Cell wall ultrastructure of flocculent and non-flocculent Schizosaccharomyces pombe strains. Effect of cell wall hydrolysing enzymes on flocculation and cell wall ultastructure

Scanning and transmission electron microscopic studies revealed the presence of slime-like, amorphous material on the surface of Schizosaccahromyces pombe RIVE 4-2-1 cells, independently, whether they were in flocculated or in non-flocculated state. Close contact of the adjacent cells via the merging outermost cell wall layers was found, however, only in the case of floc formation, which was induced by cultivating the cells in the presence of 6% (v/v) ethanol. Irreversible loss of the flocculation ability of the cells by treatment with proteinases suggests that proteinaceous cell surface molecules as lectins contribute to the cell-to-cell interaction during flocculation. Both proteinase K and pronase treatments removed a distinct outer layer of the cell wall, which indicated that the protein moieties of the phosphogalactomannan outer surface layer has a crucial role in the maintenance of cell wall integrity. In the case of lysing enzyme treatment the removal of the outermost layer was also observed as the first step of the cell wall digestion, while driselase treatment resulted in almost complete digestion of the cell wall.     

Electron Microscopic Studies on the Outer Layers of Staphylococcus aureus Using a Lytic Enzyme from Flavobacterium

The structure of the outer layers (cell wall and membrane) of Staphylococcus aureus was studied by electron microscope using a bacteriolytic enzyme from Flavobacterium sp. called the L-11 enzyme. Comparative studies on the morphology of bacteria before and after treatment with this enzyme and cell wall and membrane fractions obtained from bacteria after the enzyme treatment led to the following conclusions. (1) The cell wall of S. aureus is composed of morphologically distinct two layers which are both susceptible to the L-11 enzyme. (2) Between the cell wall and membrane, there is an electron opaque region which could not be stained using any of the methods tested. (3) Before treatment of bacteria with the enzyme the cell membrane could not be seen clearly. However, after enzyme treatment the membrane was clearly seen. (4) The infolding of the inner layer of the cell wall, forming a structure like a mesosome, was liberated by extensive enzyme treatment.     

gamma-Glutamyltranspeptidase from Proteus mirabilis: localization and activation by phospholipids.

Antiserum was prepared against the purified gamma-glutamyltranspeptidase (EC 2.3.2.2) of Proteus mirabilis. The antiserum inactivated the gamma-glutamyltranspeptidase activities of both purified enzyme and intact cells. Native cells were agglutinated with the antibody. Immunocytochemical studies with indirect immunofluorescence and electron microscopy analysis suggested that gamma-glutamyltranspeptidase is localized on the surface of the cell. Its distribution in the cell wall or periplasmic space or both was also confirmed by the treatment of cells with lysozyme-EDTA. The purified enzyme was activated by the addition of membrane phospholipids isolated from the same bacterium. The hydrolysis activity was stimulated more than the transpeptidation activity by several phospholipids.     

Morphological effect of cerulenin treatment on Streptococcus faecalis as studied by ultrastructure reconstruction.

Exponential-phase cells of Streptococcus faecalis ATCC 9790 were treated with a concentration of cerulenin (5 micrograms/ml) that has been shown to block both lipoteichoic acid and lipid synthesis and cell division within 10 min. The morphological effect of this treatment was studied by making three-dimensional reconstructions of cells based on measurements taken from axial thin sections. This analysis indicated that cerulenin interferes with cell division by inhibiting normal constriction of the division furrow and centripetal growth of the cross wall in envelope growth sites. Rather than dividing, many of the sites in treated cells apparently continue to elongate and produce abnormally large amounts of peripheral wall surface. These observations were interpreted in terms of a previously proposed model in which cerulenin would prevent the synthesis of a lipid-containing inhibitor of autolytic enzyme activity needed for division. In addition, measurements showed that the average number of envelope growth sites per cell increased during treatment, suggesting that although cerulenin treatment blocks division, it does not interfere with the formation of new envelope growth sites. It was also observed that the size and frequency of mesosomes did not decline during the 60-min period of drug treatment. This tends to decrease the likelihood that mesosomes are formed from a pool of intracellular membrane precursors that would be depleted during a period of restricted lipid biosynthesis.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Oxidative quenching of spruce thermomechanical pulp fiber autofluorescence monitored in real time by confocal laser scanning microscopy-implications for lignin autofluorescence

Confocal laser scanning microscopy (CLSM) was used to monitor real-time lignin autofluorescence intensities from different cell wall compartments of spruce fibers during oxidation by laccase-2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS) treatment. CLSM data revealed an instant emission quenching from all cell wall compartments, including those not physically accessible to enzyme or ABTS, followed by an additional decrease simultaneously in all cell wall compartments over a 45 min time course. The importance of site-to-site excitation energy transfer in lignin efficient over long distance is suggested.     

