Murein chemistry of cell division in Escherichia coli

The length distribution of the glycan strands of murein has been analysed with a novel method in filamentous and spherical cells of Escherichia coli, as well as during septum formation and cell separation. A shift to the longer glycan strands was observed in the murein of furazlocillin-induced filaments. In contrast, shorter glycan strands were increased in the murein of mecillinam-induced spherical cells. During septum formation in a chain-forming envA mutant that is defective in the splitting process of the septum, a shift to the shorter glycan strands was detected that was not seen in wild type E. coli cells. It is concluded that septum-specific murein structures of rather short glycan strands are released during splitting of the septum. This intermediate material remains present in the septum of the envA mutant. The splitting process of the septum was investigated by analysing the murein during penicillin-induced bacteriolysis, which is known to take place by strictly localized murein degradation in the equatorial zone of the cell. No changes in the length distribution of the glycan strands could be detected during penicillin-induced lysis, with the exception of an increase in disaccharides, the shortest glycan strands possible. This is explained by the action of exo-muramidases progressively digesting glycan strands, leaving disaccharide units covalently linked to the remaining murein at the sites of murein cross-linkage. It is proposed that this "zipper-like" mechanism represents the normal cutting process of the septum during cell separation.     

Evidence from a carbohydrate incorporation assay for direct activation of bone marrow myelopoietic precursor cells by bacterial cell wall constitutents.

The stimulation of incorporation of [3H]galactose into membrane glycoconjugates, measured in a precipitation test, was used as a criterion for activation of bone marrow cells. In this assay, purified bacterial lipopolysaccharide, lipoprotein, and murein monomer and dimer fragments all activated rat bone marrow cells in vitro. The response was dose dependent, followed a defined time course, and was not serum dependent. O-Acetylated murein dimer fragments from Proteus mirabilis were much less active than their unsubstituted counterparts, indicating a structural specificity for murein activation. Removal of adherent and phagocytizing cells from the marrow suspensions did not alter these results. The labeled, activated cells constituted a distinct population of buoyant density 1.064 to 1.069 g/cm3 when centrifuged on a continuous gradient of Percoll. Enrichment of the target cell population was achieved by a combination of adherent cell removal and discontinuous density gradient centrifugation to remove granulocytes and erythropoietic cells. It was concluded that a population of myelopoietic precursors could be activated by direct contact with bacterial cell wall constituents. The stimulation of galactose incorporation was not coupled to active deoxyribonucleic acid synthesis in the marrow cells. Thus, the activation was interpreted as an induction of differentiation rather than a mitotic event.     

Layered murein revisited: a fundamentally new concept of bacterial cell wall structure, biogenesis and function

The classical concept of the architecture of microbial murein assumes cross-linked glycan chains to be arranged in horizontal layers outside of the plasma membrane. It necessitates elaborate hypotheses to explain processes such as the biosynthesis, growth and division of the bacterial cell wall and provides no explanation for transenvelope macromolecular transport. Moreover, this model is difficult to reconcile with a number of basic chemical and electron microscopical data. According to a fundamentally distinct concept which is presented here, glycan strands in the microbial wall run perpendicular to the plasma membrane, each strand being cross-linked by peptide bridges with four other strands. This arrangement allows the formation of a structured matrix pierced with ordered ionophoric channels potentially harboring either lipoprotein or teichoic (lipoteichoic) acid molecules in Gram-negative and Gram-positive bacteria, respectively. New wall structures are synthesized in toto emerging from the cytoplasmic membrane as a condensed gel-like network below the old wall without being covalently attached to it, expanding due to inherent elasticity as the old wall is lyzed. This model reflects published genetic and biochemical data and offers a simple explanation for peptidoglycan biogenesis. As the biosynthesis is terminated by enzymic cleavage of all glycan strands, murein is irreversibly released from the membrane. The murein detachment prepares the membrane for de novo assembly of both the new wall synthesis machinery and the multicomponent factory for protein, DNA and phospholipid transfer. Being assembled in parallel, both new murein and the traffic complexes grow from the membrane together. This concept eliminates the necessity for the traffic complexes to penetrate intact murein. In the process of simultaneous assembly, the expanding murein functions as a lifting platform driven by the force of turgor pressure, transporting macromolecules through the perisplasmic space. Received: 5 October 1998     

Effect of mineral acids on iron bacteria

The oxidative ability of Thiobacillus ferrooxidans in the presence of different concentrations of H₂SO₄, HCl, HNO₃, and their mixtures was investigated. Bacteriological oxidation of FeSO₄ is expressed as a function of time and acid concentration. For each acid and for their mixtures lethal concentrations for T. ferrooxidans were found experimentally. On the basis of the lethal concentration for each acid separately the lethal concentrations of their mixtures were computed and it was found that they are in good agreement with the experimental data.     

