Substrate binding affinity of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B by hydrogen-deuterium exchange MALDI MS

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer, peptidoglycan, between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. With 72% amino acid sequence identity between the enzymes, the theoretical structure of the membrane-bound lytic transglycosylase B (MltB) from Psuedomonas aeruginosa was modeled on the known crystal structure of Escherichia coli Slt35, the soluble derivative of its MltB. Of the twelve residues in Slt35 known to make contacts with peptidoglycan derivatives in Slt35, nine exist in the same position in the P. aeruginosa homologue, with two others only slightly displaced. To probe the binding properties of an engineered soluble form of the P. aeruginosa MltB, a SUPREX method involving hydrogen/deuterium exchange coupled with MALDI mass spectrometry detection was developed. Dissociation constants were calculated for a series of peptidoglycan components and compared to those obtained by difference UV absorption spectroscopy. These data indicated that GlcNAc alone does not bind to MltB with any measurable affinity but it does contribute to the binding of GlcNAc-MurNAc-dipeptide. With the MurNAc series of ligands, significant binding contributions are made through both the N-acetyl and C-3 lactyl moieties of the aminosugar with additional contributions to binding provided by associated peptides.     

Crystallographic studies of the interactions of Escherichia coli lytic transglycosylase Slt35 with peptidoglycan

Lytic transglycosylases catalyze the cleavage of the beta-1, 4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydro bond in the MurNAc residue. To understand the reaction mechanism of Escherichia coli lytic transglycosylase Slt35, three crystal structures have been determined of Slt35 in complex with two different peptidoglycan fragments and with the lytic transglycosylase inhibitor bulgecin A. The complexes define four sugar-binding subsites (-2, -1, +1, and +2) and two peptide-binding sites in a large cleft close to Glu162. The Glu162 side chain is between the -1 and +1 sugar-binding sites, in agreement with a function as catalytic acid/base. The complexes suggest additional contributions to catalysis from Ser216 and Asn339, residues which are conserved among the MltB/Slt35 lytic transglycosylases.     

Role of Ser216 in the mechanism of action of membrane-bound lytic transglycosylase B: further evidence for substrate-assisted catalysis

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. Based on sequence alignments, Ser216 in Pseudomonas aeruginosa membrane-bound lytic transglycosylase B (MltB) was targeted for replacement with alanine to delineate its role in the enzyme's mechanism of action. The specific activity of the Ser216-->Ala MltB derivative was less than 12% of that for the wild-type enzyme, while its substrate binding affinity remained virtually unaltered. These data are in agreement with a role of Ser216 in orienting the N-acetyl group on MurNAc at the -1 subsite of MltB for its participation in a substrate-assisted mechanism of action.     

Assay for lytic transglycosylases: a family of peptidoglycan lyases

An assay has been developed to monitor the activity of the lytic transglycosylases which does not involve the use of radiolabel. Samples of lytic transglycosylase were incubated with isolated and purified insoluble peptidoglycan as substrate for varying lengths of time. Residual insoluble material was removed by ultracentrifugation in a microfuge and the solubilized components were treated with sodium borohydride prior to acid hydrolysis. The optimal conditions for this acid hydrolysis were established to be incubation at 96 degrees C for 1 h in 6 M HCl, in vacuo. The hydrolyzed samples were subjected to amino acid/sugar analysis by cation-exchange chromatography on a Beckman System Gold amino acid analyzer. To effect a clear resolution of muramic acid from serine and glutamic acid, the equilibration buffer was modified to be composed of 33 mM sodium citrate, pH 3.12. The product of the lyase reaction of the lytic transglycosylases are 1,6-anhydromuramyl residues, which are not reduced by the sodium borohydride treatment. On the other hand, the muramyl residues arising at the reducing ends of peptidoglycan after treatment with muramidases (hydrolyases) are reduced to muramitol residues, which elute from the amino acid analyzer prior to aspartic acid. This assay thus distinguishes the activity of the two enzymes and was applied to determine the initial activities of increasing concentrations of a soluble derivative of lytic transglycosylase B from the opportunistic pathogen Pseudomonas aeruginosa.     

