Murein Layer Research Articles

A physical basis for the precise location of the division site of rod-shaped bacteria: the Central Stress Model

There are difficulties with all published models that attempt to explain how rod-shaped bacteria locate their midpoint in preparation for the next cell division. Many bacteria find their middle quite accurately. This evenness of partition has been measured cytologically and is implied by the persistence of synchrony under certain circumstances. Previously, a number of models for the control of cell division have been proposed based on aspects of molecular genetics, ultrastructure measurements, or biochemical kinetics. This paper points out that none of the models in spite of their quite different natures can explain the precision of the location of the division site. Here, the ‘Central Stress Model’ is proposed which depends on the partition of wall tension between the cytoplasmic membrane (CM) and the murein layer in such a way that the CM at the centre of the rod experiences a higher stress than near the poles and that this peak stress increases through the cell cycle. The model assumes that: (i) murein is not incorporated at an established pole but is incorporated diffusely over the sidewall and intensely at sites of cell constriction; (ii) CM is synthesized over the entire cell wall; (iii) the murein and CM layers are attached non-covalently to each other, and interact physically with each other; (iv) this differential location of synthesis leads to a ‘tug-of-war’ that creates differential stresses that peak at the cell centre. Because of the fluid nature of the phospholipid bilayer there is a flux of lipid from the established poles towards the cell centre as the murein sidewall elongates. The flux from the pole lowers the tension in the CM at the ends of the sidewalls and creates a peak tension in the centre. Cells also have a discontinuity in the stresses in the murein at the junction of the curved pole with the cylindrical region of the cell wall (a doubling of the hoop stress above the axial stress). Thus in addition to the midpoint of the cell, these junctions between the polar caps and the cylindrical part of the cell wall are characterized by an abrupt change in the surface stress and we suggest that this can trigger cell division at these junctions to form a chromosome-less minicell. Two other assumptions of the model are that the cell has a membrane-associated system to sense the stress, and to trigger cell division locally when a threshold has been reached. It is suggested that there is a special two-component sensory system responding to tension in the CM. As the cell cycle progresses, and the stress in the centre of the cell exceeds some threshold, a system molecule triggers a cell division event at that site. Like other two-component systems, the sensory component of this two-component system is assumed to be distributed all over the CM. It is also assumed that when the sensory component is triggered it also causes local changes that ensure that division occurs at that site. Consequently, this model can explain why sister cells have very nearly the same size (length, volume, or biomass) and why genes that control a mechanism that senses cell size and initiates cell division have never been identified because they may not exist.

The Metabolic Inertness of the Pole Wall of a Gram-negative Rod

During the growth cycle of Escherichia coli, elongation takes place by insertion of new murein into the thin cylindrical side wall. Where cell division is to take place, the cell inserts new wall at a high rate. However, it is concluded here that after separation of sisters incorporation of new murein into the mature poles wall is negligibly small. By reanalysis of published high-resolution electron microscope autoradiographic studies of the pulse incorporation of tritium-labeled diaminipimelic acid, earlier interpretations are shown to be incorrect. Reanalysis of these studies is possible using a computer program that allowed the data, as published, to be corrected in two ways: (i) for the distribution of silver grains around the site of a tritium decay; and (ii) for the effects of the normalization of cell lengths which had been used in presenting the primary data. In addition, we have reanalyzed published work on LamB and lipoprotein incorporation and conclude that incorporation of LamB and the lipoprotein, like the murein layer, into the outer membrane of the completed pole is essentially nil. Thus, in contrast to our earlier papers, it is concluded that three components of the envelopes of the established poles are quite inert and therefore the poles could serve as a stable support for cylindrical side-wall elongation, consistent with the key assumption of the surface stress theory. The possibility of continued insertion of new cytoplasmic membrane in an established pole is not covered by the available experimental data. A second conclusion is that incorporation of murein may occur during the development of the constriction site of the nascent pole over a much larger area than just the tip of the constriction.

Lysis protein T of bacteriophage T4.

Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage lambda is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of lambda (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18,000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.

Bacteriophage lysis: mechanism and regulation.

Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.

Bacteriophage lysis: mechanism and regulation.

Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.

Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein.

To target recombinant antibodies to the surface of Escherichia coli, we have fused single-chain variable domains to its peptidoglycan associated lipoprotein (PAL). The fusion protein was able to bind antigen and was tightly bound to the murein layer of the cell envelope. Antibody-PAL had little effect on cell growth and viability. In contrast, the expression of single chain antibody alone eventually resulted in cell lysis. Immunofluorescence studies on unfixed cells showed that functional antibodies were accessible at the surface of intact bacteria. This could provide a means of isolating single cells producing specific antibodies from libraries in E. coli by fluorescence assisted cell sorting (FACS). Pal fusions may also be of general interest for the presentation of proteins at the surface of E. coli as, for example, in the production of live vaccines.

