Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli.

A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70). The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography. The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38. Optimal activity was found in buffers with a pH between 4.0 and 4.5. The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C. Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment. The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate. The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank. With Mlt38, the number of proteins in E. coli showing lytic transglycosylase activity rises to three.     

A murein hydrolase is the specific target of bulgecin in Escherichia coli.

A deletion in the structural gene for the soluble lytic transglycosylase, the predominant murein hydrolase in the soluble fraction of Escherichia coli, has been constructed. The mutant grows normally but exhibits increased sensitivity toward mecillinam, a beta-lactam specific for penicillin-binding protein 2. In the presence of furazlocillin or other beta-lactams with a specificity for penicillin-binding protein 3 which normally cause filamentation, bulges were formed prior to rapid bacteriolysis. Similar morphological alterations are known to develop in wild type E. coli cells when furazlocillin is combined with bulgecin, an antibiotic of unusual glucosaminyl structure. It turned out that bulgecin specifically inhibits the Sl-transglycosylase in a noncompetitive manner. Since bulgecin shows some structural analogy to the murein subunits we postulate that the soluble lytic transglycosylase, in addition to its active site, has a recognition site for specific murein structures. The possibility of an allosteric modulation of the activity of the enzyme by changes in the structure of the murein sacculus is discussed.     

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.     

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.     

Murein (peptidoglycan) binding property of the essential cell division protein FtsN from Escherichia coli

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.     

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.     

Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli

Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10(-6) M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.     

Membrane-Bound Lytic Endotransglycosylase in Escherichia coli

The gene for a novel endotype membrane-bound lytic transglycosylase, emtA, was mapped at 26.7 min of the E. coli chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22 kDa. Overexpression of the emtA gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.     

Murein segregation in Escherichia coli.

Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.     

Involvement of N-acetylmuramyl-l-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli

N-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain-forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein cross-bridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross-links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.     

Failure to trigger the autolytic enzymes in minicells of Escherichia coli

Minicells from Escherichia coli P678-54 are refractory towards procedures known to induce bacteriolysis of DNA-containing E. coli cells. Although still engaged in murein synthesis, minicells could not be lysed by penicillin G. Likewise, endogenous overproduction of the cloned soluble lytic transglycosylase, the predominant murein hydrolytic activity in E. coli, failed to lyse minicells. Furthermore, induction of the phage MS2 lysis protein, a hydrophobic protein assumed to trigger the autolytic system of the host, did not result in bacteriolysis. It is concluded that the murein hydrolases present in minicells are under a tight cellular control.     

Evidence for multisite growth of Escherichia coli murein involving concomitant endopeptidase and transpeptidase activities.

During diaminopimelic acid starvation of Escherichia coli W7, a large fraction of the preexisting murein cross-links are opened by murein endopeptidase and the resulting uncross-linked material is degraded. This is reflected morphologically in a general loss of rigidity of the murein sacculus long before lysis occurs. In growing cells, a dynamic situation is demonstrable. When cells whose murein sacculi are uniformly labeled with [14C]diaminopimelic acid were chased with unlabeled DAP, a significant, rapid shift of [14C]diaminopimelic acid from the donor to the acceptor half of dimers was observed. The shift can be explained by the presence of about 100 separate sites where new murein strands were being inserted between old radioactive strands of murein. Thus, the gradual loss of rigidity of the murein sacculus as endopeptidase continues to function during starvation of E. coli W7 suggests an even distribution of the active endopeptidases. This is consistent with the kinetic data which suggest that endopeptidase, along with murein synthetase and transpeptidase, acts at about 100 distinct sites to elongate the murein sacculus.     

Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli

The soluble lytic transglycosylase (Slt) of Escherichia coli is known to be a powerful murein hydrolase in vitro. It is shown here to act as an autolysin in vivo as well. Rapid autolysis of Slt overproducing cells was induced by protein biosynthesis inhibitors, which also block the fomration of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). When amino acid starvation was used to inhibit protein synthesis, autolysis was suppressed in relA+ but not in relA- cells. These findings indicate that the stringent control modulates the enzymatic activity of the soluble lytic transglycosylase in vivo.     

Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection

The predicted catalytic glutamate residue for transglycosylase activity of bacteriophage T7 gp16 is not essential for phage growth, but is shown to be beneficial during infection of Escherichia coli cells grown to high cell density, conditions in which murein is more highly cross-linked. In the absence of the putative transglycosylase, internalization of the phage genome is significantly delayed during infection. The lytic transglycosylase motif of gp16 is essential for phage growth at temperatures below 20 degrees C, indicating that these growth conditions also lead to increased cross-linking of peptidoglycan. Overexpression of sltY, E. coli soluble lytic transglycosylase, partially complements the defect in infection of mutant phage particles, allowing them to infect at higher efficiencies. Conversely, an sltY deletion increases the latent period of wild-type phage.     

Two different species of murein transglycosylase in Escherichia coli.

We demonstrated that Escherichia coli murein transglycosylase exists in two forms. After mechanical disruption of the cells, one form was found in the soluble fraction and the other, in the cell envelope. The two enzymes differed with respect to molecular weight, isoelectric point, solubility in aqueous buffers, and to some extent in their requirements for maximal catalytic activity. The molecular weight of the membrane-bound transglycosylase (35,000) was half that of the soluble enzyme. Whether the high-molecular-weight soluble protein is a precursor of the membrane-bound enzyme species remains to be elucidated.     

Process of cellular division in Escherichia coli growth pattern of E. coli murein

The growth pattern of the murein-sacculus which determines the shape of the Escherichia coli cell was studied by the use of high-resolution autoradiography with the electron microscope. The murein was pulse labelled with ³H-labelled diaminopimelic acid as a specific murein precursor and sacculi were prepared immediately. The radioactivity of the nascent murein appeared on the auto- radiographs at a well-defined growth zone in the central area of the sacculus. This was true regardless of the size of the cells. Pulse chase experimenta show rapid mixing of labelled murein with pre-existing murein and its even distribution over the whole surface of the sacculus.     

Arrangement of glycan chains in the sacculus of Escherichia coli.

A novel of Escherichia coli endopeptidase was used for a selective partial hydrolysis of the peptide bridges which interlink the glycan chains in E. coli sacculi. The loosening of the murein network revealed, in the electron microscope, a preferential orientation of the glycan chains, more or less perpendicular to the length axis of the cell. Control incubations with E. coli transglycosylase or egg-white lysozyme did not leave ordered structures behind.     

GNA33 from Neisseria meningitidis serogroup B encodes a membrane-bound lytic transglycosylase (MltA).

In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48-kDa lipoprotein that is 34.5% identical with membrane-bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e. Meningococcus MltA, as a lipoprotein in E. coli. The lipoprotein nature of recombinant MltA was demonstrated by incorporation of [3H]palmitate. MltA lipoprotein was purified to homogeneity from E. coli membranes by cation-exchange chromatography. Muramidase activity was confirmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands. HPLC analysis demonstrated the formation of 1,6-anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase. Optimal muramidase activity was observed at pH 5.5 and 37 degrees C and enhanced by Mg2+, Mn2+ and Ca2+. The addition of Ni2+ and EDTA had no significant effect on activity, whereas Zn2+ inhibited activity. Triton X-100 stimulated activity 5.1-fold. Affinity chromatography indicated that MltA interacts with penicillin-binding protein 2 from Meningococcus B, and, like MltA from E. coli, may form part of a multienzyme complex.     

Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface.     

Morphogenetic Aspects of Murein Structure and Biosynthesis

The shape of Escherichia coli is fixed by the form of the sacculus. This sacculus is a macromolecule made up from the polymer murein. In an investigation of the possible factors determining the shape of the sacculus, we attempted to resolve between two fundamental alternatives. (i) Is the shape of the sacculus automatically fixed by its chemical composition? or (ii) does a special morphogenetic system exist which determines the shape of the sacculus? An analysis of sacculi from cells grown in poor and rich media and harvested at different stages of growth was made. Significant variations in the composition of murein were found, whereas the general shape of the cells remained unchanged. This finding stands opposed to the assumption of a strict correlation between chemistry and shape of the sacculus. The second alternative was investigated by attempting to change artificially the shape of the sacculus by modifying the form of the hypothetical morphogenetic system. Rod-shaped cells were converted into spherical spheroplasts which were subsequently allowed to reform a new spherical sacculus. In chemical composition this spherical sacculus was found to be indistinguishable from the rod-shaped sacculus. This finding is taken as evidence for the existence of a distinct morphogenetic apparatus in the cell wall whose form is reflected by the shape of the sacculus.