The C-terminal domain of Escherichia coli YfhD functions as a lytic transglycosylase

The hypothetical Escherichia coli protein YfhD has been identified as the archetype for the family 1B lytic transglycosylases despite a complete lack of experimental characterization. The yfhD gene was amplified from the genomic DNA of E. coli W3110 and cloned to encode a fusion protein with a C-terminal His(6) sequence. The enzyme was found to be localized to the outer membrane of E. coli, as would be expected for a lytic transglycosylase. Its gene was engineered for the production of a truncated soluble enzyme derivative lacking an N-terminal signal sequence and membrane anchor. The soluble YfhD derivative was purified to apparent homogeneity, and three separate in vitro assays involving high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used to demonstrate the YfhD-catalyzed release of 1,6-anhydromuro-peptides from insoluble peptidoglycan. In addition, an in vivo bioassay developed using the bacteriophage lambda lysis system confirmed that the enzyme functions as an autolysin. Based on these data, the enzyme was renamed membrane-bound lytic transglycosylase F. The modular structure of MltF was investigated through genetic engineering for the separate production of identified N-terminal and C-terminal domains. The ability to bind peptidoglycan and lytic activity were only associated with the isolated C-terminal domain. The enzymatic properties of this lytic transglycosylase domain were found to be very similar to those of the wild-type enzyme. The one notable exception was that the N-terminal domain appears to modulate the lytic behavior of the C-terminal domain to permit continued lysis of insoluble peptidoglycan, a unique feature of MltF compared with other characterized lytic transglycosylases.     

Neisseria gonorrhoeae uses two lytic transglycosylases to produce cytotoxic peptidoglycan monomers

Peptidoglycan fragments released by Neisseria gonorrhoeae contribute to the inflammation and ciliated cell death associated with gonorrhea and pelvic inflammatory disease. However, little is known about the production and release of these fragments during bacterial growth. Previous studies demonstrated that one lytic transglycosylase, LtgA, was responsible for the production of approximately half of the released peptidoglycan monomers. Systematic mutational analysis of other putative lytic transglycosylase genes identified lytic transglycosylase D (LtgD) as responsible for release of peptidoglycan monomers from gonococci. An ltgA ltgD double mutant was found not to release peptidoglycan monomers and instead released large, soluble peptidoglycan fragments. In pulse-chase experiments, recycled peptidoglycan was not found in cytoplasmic extracts from the ltgA ltgD mutant as it was for the wild-type strain, indicating that generation of anhydro peptidoglycan monomers by lytic transglycosylases facilitates peptidoglycan recycling. The ltgA ltgD double mutant showed no growth abnormalities or cell separation defects, suggesting that these enzymes are involved in pathogenesis but not necessary for normal growth.     

Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis of both sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 from Escherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in the soluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residues were targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined. The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, were shown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays and SUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determined for the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in K(M) values, significant decreases in k(cat) values suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilization of both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 are vital for proper orientation of the substrate in the active site, and furthermore this supports the proposed role of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that would appear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity or activity.     

Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."     

Characterization of three different lytic transglycosylases in Escherichia coli

Abstract Two lytic transglycosylases, releasing 1,6-anhydromuropeptides from murein sacculi are present in a mutant deleted for the soluble lytic transglycosylase 70 (Slt70). Thus, there are three different lytic transglycosylases in Escherichia coli . One of the remaining enzymes is soluble and one is a membrane protein that can be solubilized by 2% Triton X-100 in 0.5 M NaCl. Both enzymes are exo-muramidases. Only the membrane enzyme, but not the soluble ones, hydrolyses isolated murein glycan strands (poly-GlcNAc-MurNAc). While the soluble enzymes are inhibited by the muropeptide TetraTriLysArg(dianhydro), the membrane enzyme is not. The antibiotic bulgecin that inhibits Slt70 does not inhibit the lytic transglycosylases present in the slt70 deletion mutant.     

AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system

Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.     

Peptidoglycan maturation enzymes affect flagellar functionality in bacteria

The flagellar machinery is a highly complex organelle composed of a free rotating flagellum and a fixed stator that converts energy into movement. The assembly of the flagella and the stator requires interactions with the peptidoglycan layer through which the organelle has to pass for externalization. Lytic transglycosylases are peptidoglycan degrading enzymes that cleave the sugar backbone of peptidoglycan layer. We show that an endogenous lytic transglycosylase is required for full motility of Helicobacter pylori and colonization of the gastric mucosa. Deficiency of motility resulted from a paralysed phenotype implying an altered ability to generate flagellar rotation. Similarly, another Gram‐negative pathogen Salmonella typhimurium and the Gram‐positive pathogen Listeria monocytogenes required the activity of lytic transglycosylases, Slt or MltC, and a glucosaminidase (Auto), respectively, for full motility. Furthermore, we show that in absence of the appropriate lytic transglycosylase, the flagellar motor protein MotB from H. pylori does not localize properly to the bacterial pole. We present a new model involving the maturation of the surrounding peptidoglycan for the proper anchoring and functionality of the flagellar motor.     

Mutation of a single lytic transglycosylase causes aberrant septation and inhibits cell separation of Neisseria gonorrhoeae

The function of lytic peptidoglycan transglycosylases is poorly understood. Single lytic transglycosylase mutants of Escherichia coli have no growth phenotype. By contrast, mutation of Neisseria gonorrhoeae ltgC inhibited cell separation without affecting peptidoglycan monomer production. Thus, LtgC has a dedicated function in gonococcal cell division.     

Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand.

BACKGROUND: Lytic transglycosylases are bacterial muramidases that catalyse the cleavage of the beta- 1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydrobond in the MurNAc residue. These muramidases play an important role in the metabolism of the bacterial cell wall and might therefore be potential targets for the rational design of antibacterial drugs. One of the lytic transglycosylases is Slt35, a naturally occurring soluble fragment of the outer membrane bound lytic transglycosylase B (MltB) from Escherichia coli. RESULTS: The crystal structure of Slt35 has been determined at 1.7 A resolution. The structure reveals an ellipsoid molecule with three domains called the alpha, beta and core domains. The core domain is sandwiched between the alpha and beta domains. Its fold resembles that of lysozyme, but it contains a single metal ion binding site in a helix-loop-helix module that is surprisingly similar to the eukaryotic EF-hand calcium-binding fold. Interestingly, the Slt35 EF-hand loop consists of 15 residues instead of the usual 12 residues. The only other prokaryotic proteins with an EF-hand motif identified so far are the D-galactose-binding proteins. Residues from the alpha and core domains form a deep groove where the substrate fragment GlcNAc can be bound. CONCLUSIONS: The three-domain structure of Slt35 is completely different from the Slt70 structure, the only other lytic transglycosylase of known structure. Nevertheless, the core domain of Slt35 closely resembles the fold of the catalytic domain of Slt70, despite the absence of any obvious sequence similarity. Residue Glu162 of Slt35 is in an equivalent position to Glu478, the catalytic acid/base of Slt70. GlcNAc binds close to Glu162 in the deep groove. Moreover, mutation of Glu162 into a glutamine residue yielded a completely inactive enzyme. These observations indicate the location of the active site and strongly support a catalytic role for Glu162.     

Interference with murein turnover has no effect on growth but reduces beta-lactamase induction in Escherichia coli

Physiological studies of a mutant of Escherichia coli lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC beta-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of beta-lactamase, present on a plasmid carrying the ampC and ampR genes from Enterobacter cloacae. Overproduction of MltB stimulated beta-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed ampC expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like beta-lactamases.     

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.     

Inhibition of membrane-bound lytic transglycosylase B by NAG-thiazoline

The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue. The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis. Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase. The beta-hexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance. Using surface plasmon resonance and difference absorbance spectroscopy, Kd values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.     

Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.     

LytM factors affect the recruitment of autolysins to the cell division site in Caulobacter crescentus

Most bacteria possess a peptidoglycan cell wall that determines their morphology and provides mechanical robustness during osmotic challenges. The biosynthesis of this structure is achieved by a large set of synthetic and lytic enzymes with varying substrate specificities. Although the biochemical functions of these proteins are conserved and well‐investigated, the precise roles of individual factors and the regulatory mechanisms coordinating their activities in time and space remain incompletely understood. Here, we comprehensively analyze the autolytic machinery of the alphaproteobacterial model organism Caulobacter crescentus, with a specific focus on LytM‐like endopeptidases, soluble lytic transglycosylases and amidases. Our data reveal a high degree of redundancy within each protein family but also specialized functions for individual family members under stress conditions. In addition, we identify two lytic transglycosylases and an amidase as new divisome components that are recruited to midcell at distinct stages of the cell cycle. The midcell localization of these proteins is affected by two LytM factors with degenerate catalytic domains, DipM and LdpF, which may serve as regulatory hubs coordinating the activities of multiple autolytic enzymes during cell constriction and fission respectively. These findings set the stage for in‐depth studies of the molecular mechanisms that control peptidoglycan remodeling in C. crescentus.     

Crystal Structure of the Catalytic Domain of the Bacillus cereus SleB Protein, Important in Cortex Peptidoglycan Degradation during Spore Germination

The SleB protein is one of two redundant cortex-lytic enzymes (CLEs) that initiate the degradation of cortex peptidoglycan (PG), a process essential for germination of spores of Bacillus species, including Bacillus anthracis. SleB has been characterized as a soluble lytic transglycosylase that specifically recognizes spore cortex PG and catalyzes the cleavage of glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine residues with concomitant formation of a 1,6-anhydro bond in the NAM residue. We found that like the full-length Bacillus cereus SleB, the catalytic C-terminal domain (SleB^C) exhibited high degradative activity on cortex PG in vitro, although SleB''s N-terminal domain, thought to bind PG, was inactive. The 1.85-Å crystal structure of SleB^C reveals an ellipsoid molecule with two distinct domains dominated by either α helices or β strands. The overall fold of SleB closely resembles that of the catalytic domain of the family 1 lytic transglycosylases but with a completely different topological arrangement. Structural analysis shows that an invariant Glu157 of SleB is in a position equivalent to that of the catalytic glutamate in other lytic transglycosylases. Indeed, SleB bearing a Glu157-to-Gln mutation lost its cortex degradative activity completely. In addition, the other redundant CLE (called CwlJ) in Bacillus species likely has a three-dimensional structure similar to that of SleB, including the invariant putative catalytic Glu residue. SleB and CwlJ may offer novel targets for the development of anti-spore agents.     

In vitro studies of peptidoglycan binding and hydrolysis by the Bacillus anthracis germination-specific lytic enzyme SleB

The Bacillus anthracis endospore loses resistance properties during germination when its cortex peptidoglycan is degraded by germination-specific lytic enzymes (GSLEs). Although this event normally employs several GSLEs for complete cortex removal, the SleB protein alone can facilitate enough cortex hydrolysis to produce vulnerable spores. As a means to better understand its enzymatic function, SleB was overexpressed, purified, and tested in vitro for depolymerization of cortex by measurement of optical density loss and the solubilization of substrate. Its ability to bind peptidoglycan was also investigated. SleB functions independently as a lytic transglycosylase on both intact and fragmented cortex. Most of the muropeptide products that SleB generates are large and are potential substrates for other GSLEs present in the spore. Study of a truncated protein revealed that SleB has two domains. The N-terminal domain is required for stable peptidoglycan binding, while the C-terminal domain is the region of peptidoglycan hydrolytic activity. The C-terminal domain also exhibits dependence on cortex containing muramic-δ-lactam in order to carry out hydrolysis. As the conditions and limitations for SleB activity are further elucidated, they will enable the development of treatments that stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.