Human B-cell differentiation induced by murein

Like "true" polyclonal B-cell activators (PBA) for murine B cells, crude membrane preparations of Klebsiella pneumoniae (Klebs M) and some other enterobacteriaceae stimulate human B cells to mature into immunoglobulin (Ig) secreting cells without significant prior proliferation and in the absence of T cells. To investigate the biochemically defined membrane component with this unique PBA property, we studied lipoprotein and murein isolated from E. coli, since other components (e.g., a variety of lipopolysaccharide (LPS) fragments) failed to imitate Klebs M as a PBA. Mononuclear cells (MNC) and B cell-enriched cell populations from healthy blood donors were stimulated with various doses of lipoprotein and murein and, in comparison, Klebs M and pokeweed mitogen (PWM). Cell cultures exposed to either lipoprotein, murein, or Klebs M failed to incorporate [3H]thymidine significantly after 5 days in culture. In contrast, there was significant DNA synthesis (stimulation index greater than 3) when PWM was given to the same MNC population. All stimulants, with the exception of lipoprotein, induced B-cell differentiation in MNC cultures, as measured by an ELISA quantitating secreted Ig in the culture supernatants. In cultures with B cell-enriched cell populations, however, only Klebs M and murein were able to induce the production of significant amounts of IgM. Thus, the actual PBA moiety contained in the crude membrane fraction (Klebs M) appears to be associated with murein. It is important to note that murein induced considerably weaker Ig secretion than Klebs M did.     

Studies on the deoxyribonucleic acid-bearing portion of the cell envelope of Escherichia coli

The envelope components of nuclear bodies which were obtained from Escherichia coli W7 by a mild lysis method were investigated. By using 2,6-diaminopimelic acid (DAP) as precursor which is incorporated only into peptidoglycan in this strain it was found that the particles contained about 14% of the murein layer of the cell. The percentage of phosphatidylethanolamine was enriched at the cost of the other phospholipids in the nuclear bodies compared to whole cells. If lipids were labelled with ³H-palmitic acid the cytoplasmic and the outer membrane could be found after isopycnic centrifugation; however, when the cells were incubated with chloramphenicol, only the outer membrane was seen. The peptidoglycan and the proteins could be assigned only to the outer membrane. The DNA is also bound to the outer membrane. From these results it was concluded that (1) in all lysis methods the cytoplasmic membrane is more easily dissolved than the outer layers of the envelope, and (2) that there is a firm binding between DNA and the outer membrane in vivo.     

Modulation of Myelopoiesis In Vivo by Chemically Pure Preparations of Cell Wall Components from Gram-Negative Bacteria: Effects at Different Stages

Modulation of myelopoiesis by chemically pure preparations of different cell wall components from gram-negative bacteria was investigated in vivo. The effects of lipid A, outer membrane lipoprotein, and murein were evaluated at several distinct stages: induction of colony-stimulating activity (CSA) in the serum, increase in the number of committed splenic precursor cells (CFU-C) forming granulocyte-macrophage colonies in vitro, and triggering into the cell cycle of noncommitted hemopoietic stem cells (CFU-S) from bone marrow. The results reveal different patterns of activity of the bacterial cell wall components (BCWC) tested. (i) In C57Bl/6 mice and C3H/Bom mice, all three preparations were potent inducers of CSA. In C3H/HeJ mice, CSA was only induced by lipoprotein and murein and not by lipid A. After injection of lipid A or lipoprotein, but not murein, the number of CFU-C in spleens of C57Bl/6 mice was increased up to 100-fold. In C3H/Bom and C3H/HeJ mice, not only murein but also lipoprotein were much less potent in this respect. (iii) In C57Bl/6 mice, both lipid A and lipoprotein, but not murein, were capable of inducing the proliferation of CFU-S, as demonstrated by a hot thymidine cytocide technique. Thus, induction of CSA and changes in the pool size of splenic CFU-C after administration of BCWC may be unrelated events. On the other hand, the increase of CFU-C might reflect the mitogenicity of BCWC for CFU-S.     