Electron microscopic study of the I, II phases and R-forms of Sh. sonnei]

Electron microscopic study of the microbial cells of the I, II phases and the R-form was carried out. Intact cells were examined by negative contrasting, and morphological differences of various bacterial phases were shown: cells of the I phase had a relatively smooth surface, bacteria of the II phase had a smooth surface, but many cell wall fragments were split from them; the surface of the R-form cells was coarse, folded, and cell wall fragments were split from the majority of bacteria. Antigenic determinants responsible for phasic specificity in bacteria of the I and II phases were located at some distance from the external membrane of the cell wall; as to the R-form cells--they were localized on the wall.     

Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.     

Increased trypsin sensitivity of cell surface macromolecules after malignant transformation.

The effect of treatment with 0.04% (w/v) trypsin (EC 3.4.4.4) for 3 h on the electrophoretic mobility (EPM) of polyoma-virus malignantly transformed BHK21 cells (Py6) and their normal counterparts has been investigated. These particular conditions were chosen because an earlier study had shown that such treatment released material from the Py6 cells which was not obtained from the BHK21 cells. The negative EPM of the Py6 cells at pH 7.5 was greatly increased by this treatment; whereas the EPM of the BHK21 cells remained unchanged. Active enzyme was required to produce the change. No evidence was obtained for cytolysis, cytotoxicity or uptake of the enzyme by the treated Py6 cells. Measurement of the EPM of the Py6 cells at different pH levels before and after trypsin treatment suggested that the enzyme was removing cationic groupings from the cell surface.     

A rapid method for the purification of fatty acid synthetase from the yeast Saccharomyces cerevisiae

A rapid method for the isolation and purification of small quantities of highly active fatty acid synthetase (FAS) from several strains of the yeast Saccharomyces cerevisiae, is presented. The purification procedure which is the shortest reported to this date (18 hr), involves the release of the enzyme by either cell wall digestion with Zymolyase 60000 or cell wall disruption by glass beads, followed by 35-50% ammonium sulfate fractionation, desalting by Sephadex G-25 chromatography, then calcium phosphate gel treatment, concentration by 50% ammonium sulfate precipitation, sedimentation of the enzyme in the ultracentrifuge and finally, column chromatography on DEAE Bio-Gel A. Fatty acid synthetase prepared by the cell breakage method, was found to be homogeneous according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-Tris-glycine disc gel electrophoresis and immunoelectrophoresis criteria. However, enzyme prepared from Zymolyase treated cells showed several proteolytic bands in addition to FAS bands, on SDS-PAGE. Enzyme obtained by both methods of cell breakage, showed a similar behavior throughout the purification procedure and gave a similar yield of enzyme of high specific activity (4800-7200 nmol/min/mg) that remained stable for several months at -85 degrees C.     

In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep-tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus - without radically altering their catalytic activity - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cellwall-spanning region and a standard cell-wall sorting signal.     

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.     

Expression of dimeric and tetrameric acetylcholinesterase isoforms on the surface of cultured bovine adrenal chromaffin cells

Acetylcholinesterase is a highly polymorphic enzyme, which can be anchored to the cell surface through several different mechanisms. Dimeric (G2) acetylcholinesterase isoforms are attached by a glycosylphosphatidyl-inositol (GPI) linkage, whereas tetrameric (G4) forms are linked through a 20 kilodalton hydrophobic subunit. Although cells of haemopoietic origin contain large amounts of G2 GPI-linked acetylcholinesterase, most tissues express only trace amounts of this isoform. We examined the expression of acetylcholinesterase isoforms in cultured bovine adrenal medullary chromaffin cells. Two major isoforms (G2 and G4) were identified on the cell surface. The G2 isoform, which accounted for approximately half the cell-surface enzyme activity, was linked to the membrane through a GPI anchor. After treatment with diisopropylfluorophosphate to completely inhibit cellular acetylcholinesterase, the G4 isoform was found to be resynthesised and transported to the cell surface more rapidly than the G2 isoform. As the addition of GPI anchors is known to be a very rapid step, this finding suggested that the G2 and G4 isoforms might be transported to the cell surface by two different mechanisms. This conclusion was supported by results from subcellular fractionation experiments. The ratio of G4/G2 membrane-bound acetylcholinesterase varied between different subcellular fractions. The membrane-bound G2 isoform was greatly enriched in a high-speed “microsomal” fraction. G4 acetylcholinesterase is known to be actively secreted by chromaffin cells in culture. Although the G4 isoform was present on the cell surface, most of the secreted enzyme was derived from an intracellular pool. Thus, it is unlikely that the cell-surface G4 isoform contributes significantly to the pool of secreted enzyme. Instead, the expression of two different membrane-bound isoforms may provide a means by which chromaffin cells can target the enzyme to different locations on the cell surface. © 1994 Wiley-Liss, Inc.     

Release of autolytic enzyme from Streptococcus, faecium cell walls by treatment with dilute alkali.