Unusual septum formation in Streptococcus pneumoniae mutants with an alteration in the D,D-carboxypeptidase penicillin-binding protein 3

An internal 630-bp DNA fragment of the gene encoding penicillin-binding protein 3 (PBP 3) (dacA) of Streptococcus pneumoniae was identified in a lambda gt11 gene bank screened with anti-PBP 3 antiserum. The deduced 210-amino-acid sequence showed a high degree of homology to the low-molecular-weight PBPs 5 and 6 of Escherichia coli and Bacillus subtilis PBP 5. Viable mutants lacking a C-terminal part of PBP 3 were obtained after a plasmid containing the dacA fragment was integrated into the PBP 3 gene by homologous recombination. The truncated PBP 3* was still active in terms of beta-lactam binding. Most PBP 3 was found in the growth medium, indicating that membrane anchoring of PBP 3 is provided by the C terminus, as has been shown for other D,D-carboxypeptidases. The mutant cells grew with a slower generation time than the wild type in the shape of irregular enlarged spheres. In addition, as revealed by electron microscopy, cell separation was severely affected, septa were found unevenly distributed at multiple sites within the cells, and the murein layer appeared variable in thickness.

Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane.

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.

Evidence for a net-like organization of lipopolysaccharide particles in theEscherichia coliouter membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain ASI9 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.

Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells.

Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating [3H]leucine and [3H]palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate). Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol. Nine murein-associated [3H]leucine-labeled peptides were identified. Five of the nine peptides were lipoproteins, based on labeling with [3H]palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations. The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope.

Purification and peptidase activity of a bacteriolytic extracellular enzyme from Pseudomonas aeruginosa

A bacteriolytic enzyme excreted by Pseudomonas aeruginosa Paks I was purified: samples were found to be homogeneous by gel filtration chromatography, ion exchange chromatography using CM-cellulose, immunoelectrophoresis, PAGE and SDS-PAGE. The molecular weight of the lytic enzyme was estimated to be 15,000–19,000. The enzyme was active on Gram-positive bacteria with glycine-containing interpeptide bridges in their murein layers. In addition, this lytic enzyme showed peptidase activity catalysing the hydrolysis of pentaglycine peptides into tri- and diglycine peptides.

Ultrastructure of the cell envelope and amino acid composition of the murein ofClostridium symbiosum

The cell envelope of the Gram-negative staining Clostridium symbiosum is 18 nm thick. It appears triple-layered and consists of an inner electrondense layer of about 5 nm, a lighter zone of 4 nm and an outer electron-dense layer of 9 nm. The inner layer corresponds to the murein sacculus, since the isolated peptidoglycan sacculi showed a thickness of 3–5 nm. Analysis showed that it belongs to the A2pm-direct murein type. The outer layer could be removed by sodium dodecylsulfate. It contained mainly protein, small amounts of sugars and essentially no lipid, indicative of an S-layer rather than a typical Gram-negative type of outer membrane. Furthermore, l-alanine aminopeptidase activity characteristic of Gram-negative aerobic bacteria was absent in this organism and in other anaerobic Gram-negative bacteria tested. This demonstrates that such activity is an unreliable tool for the classification of anaerobic eubacteria. In spite of the thin murein layer, which is the likely reason for the Gram-negative reaction, the anaerobic growth, peritrichous flagellation and endospore formation indicate that this organism belongs to the genus Clostridium.

Ultrastructure of the cell envelope and amino acid composition of the murein of Clostridium symbiosum

The cell envelope of the Gram-negative staining Clostridium symbiosum is 18 nm thick. It appears triple-layered and consists of an inner electrondense layer of about 5 nm, a lighter zone of 4 nm and an outer electron-dense layer of 9 nm. The inner layer corresponds to the murein sacculus, since the isolated peptidoglycan sacculi showed a thickness of 3–5 nm. Analysis showed that it belongs to the A2pm-direct murein type. The outer layer could be removed by sodium dodecylsulfate. It contained mainly protein, small amounts of sugars and essentially no lipid, indicative of an S-layer rather than a typical Gram-negative type of outer membrane. Furthermore, l-alanine aminopeptidase activity characteristic of Gram-negative aerobic bacteria was absent in this organism and in other anaerobic Gram-negative bacteria tested. This demonstrates that such activity is an unreliable tool for the classification of anaerobic eubacteria. In spite of the thin murein layer, which is the likely reason for the Gram-negative reaction, the anaerobic growth, peritrichous flagellation and endospore formation indicate that this organism belongs to the genus Clostridium.

