Bacterial growth does require peptidoglycan hydrolases

Most bacteria surround their cytoplasmic membrane with a net‐like, elastic heteropolymer, the peptidoglycan sacculus, to protect themselves from bursting due to the turgor and to maintain cell shape. It has been assumed that growing bacteria require peptidoglycan hydrolases to open meshes in the peptidoglycan net allowing the insertion of the newly synthesized material for surface expansion. However, peptidoglycan hydrolases essential for bacterial growth have long remained elusive. In this issue of Molecular Microbiology Singh et al. ( 2012 ) report the identification in Escherichia coli of three new DD‐endopeptidases (Spr, YdhO and YebA) which are collectively required for peptidoglycan growth. Cells depleted of the three enzymes fail to incorporate new peptidoglycan, indicating that the cleavage of cross‐links by the new endopeptidases is needed for surface growth of the sacculus. These results are corroborated by recent data showing that Bacillus subtilis cells require the DL‐endopeptidase activity of CwlO or LytE for growth.     

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.     

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.     

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.     

FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross‐linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross‐links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod‐shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP‐binding cassette transporter‐like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross‐link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.     

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.     

Cell shape determination in Escherichia coli

The rigid cell wall peptidoglycan (murein) is a single giant macromolecule whose shape determines the shape of the bacterial cell. Insight into morphogenetic mechanism(s) responsible for determining the shape of the murein sacculus itself has begun to emerge only in recent years. The discovery that MfreB and Mbl are cytoskeletal actin homologues that form helical structures extending from pole to pole in rod-shaped cells has opened an exciting new field of microbial cell biology. MreB (in Gram-negative rods) and Mbl (in Gram-positive species) are essential for murein synthesis along the lateral wall and hence, the rod shape of the cell. Known members of the morphogenetic system include MreB (or Mbl), MreC, MreD and PBP2, but Rod A and murein biosynthetic enzymes involved in peptidoglycan precursor synthesis and assembly are likely to be recruited to the same multimolecular apparatus. However, the actual role of MreB in assembly of the morphogenetic complex is still not clear and little is known about regulatory mechanisms controlling the switch from lateral murein elongation to septa1 murein synthesis at the time of cell division.     

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.     

The exocytoskeleton

The murein or peptidoglycan wall enclosing most bacteria is essential for the life style of most organisms in the Domain of Bacteria. Only in special situations does it not play a role in the bacterial growth cycle. When life first appeared on this planet the cellular osmotic pressure was probably low and a sacculus was probably not relevant, but became necessary as bacterial life evolved from the complex and sophisticated cell called the Last Universal Ancestor. The construction of the murein wall outside of the cytoplasmic membrane is complex and requires elaborate special biochemistry. Growth of the sacculus in some parts of the surface and not in others is important for bacteria cells and allows them to divide and grow without becoming larger and larger and for their being able to maintain a shape characteristic of individual species.     

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.     

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.     

Substrate specificity of an elongation‐specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae

The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross‐linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross‐links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth – particularly elongation – of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d ‐endopeptidase able to cleave most peptide chain cross‐links in V. cholerae's PG. PG from a ?shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate‐dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side‐wall growth are discussed.     

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.     

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.     

Partition of Old Murein in Small Patches over the Entire Wall of E. coli Cells Forced to Grow as a Coccoid

With the development of a technique to visualize the ages of different portions of the sacculus, De Pedro et al. showed that the sacculus of Escherichia coli was tripartite: (i) the establish poles contained only old wall, (ii) the nascent poles (or septa) were composed entirely of new murein, and (iii) the elongating cylindrical wall was a mixture of patches of both old and new peptidoglycan. This short note presents a computer analysis of data files of work presented in the recent paper by De Pedro et al. of the growth pattern of the wall of E. coli forced to grow in a quite unusual morphology as large spheres in the presence of mecillinam. Compared with rod-shaped cells, only very small patches (spikes) of old wall were retained interspersed with new murein during the conversion to large spheroids. This subdivision appeared to be the case for both the previous wall of the poles, which are ordinarily retained intact, and the previous patches retained within the cylindrical wall. These very small patches after the conversion to spheroids were much smaller than the sidewall patches in rod-shaped cells reported previously. This implies that the mechanism that prevents the insertion of new wall into both the wall of the poles and the old wall patches of the sidewall in the presence of mecillinam is superseded by insertion throughout the old wall. The work in the De Pedro et al. paper from 2001 was done with cells of same strain as in the earlier papers with rod-shaped cells, so the results of computer analysis of the fluorescence micrographs can be critically compared.     

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.     

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.