The composition of the murein of Escherichia coli

Escherichia coli murein, the polymer from which the shape-maintaining structure of the cell envelope is made, shows unexpected complexity. The separation of murein building blocks with high performance liquid chromatography reveals about 80 different types of muropeptides. Their behavior in high performance liquid chromatography and their chemical structure are described. The complexity of E. coli murein is due to the free combination of seven different types of side chains (L-Ala-D-Glu-R with R = -OH, -m-A2pm, -m-A2pm-D-Ala, -m-A2 pm-Gly, -m-A2pm-D-Ala-D-Ala, -m-A2pm-D-Ala-Gly, -m-A2pm-Lys-Arg) with two types of cross-bridges (D-Ala-m-A2pm, -m-A2pm-m-A2pm). The novel type of cross-bridge, A2pm-A2pm, contains an L,D-peptide bond, as shown by Edman degradation and chemical analysis of the reaction products. The A2pm-A2pm cross-bridge is assumed to play a role in the adaptation of the cross-linkage of murein to different growth conditions of the cell. The structural data of E. coli murein agree best with a model of a thin, however multilayered, murein sacculus.     

Recycling of murein by Escherichia coli.

The tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid [A2pm]), tetrapeptide (L-Ala-D-Glu-A2pm-D-Ala), and dipeptide (A2pm-D-Ala) which are shed by Escherichia coli from the murein sacculus were found to be reused by the cells to synthesize murein. The tripeptide was used directly, without degradation, to form UDP-N-acetylmuramyl-L-Ala-D-Glu-A2pm. The tetrapeptide lost its carboxy-terminal D-Ala, apparently in the periplasm, before being used. The dipeptide was degraded to D-Ala and A2pm before uptake.     

Molecular characterization of the beta-N-acetylglucosaminidase of Escherichia coli and its role in cell wall recycling

The beta-N-acetylglucosaminidase of Escherichia coli was found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame, ycfO, whose predicted amino acid sequence is 57% identical to that of Vibrio furnissii ExoII. NagZ hydrolyzes the beta-1,4 glycosidic bond between N-acetylglucosamine and anhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel beta-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.     

Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli.

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.     

Escherichia coli mutants altered in murein lipoprotein.

Mutants with alterations in the structure, biosynthesis, or assembly of murein lipoprotein were selected by a procedure based on radiation suicide of wild-type organisms by [3H]arginine under conditions where the radioactive arginine was preferentially incorporated into lipoprotein. Further screening for the potential mutants among the survivors of [3H]arginine suicide was carried out by using a sensitive immunodiffusion test, followed by radioactive double-labeling experiments. Three mutants were obtained and partially characterized.     

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.     

Temperature-sensitive murein synthesis in an Escherichia coli pdx mutant and the role of alanine racemase

The basis for disruption of morphogenesis by depletion of pyridoxine derivatives was studied using a pdxH null mutant of Escherichia coli K-12. Removal of pyridoxal from growing cultures severely inhibited murein synthesis in vivo, whereas simultaneous supplementation with d-alanine effectively prevented inhibition. Extractable alanine racemase was low following such starvation. Selection of mutants overcoming the glycine- or temperature-sensitivity imposed by pyridoxine limitation yielded a variety of phenotypes. The most effective of these extragenic suppressors conferred an elevated alanine racemase activity which was resistant to the effects of pyridoxal removal.Abbreviations Gly^s glycine-sensitive phenotype - Ts temperature-sensitive phenotype - DAP 2,6-diaminopimelic acid - SDS sodium dodecylsulfate     

Absence of oligomeric murein intermediates in Escherichia coli.

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.     

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.     

Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein

Turnover and recycling of the cell wall murein represent a major metabolic pathway of Escherichia coli. It is known that E. coli efficiently reuses, i.e., recycles, its murein tripeptide, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, to form new murein. However, the question of whether the cells also recycle the amino sugar moieties of cell wall murein has remained unanswered. It is demonstrated here that E. coli recycles the N-acetylglucosamine present in cell wall murein degradation products for de novo murein and lipopolysaccharide synthesis. Furthermore, E. coli also recycles the anhydro-N-acetylmuramic acid moiety by first converting it into N-acetylglucosamine. Based on the results obtained by studying mutants unable to recycle amino sugars, the pathway for recycling is revealed.     

Comparison of two hydrolytic murein transglycosylases of Escherichia coli

Escherichia coli has two murein transglycosylases, which are found in the soluble and the particulate fraction, respectively. The enzymes have been purified and have been shown to differ in some of their molecular properties [Mett, H., Keck, W., Funk, A. & Schwarz, U. (1980) J. Bacteriol. 144, 45-52]. We improved and simplified the purification procedure for the membrane-derived transglycosylase and characterized the two enzymes in more detail by peptide mapping and by immunological procedures. The peptide pattern obtained after tryptic digestion of the purified enzymes differed for the two enzymes. Antisera to the transglycosylases reacted only with their own antigen as shown by specific inhibition of the enzymatic activity, double immunodiffusion and by immunochemical staining of protein blots on nitrocellulose filters. Thus we conclude that the transglycosylases are two distinct proteins and that the one is not a precursor of the other.     

