Cell wall assembly in Bacillus subtilis: visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation.     

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Bacteriophage SP50 as a marker for cell wall growth in Bacillus subtilis.

When grown under conditions of phosphate limitation, Bacillus subtilis W23 lacked wall teichoic acid and did not adsorb phage SP50. During transition from growth under conditions of phosphate limitation to those of potassium limitation, the bacteria developed an ability to adsorb phage which increased exponentially in relation to their content of wall teichoic acid. During transition in the reverse direction, the bacteria retained near-maximum phage-binding properties until their content of wall teichoic acid had fallen to a fairly low level. These observations suggest that newly incorporated wall material does not immediately appear at the cell surface in a structure to which phage can adsorb. Examination of the location of adsorbed phage particles showed that recently incorporated receptor material appeared at the cell surface first along the length of the cylindrical portion of the cell. The results are consistent with models of wall assembly in which newly synthesized wall material is intercalated at a large number of sites that are distributed along the length of the cell. This newly incorporated material may be located initially at a level underlying the surface of the cell and may become exposed at the surface only during subsequent growth. Incorporation of new material may also proceed rapidly into the developing septa, but new wall material is incorporated into existing polar caps more slowly, or perhaps not at all.     

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.     

Pollen ultrastructure in anther cultures of Datura innoxia. II. The generative-cell wall.

In young pollen grains of Datura innoxia, a wall of the usual hemispherical type separates the 2 gametophytic cells initially and, in the electron microscope, appears as an electron-translucent matrix which is contiguous with the intine. Before detachment of the generative cell from the intine, the matrix decreases in thickness and in places is dispersed altogether leaving the plasmalemmae on either side of it in close apposition. A particularly prominent zone, triangular in profile, is left where the wall joins with the intine. After detachment of the cell, remnants of the matrix can be seen distributed irregularly around the cell and it is supposed that these are partly derived from material in the triangular zone as the cell is drawn away from the intine. The wall residues persist throughout the maturation phase of the pollen and are considered to be either callose resulting from incomplete digestion of the initial wall, or some other polysaccharide material which is unevenly laid down along the wall and concentrated at the junction with the intine. In pollen induced into embryogenesis by anther culture, wall material is also distributed irregularly around the detached cell in a series of discrete zones, but these are more extensive than in vivo, closer together and in many instances highly dilated. The wall profiles thus have a beaded appearance, the 'beads' being connected together by short links of the 2 apposed plasmalemmae. The contents of the swollen zones have a similar electron density to that of the matrix in vivo but also show traces of a fibrillar component. It is postulated that this unusual swelling is a prelude to dispersal of the wall by disruption of the plasmalemmal links and to the establishment of cytoplasmic continuity between the 2 cells. The significance of such binucleate pollen grains in the formation of non-haploid embryos is discussed.     

Calcification and Decalcification During Epithallial Cell Turnover in Coralline Red Algae

Epithallial cells of the coralline red algae are characterized by unusual structural specialization, which include deep invaginations of the distal cell surface, and by unique development, which culminates in senescence, shedding, and replacement of the cells. Electron microscopic study of epithelial cell differentiation in morphologically and taxonomically disparate species suggests that the unusual features of epithelial cell structure and development stem from the fact that these dynamics occur within a calcified matrix. Distal wall ingrowths begin to form on the initial cells, cells whose cleavage eventually gives rise distally to new epithelial cells. After the distal wall ingrowths form, the overlying crosswall becomes rich in organic material. For this organic wall material to be deposited into the existing crosswall, the wall must first be decalcified; therefore, the presence of abundant organic material in the crosswall provides a marker of localized decalcification. We propose that the location and time of origin of distal wall ingrowths indicate a connection between the ingrowths and two coordinated processes: localized secretion of wall material, and decalcification of the overlying cell wall in preparation for the movement of the young epithelial cell into a new location relative to the surrounding calcified matrix. The large plasmalemmal surface area associated with the distal wall ingrowths allows for a greater abundance of membrane‐associated components, such as proton pumps, that could drive localized cell wall decalcification.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidial germination and outgrowth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of conidia was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged conidia to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiogenesis. An ultrastructural study of the germination stage indicated nuclear migration into the emerging germ tube whose new cell wall was an extension of the parent conidium's innermost cell wall layer. Septal formation at the neck of the germ tube followed. The septal pore was filled with particulate material and the septal membranes possessed unusual linear elements in their median hydrophobic zones. The germ tube, which possessed a smooth-surfaced plasma membrane, continued to elongate with periodic septum formation. The parent conidium and later the proximal germ tube showed progressive vacuolation and the cytoplasm became largely occupied by electron-translucent material. In older cells the septal pore was blocked by Woronin bodies. Compared with normal conidial germination this microcycle' germination is far more synchronous and the resultant germling is morphologically simpler. In ultrastructural terms, however, germination appears to be identical with that obtained at 28 degrees C.     

