From growth to autolysis: the murein hydrolases inEscherichia coli

Murein hydrolases cleave bonds in the bacterial exoskeleton, the murein (peptidoglycan) sacculus, a covalently closed bag-shaped polymer made of glycan strands that are crosslinked by peptides. During growth and division of a bacterial cell, these enzymes are involved in the controlled metabolism of the murein sacculus. Murein hydrolases are believed to function as pacemaker enzymes for the enlargement of the murein sacculus since opening of bonds in the murein net is needed to allow the insertion of new subunits into the sacculus. Furthermore, they are responsible for splitting the septum during cell division. The murein turnover products that are released during growth are further degraded by these hydrolases to products that can be recycled by the biosynthetic enzymes. As potentially suicidal (autolytic) enzymes, murein hydrolases must be strictly controlled by the cell, Inhibition of murein synthesis, for example by penicillin, triggers an unbalanced action of murein hydrolases causing bacteriolysis. InEscherichia coli, 14 different murein hydrolases have so far been identified, includingN-acetylmuramyl-l-alanine amidases,dd-endopeptidases,dd-carboxypeptidases,ld-carboxypeptidases, andN-acetylglucosaminidases. In addition lysozyme-like enzymes, called “lytic transglycosylases,” produce (1→6)-anhydromuramic acid derivatives by an intramolecular transglycosylation reaction.     

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.     

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.     

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.     

Murein and the Outer Penetration Barrier of Escherichia coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa

A penetration barrier operating outside the periplasmic enzyme penicillinase was studied in an ampicillin-resistant mutant of Escherichia coli K-12. Growth in the presence of lysozyme and sublethal concentrations of ampicillin partially opened the barrier. This could be recorded as an increased penetration of penicillin G, sodium cholate, and rifampin to their respective targets. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and sodium cholate effectively impaired the barrier against penicillin and also caused leakage of penicillinase. Wild-type E. coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa also showed an increased sensitivity to cholate after treatment with penicillins. Electron micrographs showed that lysis by cholate was due to a distortion of the cytoplasmic membrane causing a leakage of protein and RNA from the cells to the medium. Physiological data indicated that the increased sensitivity to cholate induced by growth in the presence of ampicillin or lysozyme was due to effects upon the murein. This was supported by measurement of the incorporation of ³H-diaminopimelic acid. These results indicate that the murein sacculus either is a part of the penetration barrier or is responsible for holding the structure of the outer membrane together.     

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.     

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.     

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.     

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.     

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.     

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.     

Interactions of Escherichia coli membrane lipoproteins with the murein sacculus.

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.     

Salinity-Dependent Impacts of ProQ,Prc, and Spr Deficiencies on Escherichia coli Cell Structure

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell 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.     

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.     

Turnover of murein in a diaminopimelic acid dependent mutant ofEscherichia coli

Escherichia coli 173-25, whose cell wall was labelled with¹⁴C-diaminopimelic acid, was found to lose about 15% radioactivity during growth in a fresh medium, two thirds or more being lost during the first two generations. Degradation products of the cell wall were mostly of low-molecular type. About 5% of the cells lyzed as a result of transfer associated with filtration, washing and resuspension of the bacterial population in a diaminopimelic acid (DAP) deficient medium. The degradation was very low during the first 20 min. The amount of wall material released from the cells increased between 20–30 min and a sudden decrease of viability of the population was observed. The degradation of murein triggered by starvation for DAP continued when supplementing the deficient medium with DAP and when growth was resumed. About one-half of the cell wall material released into the medium under these conditions was macromolecular. However, lysis of the cells and release of proteins into the medium were rapidly interrupted after DAP was added to the starving culture and the differential rate of synthesis of the cell wall increased. Turnover of murein was not associated with protein turnover.     

Functioning of the cloned phage MS2 lysis protein in Escherichia coli impaired in murein synthesis

Abstract The mode of action of the phage MS2 lysis protein seems not to involve a direct interaction with the murein synthesis machinery as is the case for lysis induced by β-lactam antibiotics. Mutants with defects in various penicillin-binding proteins, which are involved in murein synthesis, were found to show normal lysis sensitivity towards the cloned MS2 lysis protein. In addition, both processes, longitudinal growth of the murein sacculus in the presence of furazlocillin, cephalexin and nalidixic acid as well as spherical growth in the presence of mecillinam were sensitive to the phage lysis protein. No change in the capacity of the binding proteins to bind ¹⁴C-labelled penicillin G was observed in the presence of the MS2 lysis gene product.     

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.     

Restricted Mobility of Cell Surface Proteins in the Polar Regions of Escherichia coli

The polar regions of the Escherichia coli murein sacculus are metabolically inert and stable in time. Because the sacculus and the outer membrane are tightly associated, we investigated whether polar inert murein could restrict the mobility of other cell envelope elements. Cells were covalently labeled with a fluorescent reagent, chased in dye-free medium, and observed by microscopy. Fluorescent material was more efficiently retained at the cell poles than at any other location. The boundary between high and low fluorescence intensity areas was rather sharp. Labeled material consisted mostly of cell envelope proteins, among them the free and murein-bound forms of Braun's lipoprotein. Our results indicate that the mobility of at least some cell envelope proteins is restrained at regions in correspondence with underlying areas of inert murein.