Biological roles of two new murein hydrolases of Streptococcus pneumoniae representing examples of module shuffling

We have found two murein hydrolases (LytB and LytC) tightly bound to the cell envelope that have completely changed the domain building plan previously reported for the murein hydrolases of Streptococcus pneumoniae. The active center of LytB and LytC is located at the C-terminal, whereas the binding domain is at the N-terminal. LytC has been characterized as the first lysozyme of S. pneumoniae and behaves as an autolysin at 30 degrees C. LytB appears as the main hydrolase responsible for cell separation since inactivation of lytB leads to the formation of long chains of more than 100 cells. These findings indicate that genetic adaptation of mobile domains is extremely efficient in pneumococcus.     

An alteration in outer membrane permeability associated with a division lesion in a strain of Salmonella typhimurium

Salmonella typhimurium strain 4a is a temperature sensitive mutant with defects in both septation and separation. The separation lesion was reversed by phenethylalcohol but this agent failed to allow septation or growth at restrictive temperature. Organisms of strain 4a grown at 42 °C were, unlike the parental strain, resistant to lysis by lysozyme plus EDTA and lipopolysaccharide was poorly extracted by EDTA from cultures of strain 4a grown at 42 °C. Such cultures may, therefore, be resistant to lysis with lysozyme plus EDTA not because the murein is altered but because the EDTA fails to permeabilize the outer membrane to lysozyme. In confirmation of this, murein isolated from strain 4a after growth at 42 °C showed the same sensitivity to lysozyme as murein from the parental strain. In spite of the altered envelope properties of strain 4a after growth at 42 °C, no major changes in protein or phospholipid composition have so far been demonstrated.     

Permeability of mycobacterial cell envelopes to sterols: Peptidoglycan as the diffusion barrier

Vancomycin, an inhibitor of peptidoglycan synthesis, depressed the growth of Mycobacterium sp. NRRL B 3805 and MB 3683, but not the β-sitosterol side chain degradation and androstane derivatives accumulation. As a result, the specific activity (products formed/cell weight unit × h) increased threefold in the peptidoglycan-deficient cells, indicating faster crossing of the sterol through the cell water barrier. Cell wall preparations: crude cell wall (CCW), purified cell wall (PCW), and peptidoglycan — enriched PCW — residue (PEPCW) were obtained and analysed in order to find a relationship between the vancomycin — induced chemical changes and the permeation rate of the sterol. The amounts of CCW, PCW and PEPCW, produced from 8 g lyophilised control cells were 445, 170 and 28 mg respectively. The respective figures were 176, 61, and 4.8 mg for vancomycin — treated cells. In addition to the lower content of the rigid layer, a distinct shift in the molar ratios of the peptidoglycan constituents: alanine, glutamic, diaminopimelic and muramic acids, and glucosamine was observed under the action of the murein inhibitor. The most significant change was that of muramic acid: diaminopimelic acid molar ratio, the compounds which are markers of glycan strands and tetrapeptides, respectively. In control cells it was approximately 1:1, and increased to 1.34–1.43: 1 in the compared preparation, which indicated a marked decrease in the tetrapeptide moieties crosslinking the main glycan strands. Together with the general lower content of murein, this modification may be responsible for the enhanced sterol permeation through the cell wall.     

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.     

