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.

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.

Bacterial lysozymes.

Lysozymes are found in many bacteria that are surrounded by a murein-(peptidoglycan) containing cell wall. Their physiological function for the bacteria is still a matter of debate. On the one hand they can autolyse the cell, on the other hand they may have an essential role during enlargement and division of the cell wall by the controlled splitting of bonds in the murein sacculus. Both beta-1.4-N,6-O-diacetylmuramidase and beta-1.4-N-acetylmuramidases have been described in bacteria. In some cases a modular design of the enzyme has been demonstrated with a catalytic domain and a substrate (murein)-binding and recognition domain consisting of repeated motifs.

Lysozyme substrates.

The natural substrate of lysozyme is the rigid layer of bacterial cell walls, the murein (peptidoglycan), which is a gigantic polymer of (GlcNAc-MurNAc)n polysaccharide strands crosslinked through short peptide bridges at the lactyl groups of the muramic acid residues. Thus, lysozyme lyses bacteria by degrading their protective exoskeleton, the murein sacculus. The high molecular weight murein is thereby hydrolysed to low molecular weight muropeptides, a process that can be followed quantitatively by different methods. However, due to the insolubility of the murein sacculus, the enzyme kinetics are rather complex. Therefore, a variety of different low molecular weight substrates have been prepared, both murein degradation products and synthetic compounds. These substrates allow a better characterization of the binding and catalytic mechanism of lysozyme. In addition, they are used in various photometric, isotopic and immunological lysozyme assays.

Lytic transglycosylases.

Although cleaving the same glycosidic bond between MurNAc and GlcNAc in murein, lytic transglycosylases differ from lysozymes by catalyzing an intramolecular transglycosylation of the glycosyl-bond onto the C6 hydroxyl group of the muramic acid residue yielding 1.6-anhydromuramic acid-carrying products. The three dimensional structure of the soluble lytic transglycosylase Slt70 of E. coli revealed a doughnut-like shape that would allow the protein to encircle the polysaccharide strands of the murein. Despite the absence of significant sequence homology, the catalytic center shows structural similarity to lysozymes, although the catalytic aspartate is missing. All lytic transglycosylases which have been characterized up until now turned out to be processive exo-glycosylases.

Murein chemistry of cell division in Escherichia coli.

The length distribution of the glycan strands of murein has been analysed with a novel method in filamentous and spherical cells of Escherichia coli, as well as during septum formation and cell separation. A shift to the longer glycan strands was observed in the murein of furazlocillin-induced filaments. In contrast, shorter glycan strands were increased in the murein of mecillinam-induced spherical cells. During septum formation in a chain-forming envA mutant that is defective in the splitting process of the septum, a shift to the shorter glycan strands was detected that was not seen in wild type E. coli cells. It is concluded that septum-specific murein structures of rather short glycan strands are released during splitting of the septum. This intermediate material remains present in the septum of the envA mutant. The splitting process of the septum was investigated by analysing the murein during penicillin-induced bacteriolysis, which is known to take place by strictly localized murein degradation in the equatorial zone of the cell. No changes in the length distribution of the glycan strands could be detected during penicillin-induced lysis, with the exception of an increase in disaccharides, the shortest glycan strands possible. This is explained by the action of exo-muramidases progressively digesting glycan strands, leaving disaccharide units covalently linked to the remaining murein at the sites of murein cross-linkage. It is proposed that this "zipper-like" mechanism represents the normal cutting process of the septum during cell separation.

Molecular interplay of murein synthases and murein hydrolases in Escherichia coli.

Affinity chromatography using different lytic transglycosylases as a specific ligand revealed an interaction of both murein hydrolases and murein synthases. This interaction is taken as evidence for the assemblage into a multienzyme complex that could function as a murein replicase precisely copying the given three-dimensional structure of the murein sacculus. The sacculus of the mother cell would function as a template, which is identically replicated by copying the lengths of the existing glycan strands and the pattern of crosslinkages. A hypothetical enzyme complex specifically involved in cell division and a complex specifically involved in cell elongation are presented. It is postulated that PBPs 1a and/or 1b are present in both complexes, whereas the presence of PBP2 or PBP3 defines the specificity of the murein-synthesizing machinery as being involved in either cell elongation or septation. Moreover, the proposed "holoenzyme" suprastructure could explain why the specific inhibition of PBPs 1a/1b results in bacteriolysis and why inhibition of PBP2 and PBP3 causes the well-known morphological alterations, spherical growth, and filamentation, respectively.

Affinity chromatography as a means to study multienzyme complexes involved in murein synthesis.

