In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors.     

Enzymes synthesizing and hydrolyzing murein in Escherichia coli. Topographical distribution over the cell envelope.

Envelopes from regions of the cell which in vivo show very little, if any, murein synthesis were isolated using the minicell-producing strain P678-54. Envelopes from minicells, representing in fact cell ends, were able to synthesize murein and to carry out transpeptidation in vitro; also all four murein hydrolase activities tested, carboxypeptidase, endopeptidase, amidase and transglycosylase, were found to be present. The specific activities of the murein synthesizing and degrading enzymes in envelopes derived from cell poles and from actively growing cells were similar. The topological distribution of murein-synthesizing enzymes and of murein hydrolases over the cell envelope is discussed.     

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.     

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.     

Incorporation of S-[3H]methyl-d-cysteine into the peptidoglycan of ether-treated cells of Escherichia coli

Certain D-amino acids can be incorporated into the murein sacculus of Escherichia coli apparently through a direct transpeptidation reaction independent of the normal biosynthetic pathway. Investigation of this process is important because it could lead to the identification of hitherto unknown enzymes involved in murein metabolism. However, a serious drawback is the lack of an appropriate in vitro assay. We have analysed the suitability of a system based on the incorporation of a radioactive substrate (S-[3H]methyl-D-cysteine) by ether-treated cells, a method successfully applied before to the study of murein biosynthesis. The results reported here indicate that ether-treated cells are indeed proficient in the incorporation of D-amino acids, matching closely the properties of the reaction in growing cells.     

In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli

PBP1B is a major bifunctional murein (peptidoglycan) synthase catalyzing transglycosylation and transpeptidation reactions in Escherichia coli. PBP1B has been shown to form dimers in vivo. The K(D) value for PBP1B dimerization was determined by surface plasmon resonance. The effect of the dimerization of PBP1B on its activities was studied with a newly developed in vitro murein synthesis assay with radioactively labeled lipid II precursor as substrate. Under conditions at which PBP1B dimerizes, the enzyme synthesized murein with long glycan strands (>25 disaccharide units) and with almost 50% of the peptides being part of cross-links. PBP1B was also capable of synthesizing trimeric muropeptide structures. Tri-, tetra-, and pentapeptide compounds could serve as acceptors in the PBP1B-catalyzed transpeptidation reaction.     

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.     

Murein biosynthesis during a synchromous cell cycle of Escherichia coli B.

The last stages of murein biosynthesis were studied in relation to the division cycle of Escherichia coli in cells synchronized by amino acid starvation (Ron et al., J. Bacteriol. 123:374--376, 1975). Murein synthesis and the activities of the D-alanine carboxypeptidase and transpeptidase were found to vary significantly during the cell cycle. Maximal synthesis and transpeptidation were observed immediately after cell division, whereas maximal D-alanine carboxypeptidase activity was detected before cell division. These results are in agreement with our earlier findings that before cell division there is a stage of increased hydrolysis of the C-terminal D-alanine moiety of newly synthesized murein strands.     

Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope

Cell envelopes of Salmonella typhimurium and Escherichia coli were disrupted in a French pressure cell and fractionated by successive cycles of sedimentation and floatation density gradient centrifugation. This permitted the identification and isolation of several membrane fractions in addition to the major inner membrane and murein-outer membrane fractions. One of these fractions (fraction OML) accounted for about 10% of the total cell envelope protein, and is likely to include the murein-membrane adhesion zones that are seen in electron micrographs of plasmolyzed cells. Fraction OML contained inner membrane, murein, and outer membrane in an apparently normal configuration, was capable of synthesizing murein from UDP-[3H]N-acetylglucosamine and UDP-N-acetylmuramylpentapeptide and covalently linking it to the endogenous murein of the preparation, and showed a labeling pattern in [3H]galactose pulse-chase experiments that was consistent with its acting as an intermediate in the movement of newly synthesized lipopolysaccharide from inner membrane to outer membrane. The fractionation procedure also identified two new minor membrane fractions, with characteristic protein patterns, that are usually included in the region of the major inner membrane peak in other fractionation procedures but can be separated from the major inner membrane fraction and from contaminating flagellar fragments by the subsequent floatation centrifugation steps.     

A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay

The bacterial cell wall is a polymer consisting of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, cross-linked via peptides appended to MurNAc. The final steps in the formation of cell wall, also referred to as murein, are catalyzed by high-molecular-weight, class A penicillin-binding proteins (PBPs). These bifunctional enzymes catalyze both glycosyltransfer, to form the carbohydrate backbone of murein, and transpeptidation, to form the interstrand peptide linkages. Using PBP1b from Eschericia coli, an in vitro kinetic characterization of the glycosyltransfer reaction was carried out. Initial studies with unlabeled substrate (Lipid II) revealed that activity is strongly influenced by DMSO, as well as metal and detergent. In addition, a continuous fluoresence assay was developed and used to determine the effect of pH on the reaction. A single basic residue was titrated, with a pK(a) of 7.0. Taken together, these data suggest a mechanism for PBP1b where the glycosyltransfer reaction is catalyzed by the concerted effect of an active site base to deprotonate the glycosyl acceptor and a divalent metal to assist departure of the leaving group of the glycosyl donor.     

The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia 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.     

Role of precursor translocation in coordination of murein and phospholipid synthesis in Escherichia coli.