Antigen Release from <i>Thermoactinomyces vulgaris</i> by Lysozyme Treatment

Lysozyme treatment was used to release antigenic material from Thermoactinomyces vulgaris, one of the microbes associated with farmer's lung. Lysozyme caused degradation of the murein layer visualized as changes and disappearance of the bacterial morphology in scanning electron microscopy. Enrichment of different antigenic components in the lysozyme extract and in the cell residue, respectively, was detected by immunoprecipitation. When lysozyme extract and cell residue antigens were used in enzyme-linked immunosorbent assay to test sera of farmer's lung patients and control persons, it became evident that there was no significant difference between the reactions against the two antigens. However, a number of sera reacted preferentially against one or the other of the two antigens.

Spherical E. coli due to elevated levels of D-alanine carboxypeptidase.

The characteristic shape of bacterial cells is maintained by the strength and form of the murein layer of the cell wall1. Thus, rod-shaped bacteria need to synthesize a rod-shaped murein. The shape of the murein may be determined by the properties of the penicillin-binding proteins (PBPs) that catalyse the insertion of murein precursors into the cell wall2. In Escherichia coli inhibition of PBPs produces characteristic effects on bacterial morphology. For example, inactivation of PBP 2 results in an inability to synthesize cylindrical murein during cell elongation and the growth of E. coli as spherical cells2. We report here that osmotically stable spherical cells of E. coli can also be produced by an increase in the level of PBP 5, a D-alanine carboxypeptidase3, and show that these cells, and those produced by inactivation of PBP 2, have the same abnormality in the structure of the newly inserted murein.

Nature of the regions involved in the insertion of newly synthesized protein into the outer membrane of Escherichia coli

Outer membrane proteins are synthesized by cytoplasmic membrane-bound polysomes, and inserted at insertion sites which cover about 10% of the total outer membrane when cells grow with a generation time of 1 h. A membrane fraction enriched in outer membrane insertion regions was isolated and partly characterized. The rate at which newly inserted proteins are transferred from such insertion regions into the rest of the outer membrane was found to be very fast; the new protein content of insertion regions and that of the remaining outer membrane equilibrate completely within about 20 s at 25°C. Given the rather rigid structure of the outer membrane and the multiple interactions between outer membane components and the murein layer, lateral diffusion of newly inserted proteins from insertion sites to the remaining outer membrane is not likely to explain this rapid equilibration. Instead, the data support a model in which mobile insertion regions move along the cell surface, leaving behind stationary, newly inserted outer membrane proteins.

Surface macromolecules and virulence in intracellular parasitism: comparison of cell envelope components of smooth and rough strains of Brucella abortus.

The surface topography of whole cells and the chemical composition of cell envelopes of a smooth-intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus was examined. Electron microscopy of whole cells and thin sections did not reveal any gross surface difference(s). Only minor quantitative differences were observed in total lipids, proteins, and the murein layer. However, the lipopolysaccharide composition of the two strains was quite different. Both phenol- and water-soluble lipopolysaccharide fractions were obtained from the strain of higher virulence (45/0), whereas only aqueous lipopolysaccharide could be isolated from the rough strain. In addition to being toxic, the phenol-soluble lipopolysaccharide may be a key virulence factor in intracellular survival of B. obortus within phagocytic cells.

Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.

The effect of osmotic shock on the accessibility of the murein layer of exponentially growing Escherichia coli to lysozyme

The restricted access of lysozyme to the murein layer of exponential phase Escherichia coli is enhanced considerably by osmotic shock. When cells suspended in Tris/EDTA/sucrose are diluted 11-fold in water or 10 mM EDTA in the presence of lysozyme, their susceptibility to lysozyme increases by a factor of 50–100, for both Escherichia coli JC411 and W3110, grown to the early exponential phase in unsupplemented or supplemented minimal media, and in Brain Heart Infusion. Since an 11-fold dilution causes lysis of lysozyme spheroplasts, the effects of a 2-fold dilution have also been investigated. A 2-fold dilution of cells suspended in Tris/EDTA/sucrose still increases their susceptibility to lysozyme by a factor of 10–50, but the resulting spheroplasts remain intact. EDTA is necessary to permit lysozyme access to the murein layer during the dilution, which is ineffective in the presence of 5 mM MgCl 2. These results are discussed in terms of the formation of lysozyme spheroplasts from young Escherichia coli.