Morpho-functional analysis of isogenic strains of Escherichia coli-- producers of penicillinacylases

A population of Escherichia coli strains producing penicillinacylase (PA) and differing in the level of their enzyme activity was studied. Their structural-functional analysis showed that selection according to the property of PA production yielded strains with aberrations in the processes of cell division. The population of microbial cells producing PA had a correlation between its morphological composition and enzyme activity, and the two characteristics depended on the conditions under which the strains were cultivated. The highest enzyme activity was exerted by normally dividing cells. A type of colonies optimal for stabilising the level of PA production was determined for the enzyme-producing strain. The structure of cell envelopes, their composition and permeability changed considerably as the ability to synthesize PA increased. The results allow one to specify further rational selection of strains superproducing the enzyme on the basis of changes in the membrane permeability.     

Penicillinamidohydrolase inEscherichia coli

Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) fromEscherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. SomeN-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial Tates of the enzyme hydrolysis of different substrates were found when using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space.N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.     

Mode of Cell Wall Growth of Bacillus megaterium

The growth of the cell wall of Bacillus megaterium KM was examined by labeling the cell wall uniformly with (3)H-alpha, epsilon-diaminopimelic acid and examining by radioautography the distribution of labeled cell wall material in daughter cells. The results indicate that the label is distributed uniformly to all daughter cells and rule out a single growth point, either central or apical, for these cells. The results strongly suggest that cell wall growth occurs by uniform deposition of new wall over the whole cell surface.     

Inhibitory activity of 1-farnesylpyridinium on the spatial control over the assembly of cell wall polysaccharides in Schizosaccharomyces pombe

The modes of actions of 1-farnesylpyridinium (FPy) on yeast cell growth were investigated on the basis of its effects on cell cycle progression, morphogenesis and the related events for construction of cell wall architecture in Schizosacchromyces pombe. FPy predominantly inhibited the growth of the yeast cells after various cycles of cell division so that cells were arrested at the phase of separation into daughter cells accompanying morphological changes to swollen spherical cells at 24 h of incubation. FPy-treated cells were osmotically stable but were susceptible to the lytic action of (1, 3) beta-D-glucanases, and characterized by serious damages to the cell wall architecture as represented by a rough and irregular surface outlook. The isolated cell wall fraction gave a similar hexose composition with or without FPy treatment, suggesting that FPy did not inhibit the synthesis of each cell wall polysaccharide. FPy was permissive for the extracellular accumulation of amorphous cell wall materials and septum development in protoplasts, but absolutely interfered with the following morphogenetic process for construction of the rod-shaped cell wall architecture. Our results suggest the inhibitory activity of FPy on the spatial control over the assembly of cell wall polysaccharides.     

Determination of the penicillin acylase activity of E. coli cultures using a method of gas-liquid chromatography]

Penicillinacylase activity was determined in E. coli by the product of benzylpenicillin destruction, i.e. phenylacetic acid formed under the effect of the enzyme. The determination was performed on a chromatograph. The immobile phase consisted of 10 per cent of ethylenglycol edipate on chromosorb A, modified with 2 per cent H3PO4. Nitrogen was used as the gaseous carrier. The method is rapid and handy for mass testing of the cultures with a purpose of detecting penicillinacylase-producing strains. It provided reliable determination of penicillinacylase in the cultures producing simultaneously beta-lactamase, another penicillin-destroying enzyme.     

Effects of reduction of auxin and destruction of microtubules on cell wall proteins and cell morphology of the BY-2 line of tobacco cells

Variations of cell wall proteins and proteins in the medium associated with changes in cell morphology were investigated in the BY-2 line of cultured cells. BY-2 cells cultured in LS medium grew as long chains of cells, with the plane of division perpendicular to the longitudinal axis. Reduction in the levels of auxin in the medium resulted in inhibition of cell division and promotion of cell elongation. Levels of cell wall proteins in cell walls decreased and relative levels of cell wall proteins and proteins in the medium changed. Upon treatment with the anti-microtubule drug, propyzamide, cells expanded laterally. Level of cell wall proteins and relative levels of individual cell wall proteins did not change very much, but levels of proteins in the culture medium increased. In both cases, levels of acid and basic peroxidases in cell walls increased and isozyme patterns of these changed.     

