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.     

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.     

The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.     

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.     

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.     

Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the L-alanine adding enzyme and the D-alanyl-D-alanine adding enzyme

A number of properties of temperature-sensitive mutants in murein synthesis are described. The mutants grow at 30 C but lyse at 42 C. One mutant possesses a temperature-sensitive d-alanyl-d-alanine adding enzyme, has an impaired rate of murein synthesis in vivo at both 30 and 42 C, and contains elevated levels of uridine diphosphate-N-acetyl-muramyl-tripeptide (UDP-MurNAc-l-Ala-d-Glu-m-diaminopimelic acid) at 42 C. The other mutant possesses an l-alanine adding enzyme with a very low in vitro activity at both 30 and 42 C. Its in vivo rate of murein synthesis is almost normal at 30 C but is much less at 42 C. When the murein precursors were isolated after incubation of the cells in the presence of (14)C-l-alanine, they contained only a fraction of the radioactivity that could be obtained from a wild-type strain. A genetic nomenclature for genes concerned with murein synthesis is proposed.     

Murein structure and lack of DD- and LD-carboxypeptidase activities in Caulobacter crescentus

High-pressure liquid chromatography of a muramidase digest of murein sacculi from Caulobacter crescentus showed that the absence of D-alanine carboxypeptidase activity in the cells was reflected by a very high content of pentapeptide in the murein. Approximately half of the pentapeptide side chains were shown to contain glycine, which replaced D-alanine as the terminal amino acid.     

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.     

Murein transglycosylase from phage lambda lysate. Purification and properties

Lysates of induced E. coli (lambda) lysogens contain two enzymes acting on murein: endopeptidase and murein transglycosylase. The transglycosylase was separated from the endopeptidase and purified to homogeneity. Its bacteriolytic activity was 200-fold higher than of hen egg lysozyme. The bacteriolytic activity of the lysate depends on the presence of the enzyme. The endopeptidase alone not lyse the cells, but it enhances the extent of lysis. The properties of the transglycosylase (molecular weight 17 500, pH optimum at 6.6, inactivation by Zn2+), show that it is entirely different from the bacterial enzyme of the same specificity described by others. Data are presented, which suggest that this enzyme is the phage lambda R-gene product.     

Autolysis of a division mutant of Escherichia coli

The pleiotropic character of the envC chain-forming mutant of Escherichia coli was found to include leakage of periplasmic enzymes and an abnormal tendency to autolyse. Washed suspensions of envC cells released murein fragments into the supernatant, and cell extracts from the mutant were richer than those of wild type in exo-beta-N-acetylglucosaminidase (187% of the wild-type value) and in soluble endopeptidase (256%) activities, but n-acetylmuramoylamidase, D,D-carboxypeptidase, L,Dj-carboxypeptidase and transglycosylase were not markedly different. When envC cells were grown in medium containing 0.58 M-sucrose, the chains broke up into rods, the L,D-carboxypeptidase activity increased about sixfold and D,Dj-carboxypeptidase 1B about twofold. It is suggested that L,D-carboxypeptidase is involved in septum splitting. The results suggest that the triggering of autolysis in E. coli envC depends on the alteration of envelope constituents rather than on an enhanced activity of murein hydrolases.     

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.     

Regulation of murein biosynthesis and septum formation in filamentous cells of Escherichia coli PAT 84.

Both the beta-lactam antibiotic, cephalexin, and the deoxyribonucleic acid synthesis inhibitor, nalidixic acid, are known to inhibit cell division in Escherichia coli and induce the formation of filaments. The biosynthesis of murein was investigated in these filaments and compared with the murein synthesized by the normally dividing rods of E. coli PAT 84. Differences were found in the extent of peptide side-chain cross-linkage. Filamentous cells had higher extents of cross-linkages in their newly synthesized murein. Quantitative analyses of the D-alanine carboxypeptidase and transpeptidase reactions in the different cells revealed that the carboxypeptidase activity of the filamentous cells was partially inhibited. These results were similar to those previously found with filaments that were obtained after growth of the thermosensitive division mutant at its restrictive temperature. We conclude that the formation of new cell ends (septa) depends on the proper balance between the activities of the D-alanine carboxypeptidase that regulates the availability of precursor doners and the transpeptidase, which catalyzes cross-linking and attachment of newly synthesized murein.     

Constitutive septal murein synthesis in Escherichia coli with impaired activity of the morphogenetic proteins RodA and penicillin-binding protein 2.

The pattern of peptidoglycan (murein) segregation in cells of Escherichia coli with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the D-cysteine-biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. H?ltje, and H. Schwarz, J. Bacteriol. 179:2823-2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes, pbpA and rodA, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of rodA mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564-2567, 1998).     

Changes in murein composition during the cell cycle of Caulobacter crescentus

The amino acid and muropeptide compositions of murein (peptidoglycan) isolated from populations of Caulobacter crescentus predominantly composed of swarmer or stalked cells were determined and compared with the structure of murein sacculi obtained from a population of unsegregated cells. It appears that in spite of vast morphological alterations in the course of the cell cycle, the murein composition of the various cell types is not markedly different.     

Structure of peptidoglycan from Thermus thermophilus HB8.

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.     

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.