Atomic force microscopy of cell growth and division in Staphylococcus aureus

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.     

Growth of the surface of Corynebacterium diphtheriae

Surface structure and growth of the surface of Corynebacterium diphtheriae mitis strain were investigated by scanning electron microscopy and the immunofluorescence technique. The surface of the cell revealed by the scanning electron microscope showed a few elevated circular zones which encompassed the cell. The cell diameter increased at this zone and this gave the club-shaped appearance to this species. The cell surface labeled with specific antibodies against the whole bacterial cell and tagged with ferritin remained at a constant length during cell division cycles and the new cell surface emerged from the polar ends of the cell. This new wall surface was completely devoid of the ferritin particles indicating that the cell wall component on the old preexistent wall was completely conserved. A similar finding was obtained by immunofluorescence microscopy. C. diphtheriae, unlike Bacillus spp., showed apical growth as has been observed in fungal cells.     

Kinetics and hydrodynamics of Agapanthus umbellatus pollen-tube growth: A structural and stereological study

Summary In vitro pollen germination of Agapanthus umbellatus follows a logistic-type curve. It has a lag phase, which corresponds to pollen grain (PG) hydration, followed by an exponential phase — initial pollentube (PT) growth. The lag phase is characterized by an increase of about 40% in the volume of the PG as a result of the hydration process. During the exponential phase the PT emerges, and 40 min later it possesses an ultrastructural organization with a typical two-layer wall and four well-defined zones: the apical, sub-apical, nuclear and vacuolar zones. In this period the material transported by the Golgi vesicles seems to be mostly incorporated into the pollen-tube wall (PTW). Stereological analysis showed that the increase in tube volume is correlated with the increase in the vacuolar compartment at the PG level. The decrease in the relative volume occupied by the mitochondria, generative cell and vegetative nucleus in the PG suggests that these organelles move to the PT. A correlation between the disappearance of lipid droplets in the lag phase and the metabolic reactions that take place during hydration is suggested.Abbreviations PT Pollen tube - Pg pollen grain - PTW pollen-tube wall     

Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery

We have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram-positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell division sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell division protein FtsZ, which recruits penicillin-binding protein (PBP) 2. Unexpectedly, in FtsZ-depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell division proteins as drug targets.     

A study of the Candida albicans cell wall proteome

Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI motif in 19 of them. We also identified 66 "atypical" cell wall proteins that lack the above-mentioned characteristics. After tryptic removal of the most accessible proteins in the cell wall, several of the same expected GPI proteins and the most commonly found "atypical" wall proteins were identified. This result suggests that proteins are located not only at the cell wall surface, but are embedded within the cell wall itself. These results, which include new identified cell wall proteins, and comparison of proteins in blastospore and mycelial walls, will help to elucidate the C. albicans cell wall architecture.     

