Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli.

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Amount of peptidoglycan in cell walls of gram-negative bacteria.

The amount of diaminopimelic acid (Dap) in the cell wall of Escherichia coli was measured in two ways. A radiochemical method first described by us in 1985 (F. B. Wientjes, E. Pas, P. E. M. Taschner, and C. L. Woldringh, J. Bacteriol. 164:331-337, 1985) is based on the steady-state incorporation of [3H]Dap during several generations. Knowing the cell concentration and the specific activity of the [3H]Dap, one can calculate the number of Dap molecules per sacculus. The second method measures the Dap content chemically in sacculi isolated from a known number of cells. With both methods, a value of 3.5 x 10(6) Dap molecules per sacculus was obtained. Combined with electron microscopic measurements of the surface area of the cells, the data indicate an average surface area per disaccharide unit of ca. 2.5 nm2. This finding suggests that the peptidoglycan is basically a monolayered structure.     

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.     

Model for the Structure of the Shape-Maintaining Layer of the Escherichia coli Cell Envelope

The surface area per repeating murein unit (i.e. per molecule of diaminopimelate) has been determined for the cell envelopes of the Escherichia coli strains K-12 and W. This area was constantly found to be 1.3 nm(2). Using this value and other previously determined properties of E. coli murein, a three-dimensional model of murein is proposed. The model specifies a monomolecular layer in which disaccharide units are each 1.03 nm long, and the polysaccharide chains, all parallel, are 1.25 nm apart. The cross-linking peptide side-chains have the same atomic coordinates and are arranged above or below the polysaccharide chains.     

Morphogenesis of Escherichia coli.

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

Enzymology of elongation and constriction of the murein sacculus of Escherichia coli

Multiple deletions in murein hydrolases revealed that predominantly amidases are responsible for cleavage of the septum during cell division. Endopeptidases and lytic transglycosylases seem also be involved. In the absence of these enzymes E. coli grows normally but forms chains of adhering cells. Surprisingly, mutants lacking up to eight different murein hydrolases still grow with almost unaffected growth rate. Therefore it is speculated that general enlargement of the murein sacculus may differ from cell division by using transferases rather than the two sets of hydrolytic and synthetic enzymes as seems to be the case for the constriction process. A model is presented that describes growth of the murein of both Gram-positive and -negative bacteria by the activity of murein transferases. It is speculated that enzymes exist that catalyze a transpeptidation of the pre-existing murein onto murein precursors or nascent murein by using the chemical energy present in peptide cross-bridges. Such enzymes would at the same time cleave bonds in the murein net and insert new material into the growing sacculus.     

The composition of the murein of Escherichia coli

Escherichia coli murein, the polymer from which the shape-maintaining structure of the cell envelope is made, shows unexpected complexity. The separation of murein building blocks with high performance liquid chromatography reveals about 80 different types of muropeptides. Their behavior in high performance liquid chromatography and their chemical structure are described. The complexity of E. coli murein is due to the free combination of seven different types of side chains (L-Ala-D-Glu-R with R = -OH, -m-A2pm, -m-A2pm-D-Ala, -m-A2 pm-Gly, -m-A2pm-D-Ala-D-Ala, -m-A2pm-D-Ala-Gly, -m-A2pm-Lys-Arg) with two types of cross-bridges (D-Ala-m-A2pm, -m-A2pm-m-A2pm). The novel type of cross-bridge, A2pm-A2pm, contains an L,D-peptide bond, as shown by Edman degradation and chemical analysis of the reaction products. The A2pm-A2pm cross-bridge is assumed to play a role in the adaptation of the cross-linkage of murein to different growth conditions of the cell. The structural data of E. coli murein agree best with a model of a thin, however multilayered, murein sacculus.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

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.     

Growth pattern of the murein sacculus of Escherichia coli

The mechanism by which the murein sacculus of Escherichia coli is being enlarged during growth was investigated by pulse and pulse-chase labeling with [3H]diaminopimelic acid. Changes in the composition of the sacculus during aging were analyzed in detail by high performance liquid chromatography separation of the muropeptide subunits released after complete muramidase digestion. After pulses as short as 10 s, a group of novel phosphorylated muropeptides was detected. The kinetics of their appearance is consistent with these structures being derived from the undecaprenylphosphate-linked growing points of murein. A complex maturation process of murein took place including a rapid decay of pentapeptide side chains and a 10-fold increase in tripeptidyl moieties. In addition, the total degree of cross-linkage increased from 16 to 25%, partly due to a 3-fold increase in the formation of LD-A2pm-A2pm cross-links. In pulse-chase experiments the cross-linkage started to decrease after a maximum at about 35 min of chase. The kinetics in the distribution of the radioactivity among acceptor and donor part in the major cross-bridges Tetra-Tetra and Tetra-Tri differed from each other substantially, indicating that the latter structure is completely cleaved within three generations, whereas only 40% of Tetra-Tetra is cleaved during the same time. Furthermore, the attachment of the lipoprotein to murein was delayed by about one generation. It is proposed that these findings reflect an inside-to-outside growth mechanism of the murein sacculus of E. coli.     

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.     

Turnover and recycling of the murein sacculus in oligopeptide permease-negative strains of Escherichia coli: indirect evidence for an alternative permease system and for a monolayered sacculus.

Turnover of murein in oligopeptide permease-negative Escherichia coli cells appeared to be minimal or nonexistent. In one strain in which it was possible to measure turnover during the first generation of chase, it was found that the rate of turnover was constant throughout a chase of three generations. This result suggests that an "inside-to-outside" mode of growth of the sacculus does not occur in E. coli. Turnover, though minimal, was significantly higher from cells labeled uniformly than from cells labeled only in the lateral wall, suggesting that a significant portion of the observed turnover is related to cell separation. Actually, turnover only appeared to be minimal in opp mutant strains. Tripeptides were being released by turnover at a rate of about 50% per generation and then were efficiently recycled. This suggests that in addition to opp, a low-affinity uptake system for tripeptide derived from the sacculus may exist.     

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.     

Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain

Exponentially growing cells of Bacillus subtilis and Escherichia coli were Gram stained with potassium trichloro(eta 2-ethylene)platinum(II) (TPt) in place of the usual KI-I2 mordant. This electron-dense probe allowed the staining mechanism to be followed and compared with cellular perturbations throughout the staining process. A crystal violet (CV)-TPt chemical complex was formed within the cell substance and at the cell surface of B. subtilis when the dye and Pt mordant were added. The ethanol decolorization step dissolved the precipitate from the cell surface, but the internal complex was retained by the cell wall and remained within the cell. This was not the case for E. coli; the ethanol decolorization step removed both surface-bound and cellular CV-TPt. During its removal, the outer membrane was sloughed off the cells until only the murein sacculus and plasma membrane remained. We suspect that the plasma membrane was also perturbed, but that it was retained within the cell by the murein sacculus. Occasionally, small holes within the murein and plasma membrane could be distinguished through which leaked CV-TPt and some cellular debris. Biochemical identification of distinct envelope markers confirmed the accuracy of these images.     

Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus.

High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with [3H]diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process. Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation. Elongation and septation were shown to be overlapping processes.     

Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli

To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.     

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.