Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa.

Lytic transglycosylases cleave the beta,1-->4 glycosidic linkages between the N-acetylmuramoyl (MurNAc) and N-acetylglucosaminyl (GlcNAc) residues of peptidoglycan with the concomitant formation of 1,6-anhydro-N-acetylmuramyl reaction products. The genes encoding two hypothetical lytic transglycosylases were identified in the genome of Pseudomonas aeruginosa PAO1 by a BLAST search using membrane-bound lytic transglycosylase B (MltB) from Escherichia coli as the query. The two genes were amplified by PCR and cloned as fusion proteins with C-terminal hexa-His sequences. Expression studies of the two genes in E. coli in the presence of [(3)H]palmitate resulted in the labeling of only one of the two enzymes. This enzyme, named MltB, was overexpressed to form insoluble inclusion bodies. Its gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which includes Cys17, the putative site of lipidation. This MltB derivative (named sMltB) was shown to not label with [(3)H]palmitate, and it was overexpressed in soluble form. The second, nonlabeled enzyme was overexpressed in soluble form and hence was named soluble lytic transglycosylase B (SltB). Both sMltB and SltB were purified to apparent homogeneity by a combination of affinity (Ni(2+)-NTA), cation-exchange (Mono S), and gel permeation (Superdex 75) chromatographies. The reaction products released by the two enzymes from purified, insoluble peptidoglycan were characterized by a novel high-performance anion-exchange chromatography (HPAEC) assay. Both enzymes produced the same three major soluble products which were identified as anhydromuropeptides based on ESI-MS analysis (cross-linked anhydrodisaccharide-tetrasaccharide, m/z obs 1824.9; anhydrodisaccharide-pentapeptide, m/z obs 922.2; and anhydrodisaccharide-tripeptide, m/z obs 851.3. The Michaelis-Menten kinetic parameters were also determined for the two enzymes using the same insoluble peptidoglycan substrate by aminosugar compositional analysis of soluble reaction products. At pH 5.8 and in the presence of 0.1% Triton, SltB was found to be more catalytically efficient, as reflected by its k(cat)/K(M) value, than sMltB.     

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."     

Structure of the bacteriophage phi KZ lytic transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.     

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.     

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.     

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.     

Murein-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase.

In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-). The mlt gene, which supposedly encodes Mlt, was cloned, and the complete nucleotide sequence was determined. The open reading frame, identified on a 1.7-kb SalI-PstI fragment, codes for a protein of 323 amino acids (M(r) = 37,410). Two transmembrane helices and one membrane-associated helix were predicted in the N-terminal half of the protein. Lysine and arginine residues represent up to 15% of the amino acids, resulting in a calculated isoelectric point of 10.0. The deduced primary structure did not show significant sequence similarity to Slt70 from E. coli. High-level expression of the presumed mlt gene was not paralleled by an increase in murein hydrolase activity. To clarify the identity of the second transglycosylase, we purified an enzyme with the specificity of a transglycosylase from an E. coli slt deletion strain. The completely soluble transglycosylase, with a molecular mass of approximately 35 kDa, was designated Slt35. Its determined 26 N-terminal amino acids showed similarity to a segment in the middle of the Slt70 primary structure. Polyclonal anti-Mlt antibodies, which had been used for the isolation of the mlt gene, were found to cross-react with Mlt as well as with Slt35, suggesting that the previously described Mlt preparation was contaminated with Slt35. We conclude that the second transglycosylase of E. coli is not a membrane-bound protein but rather is a soluble protein.     

TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.     

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.     

Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (alpha, beta, and gamma) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.     

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.     

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.     

Overproduction of penicillin-binding protein 2 and its inactive variants causes morphological changes and lysis in Escherichia coli

Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.     

Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities

The mycobacterial cell wall presents significant challenges to mycobacteriophages--viruses that infect mycobacterial hosts--because of its unusual structure containing a mycolic acid-rich mycobacterial outer membrane attached to an arabinogalactan layer that is in turn linked to the peptidoglycan. Although little is known about how mycobacteriophages circumvent these barriers during the process of infection, destroying it for lysis at the end of their lytic cycles requires an unusual set of functions. These include Lysin B proteins that cleave the linkage of mycolic acids to the arabinogalactan layer, chaperones required for endolysin delivery to peptidoglycan, holins that regulate lysis timing, and the endolysins (Lysin As) that hydrolyze peptidoglycan. Because mycobacterial peptidoglycan contains atypical features including 3→3 interpeptide linkages, it is not surprising that the mycobacteriophage endolysins also have non-canonical features. We present here a bioinformatic dissection of these lysins and show that they are highly diverse and extensively modular, with an impressive number of domain organizations. Most contain three domains with a novel N-terminal predicted peptidase, a centrally located amidase, muramidase, or transglycosylase, and a C-terminal putative cell wall binding domain.     

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.     

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.     

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.     

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.     

VirB1* promotes T-pilus formation in the vir-Type IV secretion system of Agrobacterium tumefaciens

The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.     

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.