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.     

Two different species of murein transglycosylase in Escherichia coli.

We demonstrated that Escherichia coli murein transglycosylase exists in two forms. After mechanical disruption of the cells, one form was found in the soluble fraction and the other, in the cell envelope. The two enzymes differed with respect to molecular weight, isoelectric point, solubility in aqueous buffers, and to some extent in their requirements for maximal catalytic activity. The molecular weight of the membrane-bound transglycosylase (35,000) was half that of the soluble enzyme. Whether the high-molecular-weight soluble protein is a precursor of the membrane-bound enzyme species remains to be elucidated.     

Cleavage and resynthesis of peptide cross bridges in Escherichia coli murein.

In Escherichia coli, peptide cross bridges in the murein undergo turnover after they are synthesized. Peptide cross bridges formed in the presence of [3H]diaminopimelic acid were found to lose 3H label from their donor peptides after the [3H]diaminopimelic acid was removed from the growth medium. There was a corresponding increase in the amount of 3H label in acceptor peptides so that the total amount of label in the peptide cross bridges remained constant. Our explanation of this observation is that the cross bridges are cleaved by the cell, and the original 3H-labeled donor peptides are incorporated into new cross bridges. Since these 3H-labeled peptides are now only tetrapeptides, they can only be used as acceptors when new cross bridges are formed.     

Activity of murein hydrolases in synchronized cultures of Escherichia coli.

Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited.     

Novel cell-binding activity specific for N-acetyl-D-glucosamine in an Escherichia coli strain

Escherichia coli strains isolated from patients with different levels of urinary tract infection and from healthy persons were tested for their ability to haemagglutinate endo-beta-galactosidase-treated human erythrocytes. Among the 104 strains studied one revealed a strong agglutination reaction with the enzyme-treated erythrocytes. From the monosaccharides tested N-acetyl-D-glucosamine inhibited agglutination most effectively. Orosomucoid and asialo-orosomucoid had no effect on the haemagglutination whereas beta-galactosidase treated asialo-orosomucoid was inhibitory. These findings indicate that the E. coli strain studied contains a novel cell-binding activity with specificity for terminal N-acetyl-D-glucosamine residues.     

Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli

On the basis of the published N-terminal amino acid sequence of the soluble lytic transglycosylase 35 (Slt35) of Escherichia coli, an open reading frame (ORF) was cloned from the 60.8 min region of the E. coli chromosome. The nucleotide sequence of the ORF, containing a putative lipoprotein-processing site, was shown by [³H]-palmitate labelling to encode a lipoprotein with an apparent molecular mass of 36 kDa. A larger protein, presumably the prolipoprotein form, accumulated in the presence of globomycin. Over-expression of the gene, designated mltB (for membrane-bound lytic transglycosylase B), caused a 55-fold increase in murein hydrolase activity in the membrane fraction and resulted in rapid cell lysis. After membrane fractionation by sucrose-density-gradient centrifugation, most of the induced enzyme activity was present in the outer and intermediate membrane fractions. Murein hydrolase activity in the soluble fraction of a homogenate of cells induced for MltB increased with time. This release of enzyme activity into the supernatant could be inhibited by the addition of the serine-protease inhibitor phenylmethyl-sulphonyl fluoride. It is concluded that the previously isolated Slt35 protein is a proteolytic degradation product of the murein hydrolase lipoprotein MltB. Surprisingly, a deletion in the mltB gene showed no obvious phenotype.     

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.     

Alteration of Escherichia coli murein during amino acid starvation.

We have studied the mechanisms by which amino acid starvation of Escherichia coli induces resistance against the lytic and bactericidal effects of penicillin. Starvation of E. coli strain W7 of the amino acids lysine or methionine resulted in the rapid development of resistance to autolytic cell wall degradation, which may be effectively triggered in growing bacteria by a number of chemical or physical treatments. The mechanism of this effect in the amino acid-starved cells involved the production of a murein relatively resistant to the hydrolytic action of crude murein hydrolase extracts prepared from normally growing E. coli. Resistance to the autolysins was not due to the covalently linked lipoprotein. Resistance to murein hydrolase developed most rapidly and most extensively in the portion of cell wall synthesized after the onset of amino acid starvation. Lysozymes digests of the autolysin-resistant murein synthesized during the first 10 min of lysine starvation yielded (in addition to the characteristic degradation products) a high-molecular-weight material that was absent from the lysozyme-digests of control cell wall preparations. It is proposed that inhibition of protein synthesis causes a rapid modification of murein structure at the cell wall growth zone in such a manner that attachment of murein hydrolase molecules is inhibited. The mechanism may involve some aspects of the relaxed control system since protection against penicillin-induced lysis developed much slower in amino acid-starved relaxed controlled (relA) cells than in isogenic stringently controlled (relA+) bacteria.