The interaction of murein hydrolases and synthases was studied by affinity chromatography. The lytic transglycosylases Slt70 and MltB of E. coli were purified and covalently linked to CNBr-activated Sepharose. Membrane extracts were analyzed for proteins that interact with the immobilized murein hydrolases. Slt70-Sepharose was found to retain the PBPs 1b, 1c, 2, and 3. Likewise MltB-Sepharose enriched PBP 1b, 1c, and 3. Thus both lytic transglycosylases have an affinity for a transpeptidase, PBP2 and/or 3, as well as for the bifunctional transpeptidase/transglycosylase 1b. Interestingly, in addition, the poorly characterized PBP 1c interacts strongly with both Slt70 and MltB. It is speculated that the lytic transglycosylases assemble a multienzyme complex consisting of hydrolases and synthases, which is involved in growth of the stress-bearing murein sacculus.

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

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 beta-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.

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.

One-step purification procedure for UDP-N-acetylmuramyl-peptide murein precursors from Bacillus cereus.

A method is described for the rapid isolation of the activated murein precursors UDP-N-acetyl-muramyl-pentapeptide (UDP-MurNAc-pentapeptide) and UDP-MurNAc-tripeptide from Bacillus cereus. After accumulation of the precursors by inhibition of murein synthesis either in the presence of vancomycin (for the pentapeptide precursor) or D-cycloserine (for the tripeptide precursor) the cells were extracted with boiling water. Prior to high pressure liquid chromatography the material was freed from acid precipitable material. UDP-MurNAc-penta- and tripeptide were separated from other components by reversed-phase HPLC on Hypersil ODS using isocratic elution conditions with sodium phosphate buffer. The precursors were obtained with at least 98% purity and a yield of about 50 mumol from a 10-l culture of B. cereus.

Two-step procedure for purification and separation of the essential penicillin-binding proteins PBP 1A and 1Bs of Escherichia coli.

The penicillin-binding proteins PBP 1A and 1Bs are the essential murein polymerases of Escherichia coli. Purification of these membrane-bound bifunctional transglycosylase-transpeptidases was a major obstacle in studying the details of both enzymatic reactions. Here we describe a simple, highly specific affinity chromatography method that takes advantage of the availability of the specific inhibitor of the transglycosylase site moenomycin A in order to enrich PBP 1A and 1Bs in one step from crude membrane preparations. Separation of PBP 1A from PBP 1Bs is achieved in a second step employing cation exchange chromatography yielding enzymatically active native murein polymerases.

Characterization of three different lytic transglycosylases in Escherichia coli.

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.

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.

Effect of phi X174 protein E-mediated lysis on murein composition of Escherichia coli.

Lysis of Escherichia coli by bacteriophage phi X174 is caused by the phage protein E. As protein E is devoid of enzymatic activities it has been postulated that lysis is the result of an induction of the autolytic enzymes of the host. This hypothesis was investigated by comparing the murein composition before and during lysis of either phi X174 infected cells or protein E induced lysis of E. coli. Additionally, protein E-mediated lysis was compared with induction of the autolytic system by EDTA. The analysis showed that the overall composition of murein is not changed after induction of protein E-mediated lysis. Nevertheless, murein degradation seems to be stimulated by the action of protein E as shown by an increase in the total amount of murein turnover products by about 10%. It could be shown that an intact murein sacculus prevents the phages from being released.

Analysis of the length distribution of murein glycan strands in ftsZ and ftsI mutants of E. coli.

The chain length distribution of murein glycan strands was analyzed in wild-type cells and in cells in which preseptal and/or septal murein synthesis was prevented in ftsZ84 and ftsI36 mutants of E. coli. This revealed a significant change in glycan chain lengths in newly synthesized murein associated with inactivation of the ftsZ gene product but not with inactivation of the ftsI gene product. This is the first reported abnormality in murein biosynthesis associated with mutation of an essential cell division gene.

The negative regulator of beta-lactamase induction AmpD is a N-acetyl-anhydromuramyl-L-alanine amidase.

Construction of a malE-ampD gene fusion allowed purification of biologically active fusion protein by affinity chromatography. The cloned malE-ampD gene fusion complemented a chromosomal ampD mutation. Purified MalE-AmpD fusion protein was found to have murein amidase activity with a pronounced specificity for 1,6-anhydromuropeptides, the characteristic murein turnover products in Escherichia coli. Being a N-acetyl-anhydromuranmyl-L-alanine amidase AmpD is likely to be involved in recycling of the turnover products. It is suggested that the negative regulatory effect of AmpD is due to the hydrolysis of anhydro-muropeptides which may function as signals for beta-lactamase induction.