Structure of Embryo Sac Before and After Fertilization and Distribution of Transfer Cells in Ovules of Green Gram

The structure of embryo sac before and after fertilization, embryo and endosperm development and transfer cell distribution in Phaseolus radiatus were investigated using light and transmission electron microscopy. The synergids with distinct filiform apparatus have a chalazal vacuole, numerous mitochondria and ribosomes. A cell wall exists only around the micropylar half of the synergids. The egg cell has a chalazally located nucleus, a large micropylar vacuole and several small vacuoles. Mitochondria and plasrids with starch grains are abundant. No cell wall is present at its chalazal end. There are no plasma membranes between the egg and central cell in several places. The zygote has a complete cell wall, abundant mitochondria and plastids containing starch grains. Both degenerated and persistent synergids migh.t serve as a nutrient supplement to proembryo. The wall ingrowths occur in the central cell, basal cell, inner integumentary cells, suspensor cells and endosperm cells. These transfer cells may contribute to embryo nutrition at different developmental stages of embryo.     

Modification of protoplast cell wall regeneration by membrane perturbation

Summary Protoplasts derived from cells ofBoergesenia forbesii regenerated aberrant cell walls when treated with cholesteryl hemisuccinate (CHS). Protoplasts treated with CHS, for a short period during the initial stages of cell wall regeneration, developed a patchwork cell wall, possessing regions devoid of cell wall. This effect was reversible, and treated cells ultimately developed a normal, confluent cell wall when removed from the CHS. Freeze fracture studies revealed that for CHS-treated cells, regions without microfibril impressions did possess intramembranous particles (IMP's) but that these regions contained small domains free of IMP's suggestive of lateral phase separation. The data implies that the physical characteristics of the plasma membrane lipid are important to the deposition of cell wall microfibrils during cell wall regeneration. This effect may be attributed to altered lipid-protein interactions, modified membrane fusion characteristics, or altered membrane flow.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.     

Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers.     

Structural arrangement of polymers within the wall of Streptococcus faecalis.

The structure of the cell wall of Streptococcus faecalis was studied in thin sections and freeze fractures of whole cells and partially purified wall fractions. Also, the structures of wall preparations treated with hot trichloroacetic acid to remove non-peptidoglycan wall polymers were compared with wall preparations that possess a full complement of accessory polymers. The appearance of the wall varied with the degree of hydration of preparations and physical removal of the cell membrane from the wall before study. Seen in freeze fractures of whole cells, the fully hydrated wall seemed to be a thick, largely amorphic layer. Breaking cells with beads caused the cell membrane to separate from the wall and transformed the wall from a predominantly amorphic layer to a structure seemingly made up of two rows of "cobblestones" enclosing a central channel of lower density. Dehydration of walls seemingly caused the cobblestones to be transformed into two bands which continued to be separated by a channel. This channel was also observed in isolated wall preparations treated with hot trichloroacetic acid to remove non-peptidoglycan polymers. These observations are consistent with the interpretation that both peptidogylcan and non-peptidoglycan polymers are concentrated at the outer and inner surfaces of cell walls. These observations are discussed in relation to possible models of wall structure and assembly.     

Influence of growth conditions on the composition of cell wall polysaccharides from cultured tobacco cells

The sugar composition of cell wall polysaccharides of two tobacco varieties obtained from mesophyll, regenerating protoplasts and cells grown under various conditions were compared. Regenerating protoplasts developed an unusual cell wall with a low cellulose and a high non-cellulosic glucan content. In the presence of different phytohormones compact and friable calli were obtained with cell walls containing low and high arabinose/xylose ratios. The cell walls of compact calli were comparable to those of genuine mesophyll cells. The sugar constituents of cell walls obtained from cells grown in liquid media were different from those of solid calli. The cell wall composition of suspension cultured cells was hardly affected by various combinations of phytohormones, but was altered by high osmolarity of the medium.     