The autolytic enzyme (endo-beta-1,4-N-acetylmuramoylhydrolase) of Streptococcus faecium (S. faecalis ATCC 9790) was released in a soluble form from insoluble cell wall-autolytic enzyme complexes by treatment with dilute NaOH at 0 degree C. Treatment of cell wall-enzyme complexes, obtained from either exponential- or stationary-phase cells, with 0.008 to 0.01 N NaOH gave maximum yields of autolytic enzyme activity. At a fixed concentration of NaOH, the yield of autolysin increased with increasing wall densities and was accompanied by the release of methylpentose and phosphorus in amounts proportional to the autolysin. Since extraction of wall-enzyme complexes with 4.5 M LiCl at 0 degree C also removed methylpentose and phosphorus, release of enzyme with NaOH did not appear to result from hydrolysis of covalent linkages. The autolytic enzyme activity released from intact cells, or cell walls, was predominantly in the later (proteinase activable) form which could be activated by trypsin or a proteinase present in commerical bovine plasma albumin.     

A cell surface/plasma membrane antigen of Candida albicans.

Antibody from BALB/cByJ mice immunized against a membranous fraction of Candida albicans agglutinated whole cells as well as the membranous fraction. Hybridoma techniques were used to isolate an IgM monoclonal antibody (mAb) designated 10G which agglutinated whole cells and reacted with the subcellular fraction. Yeast cells of 15 additional C. albicans strains and isolates of C. stellatoidea, C. tropicalis, C. intermedia and C. lusitaniae were also agglutinated by mAb 10G. The antigen was not detected on other fungi, including Candida krusei, C. utilis, Cryptococcus neoformans, Cr. albidus, Torulopsis glabrata, Rhodotorula spp. and Saccharomyces cerevisiae. To determine the cellular location of the epitope to which mAb 10G is specific, freeze-substitution was compared with traditional chemical fixation methods in preparation of samples for immunocolloidal gold electron microscopy (IEM). With both fixation procedures, the antigen recognized by mAb 10G was found randomly and densely concentrated on the plasma membrane on exponential-phase yeast-form cells and had a patchy distribution on the cell wall surface. Association of the antigen with the plasma membrane was confirmed by IEM of isolated membranes. On developing hyphal cells, antigen appeared first on the plasma membrane and later on the cell wall surface. Treatment of yeast cells with beta-mercaptoethanol and Zymolyase before fixation removed the antigen from the surface but left the cytoplasmic antigen undisturbed. Treatment of yeast cells or solubilized antigen with heat or proteolytic enzyme (trypsin, Pronase B, proteinase K) did not remove or destroy the antigen, suggesting a non-protein nature of the epitope.     

Effect of cell surface trypsinization on ethanolamine base exchange enzymatic activity of astrocyte primary cultures and derived spontaneously transformed cell lines

Trypsinization of neonatal rat astrocyte primary cultures (normal cells) inhibited the activity of ethanolamine base exchange enzyme (EBEE) by 80%, whereas ethanolamine phosphotransferase (EPT) and choline base exchange (CBEE) enzymatic activities were not affected; subcellular fractionation demonstrated that trypsin treatment affected the intracellular EBEE activity. During trypsinization the enzyme was not taken up by cultured astrocytes but the cell surface was affected. In contrast, the same treatment did not alter EPT, CBEE and EBEE activities of spontaneously transformed cell lines derived from the primary cultures. However, treatment of the transformed cells with db-cAMP prior to trypsin, restored the pattern found in the primary culture, i.e. only EBEE activity was affected. These data suggest that a relationship exists between cell surface organization and intracellular EBEE activity in a culture system which possesses the property to control its own cell division or/and differentiation.     

Cell wall attachment of a widely distributed peptidoglycan binding domain is hindered by cell wall constituents

The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highly similar repeated regions of 45 amino acid residues (LysM domains), which are separated by nonhomologous sequences. The cA domain could be deleted without destroying the cell wall-hydrolyzing activity of the enzyme in vitro. This AcmA derivative was capable neither of binding to lactococcal cells nor of lysing these cells while separation of the producer cells was incomplete. The cA domain and a chimeric protein consisting of cA fused to the C terminus of MSA2, a malaria parasite surface antigen, bound to lactococcal cells specifically via cA. The fusion protein also bound to many other Gram-positive bacteria. By chemical treatment of purified cell walls of L. lactis and Bacillus subtilis, peptidoglycan was identified as the cell wall component interacting with cA. Immunofluorescence studies showed that binding is on specific locations on the surface of L. lactis, Enterococcus faecalis, Streptococcus thermophilus, B. subtilis, Lactobacillus sake, and Lactobacillus casei cells. Based on these studies, we propose that LysM-type repeats bind to peptidoglycan and that binding is hindered by other cell wall constituents, resulting in localized binding of AcmA. Lipoteichoic acid is a candidate hindering component. For L. lactis SK110, it is shown that lipoteichoic acids are not uniformly distributed over the cell surface and are mainly present at sites where no MSA2cA binding is observed.     

Experimental inhibition of cell wall formation and of reversion inNadsonia elongata andSchizosaccharomyces pombe protoplasts

Summary The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.