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1.
The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes. 相似文献
2.
On the control of septation in Escherichia coli. 总被引:1,自引:0,他引:1
Mutants of E. coli defective in cell septation (ftsA to ftsG, conditional thermosensitive mutants isolated by Ricard and Hirota) were studied with respect to their membrane protein composition, murein hydrolase activities and rates of synthesis of murein and phospholipids. Three classes of mutants have been distinguished: 1) those affected in both murein and phospholipid synthesis; 2) those affected in either murein or phospholipid synthesis and 3) those affected in neither of these parameters. Overall murein hydrolase activities, after activation, is of the same order in all the mutants screened. In addition to soluble products of murein splitting, we have found insoluble products that appear to be in dynamic equilibrium with the murein of the sacculus. Endogenous levels of cyclic adenosine 3',5'-monophosphate measured after blocking septation showed no variation. This suggests that the cyclic nucleotide is not involved in the metabolic control of septation. 相似文献
3.
Effects of furazlocillin, a beta-lactam antibiotic which binds selectively to penicillin-binding protein 3, on Escherichia coli mutants deficient in other penicillin-binding proteins. 总被引:11,自引:6,他引:5
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Furazlocillin binds selectively to penicillin-binding protein 3 (PBP-3), prevents septation of Escherichia coli, and allows the cells to form long filaments without lysis. The effect of furazlocillin on the morphology, autolysis, and murein synthesis of E. coli mutants deficient in either PBP-1A, PBP-1Bs, or PBP-2 was studied. The results reveal that PBP-1A and PBP-1Bs functions are not equivalent since furazlocillin affects the morphology, autolysis, and murein synthesis of PBP1A- mutants quite differently from that of PBP-1Bs mutants. Different "PBP-2-" mutants were found to respond to furazlocillin in dramatically different ways: strain LS-1 cells formed elongated rods with a central bulge which eventually lysed, whereas SP6 cells formed stable "barbells" in which the two daughter cells were well separated but remained connected by a thick central region. 相似文献
4.
Role of precursor translocation in coordination of murein and phospholipid synthesis in Escherichia coli.
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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. 相似文献
5.
Park JT 《Journal of bacteriology》2001,183(13):3842-3847
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. 相似文献
6.
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 总被引:15,自引:12,他引:3
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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. 相似文献
7.
Attachment of lipoprotein to murein (peptidoglycan) of Escherichia coli in the presence and absence of penicillin FL 1060. 总被引:11,自引:9,他引:2
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In vivo studies on the attachment of lipoprotein to the murein (peptidoglycan) of Escherichia coli showed that it takes several generations of growth until the amount of lipoprotein on newly made murein is equilibrated. The technique used involves degradation of the sodium dodecyl sulfate-insoluble murein-lipoprotein complex (sacculus, rigid layer) with lysozyme and separation of the labeled products on paper. No lipoprotein was found on murein subunits incorporated during a pulse of [3H]diaminopimelate for 1 min in logarithmically growing cells at 37 C. Even after one doubling of the cell mass, only 4 to 8% of the labeled murein was isolated as bound to lipoprotein. With uniformly labeled murein, 30% remains bound to lipoprotein after lysozyme treatment, corresponding to three murein subunits. Therefore it can be concluded that during pulse labeling either no lipoprotein is incorporated into the newly synthesized murein or no murein subunits are inserted into existing murein around lipoprotein attachment sites. Longer pulse and pulse-chase experiments argue for the latter interpretation. It is therefore concluded that incorporation of murein subunits into the growing murein polymer is not at all a random process. Instead, quite large areas of murein, on which lipoprotein is situated, seem to be preserved. Under the influence of penicillin FL 1060 murein synthesis is 50% inhibited. The rate of lipoprotein attachment is less affected so that increasing amounts of lipoprotein become attached during spheroplast formation. By the time the stationary growth phase has been reached, the lipoprotein content of the murein has doubled. Diaminopimelate auxotrophic mutants require, in the presence of penicillin FL 1060, more diaminopimelate for full growth than in the absence of penicillin FL 1060. This finding and the fact that murein synthesis is always inhibited by 50% over a wide range of penicillin concentration (1 to 1,000 mug/ml) point to the inhibition of an enzymatic step of murein synthesis which can be partially bypassed by a second enzyme, less efficient but resistant to penicillin FL 1060. 相似文献
8.
Effects of multiple deletions of murein hydrolases on viability,septum cleavage,and sensitivity to large toxic molecules in Escherichia coli
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The multiplicity of murein hydrolases found in most bacteria presents an obstacle to demonstrating the necessity of these potentially autolytic enzymes. Therefore, Escherichia coli mutants with deletions in multiple murein hydrolases, including lytic transglycosylases, amidases, and DD-endopeptidases, were constructed. Even a mutant from which seven different hydrolases were deleted was viable and grew at a normal rate. However, penicillin-induced lysis was retarded. Most of the mutants were affected in septum cleavage, which resulted in the formation of chains of cells. All three enzymes were shown to be capable of splitting the septum. Failure to cleave the septum resulted in an increase in outer membrane permeability, and thus the murein hydrolase mutants did not grow on MacConkey agar plates. In addition, the hydrolase mutants not only could be lysed by lysozyme in the absence of EDTA but also were sensitive to high-molecular-weight antibiotics, such as vancomycin and bacitracin, which are normally ineffective against E. coli. 相似文献
9.