Inhibition of phospholipid synthesis in Escherichia coli by either cerulenin treatment or glycerol starvation of a glycerol-auxotrophic mutant resulted in a concomitant block of murein synthesis. The intracellular pool of cytoplasmic and lipid-linked murein precursors was not affected by an inhibition of phospholipid synthesis, nor was the activity of the penicillin-binding proteins. In addition, a decrease in the activity of the two lipoprotein murein hydrolases, the lytic transglycosylases A and B, could not be demonstrated. The indirect inhibition of murein synthesis by cerulenin resulted in a 68% decrease of trimeric muropeptide structures, proposed to represent the attachment points of newly added murein. Importantly, inhibition of phospholipid synthesis also inhibited O-antigen synthesis with a sensitivity and kinetics similar to those of murein synthesis. It is concluded that the step common for murein and O-antigen synthesis, the translocation of the respective bactoprenolphosphate-linked precursor molecules, is affected by an inhibition of phospholipid synthesis. Consistent with this assumption, it was shown that murein synthesis no longer depends on ongoing phospholipid synthesis in ether-permeabilized cells. We propose that the assembly of a murein-synthesizing machinery, a multienzyme complex consisting of murein hydrolases and synthases, at specific sites of the membrane, where integral membrane proteins such as RodA and FtsW facilitate the translocation of the lipid-linked murein precursors to the periplasm, depends on ongoing phospholipid synthesis. This would explain the well-known phenomenon that both murein synthesis and antibiotic-induced autolysis depend on phospholipid synthesis and thereby indirectly on the stringent control.     

Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells

Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating [3H]leucine and [3H]palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate). Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol. Nine murein-associated [3H]leucine-labeled peptides were identified. Five of the nine peptides were lipoproteins, based on labeling with [3H]palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations. The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope.     

The complete sequence of murein synthesis in ether treated Escherichia coli

The in vitro synthesis of murein from the precursors UDP-N-acetylglucosamine, L-alanine, D-glutamic acid and meso-diaminopimelic acid was performed with the aid of ether treated Escherichia coli. This synthesis was sensitive to representative inhibitors of early reactions in the cytoplasm as well as of late reactions in the membrane or the cell wall. The sensitivity was higher than in in vitro systems starting with UDP-N-acetylmuramic acid or UDP-N-acetylmuramyl-pentapeptide.     

Two distinct transpeptidation reactions during murein synthesis in Escherichia coli.

Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.     

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.     

On the control of septation in Escherichia coli.

Mutants of E. coli defective in cell septation (ftsA to ftsG, conditional thermosensitive mutants isolated by Ricard and Hirota) were studied with respect to their membrane protein composition, murein hydrolase activities and rates of synthesis of murein and phospholipids. Three classes of mutants have been distinguished: 1) those affected in both murein and phospholipid synthesis; 2) those affected in either murein or phospholipid synthesis and 3) those affected in neither of these parameters. Overall murein hydrolase activities, after activation, is of the same order in all the mutants screened. In addition to soluble products of murein splitting, we have found insoluble products that appear to be in dynamic equilibrium with the murein of the sacculus. Endogenous levels of cyclic adenosine 3',5'-monophosphate measured after blocking septation showed no variation. This suggests that the cyclic nucleotide is not involved in the metabolic control of septation.     

Membrane-bound DD-carboxypeptidase and transpeptidase activities from Bacillus megaterium KM at pH 7. General properties, substrate specificity and inhibition by beta-lactam antibiotics.

1. The membranes from Bacillus megaterium KM contained a DD-carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 degrees C and below 20 degrees C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2-mercaptoethanol and low concentrations of p-chloromercuribenzoate. 2. The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors D-alanine or glycine. 3. The conditions for optimum activity, temperature-inactivation, temperature-dependence of the activity, carboxyl donor specificity, sensitivity to beta-lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The DD-carboxypeptidase showed inhibition by D-alanine and Ac2-L-Lys-D-Ala. 4. The inhibition by beta-lactam antibiotic was reversible for both enzymic activities and the time-dependence for their recovery was identical. 5. The DD-carboxypeptidase was very sensitive to changes in the configuration and size of the side-chains of the C-terminal dipeptide of the substrate. Amino acid residues at the C-terminus that precluded the peptide from being a DD-carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the 'model transpeptidase'. 6. It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis.     

Induction of coordinated movement of Myxococcus xanthus cells.

Rhythmically advancing waves of cells, called ripples, arise spontaneously during the aggregation of Myxococcus xanthus into fruiting bodies. Extracts prepared by washing rippling cells contain a substance that will induce quiescent cells to ripple. Three lines of evidence indicate that murein (peptidoglycan) is the ripple-inducing substance in the extracts. First, ripple-inducing activity is associated with the cell envelope of sonically disrupted M. xanthus cells. Second, whole cells, cell extracts, or purified murein from a variety of different bacteria are capable of inducing ripples. In contrast, extracts prepared from Methanobacterium spp. which contain pseudomurein instead of typical bacterial murein fail to induce ripples. Third, four components of M. xanthus murein, N-acetylglucosamine, N-acetylmuramic acid, diaminopimelate, and D-alanine, are able to induce ripples. Ripples produced by aggregating cells have a wavelength of 45 micrometers and a maximum velocity of 2 micrometers/min. Both of the multigene systems that control gliding motility appear to be required for rippling, and all known mutations at the spoC locus eliminate both rippling and sporulation.