Formation of the Pollen-aggregating Threads inStrelitzia reginae

Morphogenesis of the specialized thread-forming (TF) cells in theStrelitzia reginaeanther was investigated; particular attention was given to the cell walls and the degree of vacuolation. The mass of both cell wall and cytoplasm increased until just before dehiscence. However, cell growth and degradation were largely synchronous processes in the TF cells: before any wall thickening could be observed, degradation of primary cell wall material was already initiated. This degradation continued, with the result that the mature thread cells were eventually fully separated from their surrounding cells.Four stages of development, mainly relating to the degree of cell separation, were established. At stage 1, TF cells began to separate from the subepidermis, while at stage 2 some initial cell wall thickening was taking place. The walls of the TF cell were, at stage 3, thickened considerably (about 1 μm), especially along the radial axes. The texture of these walls was loose due to the presence of large intermicrofibrillar regions, and the previously vacuolated cells were filled with cytoplasm. Longitudinal sections revealed conical gaps in the thick cell wall over the plasmodesmata. Just before dehiscence (late stage 3), the TF cells separated from each other and the subepidermis to such an extent that only plasmodesmata and fibrillar wall remnants kept the files of TF cells in place. The released uniseriate threads were classified as stage 4. (Occasionally the threads were multicellular but only where the transverse walls had not separated from each other.) The threads had thinner cell walls than the TF cells at stage 3 and were vacuolated.     

X- ray Energy Spectra Analysis of Lanthanum in Tissue Cells of Defferent Parts of Wheat Plant

Lanthanum entered plants through the root and accumulated mostly in the cell wall of the root tip. Only a few accumulated in the cortex cell of the root elongation zone and in the cell wall of the mesophyllous cells. There was no detectable lanthanum in the cytoplasm. Observation with TEM (transmission electronic microscopy) indicated that lanthanum, which appeared as electron opaque deposits, was present in the cell wall of tissues in every part of the plant. The higher lanthanum contents occurred in the cell wall of root tips caused a dark appearance of the cell wall. The content of lanthanum in the cell wall of the cortex of elongation zone appeared as granules of different size. Those in the mesophyllous cell wall appeared as much less small granules. X-ray energy spectnnn analysis of the deposits confirmed the induction of lanthanum into the plant tissue cells and that its intensity correlated with the density of the deposits.     

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Background and Aims

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells.

Methods

Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins.

Key Results

The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls.

Conclusions

The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.     

Cell wall-associated peroxidase in cultured cells of liverwort,Marchantia polymorpha L. Changes of peroxidase level and its localization in the cell wall

Most of the peroxidase activities from cultured cells of Marchantia polymorpha L. were found in the cell wall. The activities increased markedly after the beginning of stationary growth. Cytochemical examination using an electron microscope indicated that the peroxidase was localized in the layers of the cell wall. The increase of peroxidase released from the cells into the culture medium was closely correlated with the increase of the peroxidase level in the cell wall. The release of peroxidase seemed to be caused by fragmentation of the cell wall stripped from cell.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

Changing extracellular matrix of cells during development of suspension culture of Triticum timopheevii Zhuk

A study was made of the contents of the main polysaccharide fractions in the cell wall, and extracellular polysaccharides, and of the activity of cell wall enzymes during cultivation of suspension culture of cells of the winter wheat Triticum timopheevii Zhuk. It was shown that within 3 days of cultivation (a phase enriched in dividing cells), on the background of increased callose contents in plant cells, amounts of pectins and hemicelluloses extracted by 4N alkali decreased. The content of polysaccharides reached its initial level by the end of culturing. A parallel analysis of glycosidase activity in cell walls has shown their considerable activation at the stage enriched by dividing cells, which decreased at a transition of culture into the stationary level. The increased activity of hydrolyzing enzymes was combined with an increased efflux of extracellular polysaccharides into culture medium. The detected changes in polysaccharide composition of the cell wall at the first phase indicate its qualitative changes during cell wall reconstruction at the beginning of cytokines, whereas extensive expansion of cell wall was seen on the phase of elongation.