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.     

Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus.

High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with [3H]diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process. Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation. Elongation and septation were shown to be overlapping processes.     

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.     

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.     

The effect of substitution of diaminopimelic acid by 4-hydroxy-diaminopimelic acid on the synthesis and degradation of murein inEscherichia coli 173-25

Defects in the formation of the septum and gradually autolysis of cells occur when the dapdependent mutant ofEscherichia coli is grown in a medium with 4-hydroxy-diaminopimelic acid. When the culture grown in the presence of the labelled analogue is supplemented with the non-radioactive diaminopimelic acid a portion of the TCA-soluble radioactivity is released from the cells during 20 min after the addition of diaminopimelic acid. During this time interval the elongated forms formed in the presence of the analogue divide, however, only on the condition that the above forms are not irreversibly damaged. The increased concentration of the analogue in the medium substantially suppresses the irregularities in the development of the septum as well as the degradation of analogue containing cell wall. However, the growth rate in the presence of the analogue is always slightly lower than that in the presence of diaminopimelic acid. The cell wall pulse-labelled with diaminopimelic acid or its analogue for a time interval shorter than 1/4 of the generation time exhibits the same or only slightly higher rate of turnover than the wall labelled with dap during two generations. It can be assumed that 4-hydroxydiaminopimelic acid is probably utilized less effectively for the synthesis of murein than diaminopimelic acid. However, its incorporation into the wall does not result in pronounced damage of the cell.     

Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli

Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10(-6) M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.     

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.     

Cell wall growth of Bacillus megaterium: cytoplasmic radioactivity after pulse-labeling with tritiated diaminopimelic acid.

Study of the cell wall growth in Bacillus megaterium by pulse-labeling a DAP- Lys- mutant with tritiated diaminopimelic acid (DAP) had revealed the presence of intracytoplasmic radioactivity. The nature of this radioactivity was studied on one hand by autoradiographic analysis of bacteria treated in different ways and on the other hand by chromatography of the radioactive compounds extracted with boiling water. It is shown that cytoplasmic radioactivity corresponds neither to free DAP nor to DAP metabolized into lysine, but to murein precursors. Autoradiographic analysis of bacteria in which all murein precursors were removed gives exactly the same cell wall growth pattern as the one previously obtained for untreated bacteria. It can be concluded that, in B. megaterium, cell wall elongation occurs by diffuse intercalation of newly synthesized murein along the cylindrical part of the cell wall and that only cross wall formation occurs in a precise growth zone.     

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.     

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.     

Mode of action of pesticin: N-acetylglucosaminidase activity.

Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in Escherichia coli phi nor caused detectable degradation of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not resemble that of colicin E2 as suggested by others. However, incorporation of cell wall-specific label ([14C]diaminopimelic acid) into trichloroacetic acid-insoluble material of growing cells was inhibited by pesticin which also promoted release of such radioactivity from both resting cells and purified mureinlipoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine comigrated either with [14C]diaminopimelic acid in the murein peptide or with [14C]isoleucine of the Braun lipoprotein. As judged by these findings and pesticin-dependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars, the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of murein-lipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by the bacteriocin, indicating that its antibacterial specificity resides at the level of absorption.     

Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (alpha, beta, and gamma) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.     

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.     

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.     

Changes in the composition of Escherichia coli murein as it ages during exponential growth

Escherichia coli murein was specifically labeled with [14C]diaminopimelic acid in the mutant strains W7 (dap lysA) and BUG6. Pulse-labeled heat-denatured E. coli cells were digested with 2 mg of egg-white lysozyme per ml to degrade the murein completely and free any lipoprotein-bound muropeptide trimers, dimers, and monomers. Pulse-chase experiments showed that the relative percentage of trimers and dimers found in the newly synthesized murein increased somewhat with time at the expense of monomers. The increase in cross-links indicated that the radioactive monomers served as acceptors in multisite transpeptidations occurring after the labeling period. The content of nonreducing monomers (C7 and C8) remained unaltered, indicating that the oligosaccharide chain length did not change with time. A gradual conversion of the reducing disaccharide tetrapeptide monomer to its tripeptide analog occurred during chasing. Braun lipoprotein was linked to about 2% of the murein subunits within 30 s of the incorporation of subunits into insoluble murein, and after one-half a generation of chase, lipoprotein-associated muropeptides had approached the maximum (16% of the total murein subunits). The distribution of muropeptides was similar in lipoprotein-linked and lipoprotein-free murein, showing that the enzyme that links Braun lipoprotein to murein does not discriminate between monomers, dimers, and trimers. No evidence for a chasable, soluble polymer of murein was found in our experiments. Hence, our data support the idea that new murein is incorporated directly into the sacculus without first existing as a soluble intermediate.     

