Transcriptome analysis indicated that ACh treatment under salt stress promoted the differential expression of 658 genes in leaves of N. benthamiana (527 were upregulated and 131 were downregulated). Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that exogenous ACh application was associated with a substantial increase in the transcripts of genes related to cell wall peroxidases, xyloglucan endotransglucosylases or hydrolases, and expansins, indicating that ACh activates cell wall biosynthesis in salt-stressed plants. ACh also enhanced the expression of genes associated with the auxin, gibberellin, brassinosteroid, and salicylic acid signalling pathways, indicating that ACh induces the activation of these pathways under salt stress. Collectively, these findings indicate that ACh-induced salt tolerance in N. benthamiana seedlings is mediated by the inhibition of antioxidant enzymes, activation of cell wall biosynthesis, and hormone signalling pathways. Stress-induced genes involved in osmotic regulation and oxidation resistance were induced by ACh under salt stress. The genes whose transcript levels were elevated by ACh treatment in salt-stressed N. benthamiana could be used as molecular markers of the physiological status of plants under salt stress.

Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization.

Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.