Murein Synthesis Research Articles

Interference with murein turnover has no effect on growth but reduces beta-lactamase induction in Escherichia coli.

Physiological studies of a mutant of Escherichia coli lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC beta-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of beta-lactamase, present on a plasmid carrying the ampC and ampR genes from Enterobacter cloacae. Overproduction of MltB stimulated beta-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed ampC expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like beta-lactamases.

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.

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.

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 sfiX, rfe and metN genes of Salmonella typhimurium and their involvement in the His(c) pleiotropic response.

Two loci involved in the pleiotropic response of His(c) strains of Salmonella typhimurium (sfiX and sfiY) have been characterized at the molecular level. The sfiX gene (CS 44) has been identified as a homolog of the E. coli gene sanA, located downstream of the cytidine deaminase gene (cdd). The cdd-sanA (or cdd-sfiX) operon shows a highly conserved structure in E. coli and Salmonella. Like its E. coli homolog, the sfiX gene of S. typhimurium is required for vancomycin resistance at high temperature. The dual effect of sfiX mutations (induction of vancomycin sensitivity and suppression of cell division inhibition) suggests a link between SfiX function and murein synthesis. The sfiY locus (CS 85), contains two genes arranged in a single transcriptional unit. The upstream gene is a homolog of the E. coli gene rfe; mutations in this gene suppress the cell division defect of His(c) strains. The suppressor effect of rfe mutations can be reproduced by tunicamycin, suggesting that suppression of filamentation results from an increase in the intracellular concentration of UDP-N-acetyl-D-glucosamine. The gene located downstream of rfe is also found in E. coli but its function is unknown. Insertions in rfe suppress the methionine requirement of His(c) strains of S. typhimurium by a polar effect on the downstream gene, tentatively designated metN. Complementation with a rfe+ clone indicates that the rfe gene is not involved in the methionine requirement of His(c) strains. Thus metN expression appears to cause methionine auxotrophy in a His(c) background.

Membrane-bound lytic endotransglycosylase in Escherichia coli.

The gene for a novel endotype membrane-bound lytic transglycosylase, emtA, was mapped at 26.7 min of the E. coli chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22kDa. Overexpression of the emtA gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.

Characterization of ftsZ, the cell division gene of Buchnera aphidicola (endosymbiont of aphids) and detection of the product.

Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, contains the gene ftsZ, which codes for a protein involved in the initiation of septum formation during cell division. With immunological techniques, this protein has been detected in cell-free extracts of the endosymbiont. Nucleotide sequence determination of a 6.4-kilobase B. aphidicola DNA fragment has indicated that, as in E. coli, ftsZ is adjacent to genes coding for other cell division proteins as well as genes involved in murein synthesis (murC-ddlB-ftsA-ftsZ). Although B. aphidicola ftsZ is expressed in E. coli, it cannot complement E. coli ftsZ mutants. High levels of B. aphidicola FtsZ results in the formation of long filamentous E. coli cells, suggesting that this protein interferes with cell division. The presence of FtsZ indicates that in this, as well as in many other previously described properties, B. aphidicola resembles free-living bacteria.

Murein segregation in Escherichia coli.

Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.

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.

Genetics of subtilin and nisin biosyntheses: biosynthesis of lantibiotics.

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be divided into two classes: (i) those that are synthesized by a non-ribosomal mechanism, and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyllanthionine. They are derived from prepeptides which are post-translationally modified and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A, and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.

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.

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 fromBacillus cereus

A method is described for the rapid isolation of the activated murein precursors UDP-N-acetylmuramyl-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 μmol from a 10–1 culture of B. cereus.

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 constrained hoop: an explanation of the overshoot in cell length during a shift-up of Escherichia coli.

This review presents an explanation of length overshoot in light of a recent explanation of cell wall synthesis during the division cycle. First, our current understanding of murein structure and synthesis in gram-negative bacteria will be described. This forms the basis of the explanation of how the cell wall increases in area during the division cycle. This will be followed by a discussion of the steady-state shapes of bacteria at different growth rates. Once the shapes and sizes of bacteria at different growth rates and how these shapes are produced during steady-state growth are understood, the length overshoot during a shift-up can be explained

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.

Functioning of the cloned phage MS2 lysis protein inEscherichia coliimpaired 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.

Induction of the autolytic system of Escherichia coli by specific insertion of bacteriophage MS2 lysis protein into the bacterial cell envelope.

Bacterial lysis induced by the expression of the cloned lysis gene of the RNA bacteriophage MS2 in Escherichia coli was shown to be under the same regulatory control mechanisms as penicillin-induced lysis. It was controlled by the stringent response and showed the phenomenon of tolerance when E. coli was grown at pH 5. Changes in the fine structure of the murein were found to be the earliest physiological changes in the cell, taking place 10 min before the onset of cellular lysis and inhibition of murein synthesis. Both the average length of the glycan strands and, with a time lag, the degree of cross-linkage were altered, indicating that a lytic transglycosylase and a DD-endopeptidase had been triggered. After extensive separation of the membranes by isopycnic sucrose gradient centrifugation, the lysis protein was present predominantly in the cytoplasmic membrane and in a fraction of intermediate density and, to a lesser degree, in the outer membrane, irrespective of the conditions of growth. However, only under lysis-permissive conditions could a 17% increase in the number of adhesion sites between the inner and outer membranes be observed. Thus, a casual relationship between lysis and the formation of lysis protein-induced adhesion sites seems to exist.

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.

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.