Cell Division in Yeasts: Movement of Organelles Associated with Cell Plate Growth of Schizosaccharomyces pombe

Electron microscopy of dividing fission yeast cells shows establishment of an annular rudiment (AR) of electron-transparent material under the old cell wall as the first sign of elaboration of the cell plate. The AR grows centripetally, finally closing at the mid-point of the cell. During the inward growth of the AR it is thickened by addition of denser material which becomes the scar plug after fission; the electron-transparent material is lost at fission. Lying always between the cytoplasmic membrane and the cell wall is a dark layer of variable thickness. This layer becomes markedly thickened into a fillet at the base of the centripetally growing cell plate. The fission process begins after the cell plate is completely elaborated. One striking feature of fission is the migration of dense material from the fillet at the base of the cell plate outwardly through the matrix of the cell wall to its final resting place as a dark ring, a "fuscannel," adjacent to the fission scar. The inclusion of Golgi bodies in many sections suggests their involvement in cell plate elaboration, presumably through production of the dense bodies which are seen to fuse with the dark layer proximal to the growing cell plate.     

Development of cycad ovules and seeds. 1. Implication of the ER in primary cellularisation of the megagametophyte in <Emphasis Type="Italic">Encephalartos natalensis</Emphasis> Dyer and Verdoorn

Very little is known about the pre- and post-shedding megagametophyte development and utilisation of accumulated reserves, respectively, in cycads (Zamiaceae). In the present study on developing ovules of the recalcitrant-seeded species, Encephalartos natalensis, cells of the megagametophyte were found to become progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in a homogeneous matrix. ER-derived vesicles (and not Golgi-derived vesicles) appeared to be the principal contributors of the primary cell wall components, pectin and xylan, during megagametophyte cellularisation. This was confirmed by the use of enzyme-gold localisation. High-pressure freezing (HPF) and freeze substitution (FS) of samples the following season showed that while the apparently featureless cytomatrix of the megaspore was an artefact of conventional fixation, there was still an insignificant occurrence of Golgi bodies during primary wall formation. When enzyme-gold localisation was employed on the HPF-FS material, label for pectin and xylan was found only in the regions of ER and vesicles and not in any of the few Golgi bodies or their associated vesicles. Immunocytochemistry revealed that pectin and xylan were restricted to the ER and not to any vesicles or to the occasional Golgi body that was found. This suggests that the ER exclusively, is involved in the deposition of these primary cell wall components during the cellularisation of the E. natalensis megagametophyte. While cellularisation took place over approximately 1–2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months—after which the seeds were shed whether pollination/fertilisation had recently occurred, or not.     

The role of surface stress in the morphology of microbes

The shapes of many prokaryotes can be understood by the assumption that the cell wall expands in response to tension created by the osmotically derived hydrostatic pressure. Different organisms have different shapes because wall growth takes place in different regions. A previous paper (Koch et al., 1981 a) considered the simplest case of prokaryotic growth, i.e. that of Streptococcus faecium. In the present paper, an elaboration of this theory is applied to two further cases - the more perfectly spherical cocci and the rod-shaped bacteria. These cases are more complex mathematically, because growth over a considerable fraction of the surface must be considered. Such diffuse growth cannot be treated analytically, but can be simulated on a computer or handled by geometric arguments. The spherical form of the cocci may result from either diffuse growth over their entire external surface, or from zonal growth in which the addition of new material only occurs in the immediate vicinity of the splitting septum. In the zonal model, it must be assumed that the least amount of previously laid down septal peptidoglycan consistent with wall growth is reworked in the formation of the new external wall. For Gram-positive rods, where the body of the rod is truly cylindrical, three kinds of growth zones are required: (1) the inward edge of the ingrowing septum, (2) the junction of septum and nascent pole, and (3) the cylindrical walls. Two modes for cylindrical elongation ara possible: (a) new wall is added in one or a few narrow annular zones, or (b) new wall material is added continuously all over the innermost surface and the outer layer is degraded. It is shown that the latter case applies to Bacillus subtilis. Also summarized in this paper are results, developed in more detail elsewhere, concerning the morphology of fusiform bacteria, Gram-negative rods and the hyphal tips of fungi.     