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. 相似文献
10.
Regulation of murein biosynthesis and septum formation in filamentous cells of Escherichia coli PAT 84. 总被引:15,自引:7,他引:8
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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. 相似文献
11.
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. 相似文献
12.
The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes. 相似文献
13.
hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. 总被引:8,自引:4,他引:8
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Except for a small fraction of persisters, 10(-6) to 10(-5), Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis. The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells. Mutants have been isolated in which a larger fraction, 10(-2), persists. The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid). Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h. The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min. Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence. Two of the mutations map at 33.8 min in a region containing few other recognized functions. This position and the phenotypes define hipA as a newly recognized gene. Transposons Tn5 and Tn10 have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon. 相似文献
14.
Bertsche U Breukink E Kast T Vollmer W 《The Journal of biological chemistry》2005,280(45):38096-38101
PBP1B is a major bifunctional murein (peptidoglycan) synthase catalyzing transglycosylation and transpeptidation reactions in Escherichia coli. PBP1B has been shown to form dimers in vivo. The K(D) value for PBP1B dimerization was determined by surface plasmon resonance. The effect of the dimerization of PBP1B on its activities was studied with a newly developed in vitro murein synthesis assay with radioactively labeled lipid II precursor as substrate. Under conditions at which PBP1B dimerizes, the enzyme synthesized murein with long glycan strands (>25 disaccharide units) and with almost 50% of the peptides being part of cross-links. PBP1B was also capable of synthesizing trimeric muropeptide structures. Tri-, tetra-, and pentapeptide compounds could serve as acceptors in the PBP1B-catalyzed transpeptidation reaction. 相似文献
15.
Constitutive septal murein synthesis in Escherichia coli with impaired activity of the morphogenetic proteins RodA and penicillin-binding protein 2.
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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). 相似文献
16.
Christoph Heidrich Markus F. Templin Astrid Ursinus Melisa Merdanovic Jürgen Berger Heinz Schwarz Miguel A. de Pedro Joachim-Volker Höltje 《Molecular microbiology》2001,41(1):167-178
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. 相似文献
17.
Colicin M inhibited the incorporation of DL + meso-2,6-diamino[3,4,5-3H]pimelic acid into the murein (peptidoglycan) of growing cells of Escherichia coli W7 dap lys. The inhibition of the UDP-N-acetylmuramyl pentapeptide-dependent incorporation of UDP-N-acetyl-D-[U-14C]glucosamine into isolated cell envelopes indicated interference with a late step of murein biosynthesis. After the inhibition of murein biosynthesis, cells lysed, and they released lysis products of murein. In vitro, the murein biosynthesis of colicin M-tolerant mutants (tolM) was inhibited by colicin M. Therefore, tolerance is probably conferred by an impaired uptake of an altered fixation close to the target site and not by a mutation of the target itself. Preliminary studies with beta-lactam antibiotics and with mutants in penicillin-binding proteins did not reveal a specific enzymatic step inhibited by colicin M. The unique action among the colicins renders colicin M a potentially useful tool for studying murein biosynthesis. 相似文献
18.
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. 相似文献
19.
Some strains of Escherichia coli defective for dacA, the gene coding for penicillin-binding protein 5, exhibit a strong branching phenotype when cell division is blocked. Since such branch formation implies a differentiation of polar caps at ectopic locations in the cell envelope, we analyzed murein segregation and observed a strong correlation between areas of inert murein and these morphological anomalies. In particular, the tips of branches exhibited the same properties as those described for polar caps of wild-type cells, i.e., the synthesis and turnover of murein were inhibited. Also, the mobility of cell envelope proteins was apparently constrained in areas with morphological defects. Polar regions of branching cells and sacculi had aberrant morphologies with a very high frequency. Of special interest was that areas of inert murein at polar caps were often split by areas of active synthesis, a situation unlike that observed in wild-type cells. These observations suggest that in dacA mutants, branches and other morphological anomalies may arise from split polar caps or by de novo generation of new poles built around inert peptidoglycan patches in the side walls of the cell. 相似文献
20.
Globomycin sensitivity of Escherichia coli and Salmonella typhimurium: effects of mutations affecting structures of murein lipoprotein. 总被引:7,自引:4,他引:3
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J S Lai W M Philbrick S Hayashi M Inukai M Arai Y Hirota H C Wu 《Journal of bacteriology》1981,145(1):657-660
The sensitivity of strains of Escherichia coli and Salmonella typhimurium to globomycin is increased in mutants defective in the lipopolysaccharide structure. E. coli mutants altered in the structures or biosynthesis of murein lipoprotein are more resistant to globomycin than the parental strains. 相似文献