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J V Höltje

Publications and source records attributed to J V Höltje.

At least 19 recordsLinked to original sources
PubMed2001

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.

Cell Wall↗
PubMed2001

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 Division↗
PubMed2001

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.

Anti-Bacterial Agents↗
PubMed2001

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. Höltje, 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).

Amdinocillin↗
PubMed2000

Two-step procedure for purification and separation of the essential penicillin-binding proteins PBP 1A and 1Bs of Escherichia coli.

The penicillin-binding proteins PBP 1A and 1Bs are the essential murein polymerases of Escherichia coli. Purification of these membrane-bound bifunctional transglycosylase-transpeptidases was a major obstacle in studying the details of both enzymatic reactions. Here we describe a simple, highly specific affinity chromatography method that takes advantage of the availability of the specific inhibitor of the transglycosylase site moenomycin A in order to enrich PBP 1A and 1Bs in one step from crude membrane preparations. Separation of PBP 1A from PBP 1Bs is achieved in a second step employing cation exchange chromatography yielding enzymatically active native murein polymerases.

Bacterial Proteins↗
PubMed2000

A simple screen for murein transglycosylase inhibitors.

A simple assay for detection of compounds that bind to the active site in the transglycosylation domain of the essential bifunctional transglycosylase and transpeptidase penicillin-binding proteins (PBPs) is reported. The method is based on a competition with the specific transglycosylase inhibitor moenomycin. With moenomycin coupled to Affi-Gel beads, a simple filtration procedure allows the amount of labeled PBPs that bind to moenomycin beads in the presence of test substances to be determined. The PBPs can easily be labeled by the covalent binding of penicillin derivatives. Crude membrane extracts can be used as a source for the PBPs, and different kinds of labels for the penicillin-PBP complexes can be used. The assay can be adapted to high-throughput screens.

Bacterial Proteins↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli.

The first gene of a family of prokaryotic proteases with a specificity for L,D-configured peptide bonds has been identified in Escherichia coli. The gene named ldcA encodes a cytoplasmic L, D-carboxypeptidase, which releases the terminal D-alanine from L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine containing turnover products of the cell wall polymer murein. This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary growth phase. Owing to a defect in muropeptide recycling the unusual murein precursor uridine 5'-pyrophosphoryl N-acetylmuramyl-tetrapeptide accumulates in the mutant. The dramatic decrease observed in overall cross-linkage of the murein is explained by the increased incorporation of tetrapeptide precursors. They can only function as acceptors and not as donors in the crucial cross-linking reaction. It is concluded that murein recycling is a promising target for novel antibacterial agents.

Amino Acid Sequence↗
PubMed1999

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.

Bacterial Proteins↗
PubMed1999

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.

Bacterial Proteins↗
PubMed1998

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.

Bacterial Proteins↗
PubMed1998

Effect of phi X174 protein E-mediated lysis on murein composition of Escherichia coli.

Lysis of Escherichia coli by bacteriophage phi X174 is caused by the phage protein E. As protein E is devoid of enzymatic activities it has been postulated that lysis is the result of an induction of the autolytic enzymes of the host. This hypothesis was investigated by comparing the murein composition before and during lysis of either phi X174 infected cells or protein E induced lysis of E. coli. Additionally, protein E-mediated lysis was compared with induction of the autolytic system by EDTA. The analysis showed that the overall composition of murein is not changed after induction of protein E-mediated lysis. Nevertheless, murein degradation seems to be stimulated by the action of protein E as shown by an increase in the total amount of murein turnover products by about 10%. It could be shown that an intact murein sacculus prevents the phages from being released.

Bacterial Outer Membrane Proteins↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

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.

Carbohydrate Sequence↗
PubMed1997

Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli.

Lytic transglycosylases are a unique lysozyme-like class of murein hydrolases believed to be important for growth of Escherichia coli. A membrane-bound lytic transglycosylase with an apparent molecular mass of 38 kDa, which was designated Mlt38, has previously been purified and characterized (A. Ursinus and J.-V. Höltje, J. Bacteriol. 176:338-343, 1994). On the basis of four tryptic peptides, the gene mltA was mapped at 63 min on the chromosomal map of E. coli K-12 and cloned by reverse genetics. The open reading frame was found to contain a typical lipoprotein consensus sequence, and the lipoprotein nature of the gene product was demonstrated by [3H]palmitate labeling. On the basis of the distribution of MltA in membrane fractions obtained by sucrose gradient centrifugation, a localization in the outer membrane is indicated. Overexpression of MltA at 30 degrees C, the optimal temperature for enzyme activity, but not at 37 degrees C results in the formation of spheroplasts. Not only a deletion mutant in mltA, but also double mutants in mltA and one of the two other well-characterized lytic transglycosylases (either sltY or mltB), as well as a triple mutant in all three enzymes, showed no obvious phenotype. However, dramatic changes in the structure of the murein sacculus indicate that lytic transglycosylases are involved in maturation of the murein sacculus.

Amino Acid Sequence↗
PubMed1997

Pesticin displays muramidase activity.

Pesticin of Yersinia pestis is the only bacteriocin that converts sensitive cells to stable spheroplasts. The amino acid sequence of pesticin as derived from the nucleotide sequence shows no similarity to those of any of the bacteriocins. The unique properties of pesticin prompted an investigation of its mode of action. Since the pesticin plasmid does not encode a lysis protein for release of pesticin into the culture medium, pesticin was isolated from cells and purified to electrophoretic homogeneity. Highly purified pesticin degraded murein and murein glycan strands lacking the peptide side chains to products that were similar to those obtained by lysozyme, as revealed by high-resolution high-pressure liquid chromatography. After reduction of the murein degradation products with tritium-labeled sodium borohydride, acid hydrolysis, and separation of the products by thin-layer chromatography, radiolabeled muraminitol was identified. This indicates that pesticin is a muramidase, and not an N-acetyl-glucosaminidase, that converts cells into stable spheroplasts by slowly degrading murein.

Bacteriocins↗
PubMed1997

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.

Bacterial Proteins↗