Study of cell wall growth in Bacillus megaterium by high-resolution autoradiography.

Growth of the cell wall of Bacillus megaterium was studied by pulse-labeling the cell wall of a DAP- Lys- mutant for a very short time with tritium-labeled diaminopimelic acid. The distribution of radioactivity along the cell wall was examined by high-resolution autoradiography on isolated cell walls and thin sections of bacteria. The results indicate that cell wall elongation occurs by diffuse intercalation of newly synthesized murein into the expanding cell wall during exponential growth, as well as during germination, and that the only zone of highly localized diaminopimelic acid incorporation is found at the cross wall during its synthesis. This zone contains about 30% of the radioactivity incorporated into the cell wall. Analysis of autoradiographs of thin sections of bacteria shows that the total radioactivity incorporated per bacterium doubles during the life cycle. This doubling occurs in the cylindrical part of the cell wall but not in the polar caps. This seems to indicate that elongation of the bacterium is not constant during the life cycle but increases with the length of the cell.     

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.     

Turnover of murein in a diaminopimelic acid dependent mutant ofEscherichia coli

Escherichia coli 173-25, whose cell wall was labelled with¹⁴C-diaminopimelic acid, was found to lose about 15% radioactivity during growth in a fresh medium, two thirds or more being lost during the first two generations. Degradation products of the cell wall were mostly of low-molecular type. About 5% of the cells lyzed as a result of transfer associated with filtration, washing and resuspension of the bacterial population in a diaminopimelic acid (DAP) deficient medium. The degradation was very low during the first 20 min. The amount of wall material released from the cells increased between 20–30 min and a sudden decrease of viability of the population was observed. The degradation of murein triggered by starvation for DAP continued when supplementing the deficient medium with DAP and when growth was resumed. About one-half of the cell wall material released into the medium under these conditions was macromolecular. However, lysis of the cells and release of proteins into the medium were rapidly interrupted after DAP was added to the starving culture and the differential rate of synthesis of the cell wall increased. Turnover of murein was not associated with protein turnover.     

Cloning and characterization of PBP 1C, a third member of the multimodular class A penicillin-binding proteins of Escherichia coli.

All proteins of Escherichia coli that covalently bind penicillin have been cloned except for the penicillin-binding protein (PBP) 1C. For a detailed understanding of the mode of action of beta-lactam antibiotics, cloning of the gene encoding PBP1C was of major importance. Therefore, the structural gene was identified in the E. coli genomic lambda library of Kohara and subcloned, and PBP1C was characterized biochemically. PBP1C is a close homologue to the bifunctional transpeptidases/transglycosylases PBP1A and PBP1B and likewise shows murein polymerizing activity, which can be blocked by the transglycosylase inhibitor moenomycin. Covalently linked to activated Sepharose, PBP1C specifically retained PBP1B and the transpeptidases PBP2 and -3 in addition to the murein hydrolase MltA. The specific interaction with these proteins suggests that PBP1C is assembled into a multienzyme complex consisting of both murein polymerases and hydrolases. Overexpression of PBP1C does not support growth of a PBP1A(ts)/PBP1B double mutant at the restrictive temperature, and PBP1C does not bind to the same variety of penicillin derivatives as PBPs 1A and 1B. Deletion of PBP1C resulted in an altered mode of murein synthesis. It is suggested that PBP1C functions in vivo as a transglycosylase only.     

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.