Growth and cell division of Escherichia coli 173-25 in the presence of some analogues of diaminopimelic acid

The dap-dependent mutant of Escherichia coli could grow in the presence of several analogues of diaminopimelic acid (dap). The multiplication proceeded without any apparent difference for several hours when dap was replaced with 2,6-diamino-4-fluoro-pimelic acid or lanthionine. Only growth without accompanying division could be detected in the presence of 2,6-diamino-4-hydroxypimelic acid (hydroxy-dap). ³H-hydroxy-dap was incorporated into the TCA precipitable material under these conditions. 2,4,6-triaminopimelic acid, probably also 2,6-diamino-5-oxo-pimelic acid could not substitute dap in the synthesis of murein. The synthesis of DNA measured by the incorporation of ³H-thymidine was not inhibited by hydroxy-dap but the formation of the cross wall was impaired. The rods were longer, formed bulges in the central part of the cell and ultimately lyzed. Selenium-lanththionine was more toxic but even in its presence some growth occurred and DNA synthesis took place. The replacement of dap with hydroxy-dap stimulated the degradation of the wall mucopeptide during the growth.     

Functional and structural analysis of the major amidase (Atl) in Staphylococcus

The cytoplasmic membrane of most bacteria is surrounded by a more or less thick murein layer (peptidoglycan) that protects the protoplast from mechanical damage, osmotic rupture and lysis. When bacteria are dividing processes are initiated stepwise that involve DNA replication, constriction of the membranes, cell growth, biosynthesis of new murein, and finally the generation of two daughter cells. As the daughter cells are still covalently interlinked by the murein network they must be separated by specific peptidoglycan hydrolases, also referred to as autolysins. In staphylococci, the major autolysin (Atl) and its processed products N-acetylmuramoyl-l-alanine amidase (AM) and endo-β-N-acetylglucosaminidase (GL) have been in the research focus for long time. This review addresses phenotypic consequences of atl mutants, impact of Atl in virulence, the mechanism of targeting to the septum region, regulation of atl, the structure of the amidase and the repeat regions, as well as the phylogeny of Atl and its use in Staphylococcus genus and species typing.     

Geomicrobiological leaching of the minerals by Thiobacillus ferrooxidans and organic agents

Laboratory investigations confirm that it is possible to leach tin from synthetic minerals like stannite, kesterite, stannoidite, herzenbergite, ottemannite and berndtite, and from natural tin minerals which include stannite, cassiterite and varlamoffite, in the presence of Thiobacillus ferrooxidans, and with organic agents of biological origin, especially oxalic acid and oxalic-citric acids mixture. Over a leaching period of 35 days with 0.5% pulp density, initial pH of 2.5, using minus 0.16 mm size fraction at 32°C, as much as 54.45, 72.66, 97.13, and 31.30% Sn were extracted from synthetic stannite, kesterite, stannoidite and natural stannite, respectively. Varlamoffite, found in the dried, leached residues of the tin sulphides, provides evidence that bacterial action can be responsible for the genesis of supergene varlamoffite.     

Current status of age altered enzymes: alternative mechanisms.

The occurrence of inactive enzyme molecules in a variety of tissues and animal species has been shown to be of a general nature. The levels of inactive enzyme molecules found in old animals were produced by amino acid analogs in young animals. These levels have been shown to be initially detrimental but subsequently the young system shows recovery by efficiently disposing of the analog-modified proteins. In old animals this disposal is considerably less efficient. Evidence is presented which suggests that post-translational modifications of proteins are the main cause of enzyme inactivation in old animals. Amino acid substitutions and modifications involving charge differences apparently do not contribute significantly to this phenomenon.     

Characterization of Extracellular and Nascent Dentin Phosphoproteins

Experiments were designed to compare extracellular dentin phosphoprotein (DPP) and nascent DPP prior to post-translational modifications from several vertebrate species. Dental matrix proteins were extracted with acetic acid, followed by GuHCl-EDTA, and precipitated with CaCl₂. Cross-reactivity of the DPPs with a mouse DPP antibody was determined by a dot-immunobinding assay. To analyze nascent DPP, mRNA was isolated from developing tooth organs and the mRNA-directed translation products were immunoprecipitated with the DPP specific antibody. All DPP components identified in the species which contained a DPP were shown to cross-react with the polyclonal mouse DPP antibody. The extracellular matrix DPPs were found to exhibit as much as a 30 kDa size difference using the same SDS PAGE system. In contrast, nascent DPPs were found to be the same size for all species examined. Our results indicate that differences in the molecular weight size of DPPs between species may be due to the degree of post-translational modifications such as phosphorylation.     

GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence

GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from Escherichia coli and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a Neisseria meningitidis serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence.     