Cellulose synthesis is required for deposition of reticulate wall ingrowths in transfer cells

Despite the recognized physiological importance of transfer cells, little is known about how these specialized cells achieve localized deposition of cell wall material, leading to amplification of plasma membrane surface area and enhanced membrane transport capacity. This study establishes that cellulose synthesis is a key early factor in the construction of 'reticulate' wall ingrowths, an elaborate but common form of localized wall deposition characteristic of most transfer cells. Using field emission scanning electron microscopy, wall ingrowths were first visible in epidermal transfer cells of Faba bean cotyledons as raised 'patches' of disorganized and tangled cellulosic material, and, from these structures, ingrowths emerged via further deposition of wall material. The cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile and isoxaben both caused dramatic reductions in the number of cells depositing wall ingrowths, altered wall ingrowth morphology and visibly disrupted microfibril structure. The restriction of cellulose deposition to discrete patches suggests a novel mechanism for cellulose synthesis in this circumstance. Overall, these results implicate a central role for cellulose synthesis in reticulate wall ingrowth morphology, especially at the initial stage of ingrowth formation, possibly by providing a template for the self-assembly of wall polymers.     

Kinetics of Aluminum Uptake in Triticum aestivum L: Identity of the Linear Phase of Aluminum Uptake by Excised Roots of Aluminum-Tolerant and Aluminum-Sensitive Cultivars

The identity of a linear phase of aluminum (Al) uptake in Triticum aestivum was investigated by analysis of the kinetics of Al uptake by excised roots and purified cell wall fractions. Classical interpretation of kinetic data suggests that a linear phase of uptake with time reflects uptake across the plasma membrane; however, in studies with Al the possibility that the linear phase of uptake includes accumulation of Al in both the symplasm and the apoplasm has not been discounted. In our experiments, we observed a linear phase of Al uptake at both ambient and low temperatures, although the rate of uptake at 0°C was 53 to 72% less than at 23°C, depending on cultivars. This nonsaturable phase of uptake at low temperature suggests that a portion of the linear phase of Al uptake is nonmetabolic. Furthermore, analysis of Al in cell wall fractions isolated from excised roots pretreated with Al suggests that the linear phase of uptake includes a cell wall component. When excised roots were pretreated with Al, accumulation of Al in purified cell wall material included a linear phase that could not be desorbed with a 30 minute wash in citrate. The rates of linear-phase accumulation of Al by cell wall material and cell contents were similar. In contrast, the linear phase of in vitro uptake of Al by purified cell wall material was completely desorbed by a 30 minute wash with citrate. These results suggest that the linear phase of Al uptake observed in excised roots of T. aestivum included metabolism-dependent binding of Al in apoplasm.     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

Inhibition of cell-wall autolysis and pectin degradation by cations.

Modification of cell wall components such as cellulose, hemicellulose and pectin plays an important role in cell expansion. Cell expansion is known to be diminished by cations but it is unknown if this results from cations reacting with pectin or other cell wall components. Autolysis of cell wall material purified from bean root (Phaseolus vulgaris L.) occurred optimally at pH 5.0 and released mainly neutral sugars but very little uronic acid. Autolytic release of neutral sugars and uronic acid was decreased when cell wall material was loaded with Ca, Cu, Sr, Zn, Al or La cations. Results were also extended to a metal-pectate model system, which behaved similarly to cell walls and these cations also inhibited the enzymatic degradation by added polygalacturonase (EC 3.2.1.15). The extent of sugar release from cation-loaded cell wall material and pectate gels was related to the degree of cation saturation of the substrate, but not to the type of cation. The binding strength of the cations was assessed by their influence on the buffer capacity of the cell wall and pectate. The strongly bound cations (Cu, Al or La) resulted in higher cation saturation of the substrate and decreased enzymatic degradability than the weakly held cations (Ca, Sr and Zn). The results indicate that the junction zones between pectin molecules can peel open with weakly held cations, allowing polygalacturonase to cleave the hairy region of pectin, while strongly bound cations or high concentrations of cations force the junction zone closed, minimising enzymatic attack on the pectin backbone.