Bcl-2, Bax and Bcl-x expression following kainic acid administration at convulsant doses in the rat.

Neuronal death was produced in the CA1 and CA3 areas of the hippocampus, amygdala, and piriform and entorhinal cortices after intraperitioneal administration of kainic acid at convulsant doses to adult rats. To assess the involvement of members of the Bcl-2 family in cell death or survival, immunohistochemistry, western and northern blotting to Bcl-2, Bcl-x and Bax, and in situ hybridization to Bax were examined at different time-points after kainic acid treatment. Members of the Bcl-2 family were expressed in the cytoplasm of pyramidal neurons in the hippocampus, and in a subset of neurons of the piriform and the entorhinal cortices, amygdala and neocortex in the normal adult brain. Dying neurons in the pyramidal cell layer of CA1 and CA3 areas, entorhinal and piriform cortices, and amygdala also expressed Bcl-2, Bax and Bcl-x following excitotoxicity, although many dying cells did not. In addition, a number of cells in the affected areas showed Bax immunoreactivity in their nuclei at 24-48 h following kainic acid administration, thus indicating Bax nuclear translocation in a subset of dying cells. Western blots disclosed no modifications in the intensity of the bands corresponding to Bcl-2, Bcl-x and Bax, between control and kainic acid-treated rats. No modifications in the intensity of the bcl-2 messenger RNA band on northern blots was observed in kainic acid-treated rats. However, a progressive increase in the intensity of the bax messenger RNA band was found in kainic acid-treated rats at 6 h, 12 h and 24 h following kainic acid administration. Interestingly, a slight increase in Bax immunoreactivity was observed in the cytoplasm of neurons of the dentate gyrus at 24-48 h, a feature which matches the increase of bax messenger RNA in the same area, as shown by in situ hybridization at 12-24 h following kainic acid injection. The present results suggest that cell death or survival does not correlate with modifications of Bcl-2, Bax and Bcl-x protein, and messenger RNA expression, but rather that kainic acid excitotoxicity is associated with Bax translocation to the nucleus in a subset of dying cells.     

Metabolism of oxidized and chemically modified low density lipoproteins in rainbow trout--clearance via scavenger receptors

Oxidative modifications of low density lipoprotein (LDL) convert LDL into a ligand recognized by a variety of scavenger receptors (SR) in mammals. This oxidized LDL (oxLDL) activate several cell types, and have been shown to induce expression of a variety of genes in mammals. Lipoproteins of poikilothermic animals like salmonid fishes contain high levels of polyunsaturated fatty acids susceptible to oxidative modifications. We have investigated, and found trout LDL to be susceptible to oxidation in the presence of Cu(2+). When oxidized or acetylated trout LDL was injected intravenously, the clearance rate was increased compared to that of native LDL. Modified LDL was taken up almost exclusively in the kidney, whereas native LDL was also taken up in the liver. Uptake of both oxLDL and acetylated LDL in the kidney was significantly inhibited by lipoteichoic acid (LTA) and formaldehyde treated BSA (fBSA), both of which are known ligands of SR.     

Altered immunogenicity of isoaspartate containing proteins

The development of immune tolerance is dependent on the expression of self-peptides in the thymus and bone marrow during lymphocyte development. However, not all self-antigens are expressed in the thymus, particularly for proteins that become post-translationally modified during other biological processes in a cell. We have found that one such post-translational modification, the spontaneous conversion of an aspartic acid to isoaspartic acid (isoAsp), causes ignored self-antigens to become immunogenic. In order to determine the mechanism for this autoimmune response, pigeon cytochrome c peptide 88-104 (PCC p88-104) was synthesized with and without an isoaspartyl residue. Each form was digested with cathepsin D, an enzyme involved in antigen processing. The products of cathepsin digestion were dramatically different between the two forms of self-protein suggesting that cryptic self-peptides may be revealed to the immune system by natural modifications to self-proteins. This observation also held true if whole PCC protein contained isoaspartyl residues was digested with cathespsin D. Additionally, AND transgenic TCR T cells (recognizing PCC 88-104) proliferated to a greater extent in response to isoaspartyl PCC as compared to the normal form of PCC. These finding demonstrate the importance of post-translational modifications in shaping autoimmune responses in and the development of tolerance to self-proteins.