The structure of cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus.

The structure of the pellicles and cells of the cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus, was studied by transmission electron microscopy of thin sections and freeze-etch replicas of glucose-stimulated cell suspensions, quiescent cell suspensions, and discrete pellicles. These bacteria have a relatively thin cell wall in section, with several irregular features superimposed on an otherwise simple, Gram-negative morphology. There are no flagella or pili. Unfixed, unextracted cells, viewed as whole mounts, show spherical or ellipsoidal bodies of undetermined composition which disappear after extraction with water or ethanol and propylene oxide. For both species, there are several kinds of cell surface irregularities, some of which are localized protrusions of the cell envelope. A variety of irregularities is seen frequently on cells in the first minutes of glucose incubation, on cells in a discrete pellicle, on quiescent cells, and on starved cells. Immediately after the addition of glucose to cellulose-free cells in suspension culture, fine fibrils appear on and (or) near the cell envelope. The fine fibrils are frequently as small as 3 nm in diameter in both freeze-etch and thin-section preparations and are frequently associated with freshly synthesized cellulose fibrils. Starved cells in suspensions free of (classical) microfibrils sometimes reveal stubs of an extracellular structure whose morphology resembles that of a nascent cellulose fibril.     

On the cross-sectional shape of cellulose crystallites in Valonia ventricosa

The cross-sectional shape of the cellulose crystallites from the Valonia ventricosa cell wall has been investigated by electron microscopy, using negative staining and Bragg contrast in the bright field mode, with emphasis on this latter technique. The appearance of the cellulose crystallites in the electron microscope depends on their orientation. The cross-section of each cellulose crystal is almost square, with an average side of 18 nm. Sub-units corresponding to elementary fibrils were not detectable within the crystals. The differences between these results and those of earlier workers are discussed.     

Cellulose component of Acetobacter xylinum pellicle: morphology and its significance for the mechanism of microfibril formation

The cellulose component of native, minimally disturbed pellicles of Acetobacter sylinum has a three-dimensional, microfibrillar, interconnected, ‘brush-wood’ structure. This structure could not originate from a spinneret or extrusion mechanism of cellulose microfibril formation. It may be produced by a mechanism of spontaneous association and post factor crystallization of preformed, transient I → 4β glucans.     

Cellulose orientation in the outer epidermal wall of angiosperm roots: implications for biosystematics

The net orientation of cellulose fibrils in the outer epidermal wall of the root elongation zone of 57 angiosperm species belonging to 29 families was determined by means of Congo Red fluorescence and polarization confocal microscopy. The angiosperms can be divided in three groups. In all but four plant families, the net orientation of the cellulose fibrils is transverse to the root axis. Three families, the Poaceae, Juncaceae and Cyperaceae, have a totally different organization. In the root elongation zone of these plants, the net orientation of cellulose fibrils in the outer epidermal wall is parallel with the root axis. In roots of one family, the Arecaceae, an elongation zone in the literal sense of the word is absent and cellulose fibrils are randomly oriented.     

Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

Cellulose orientation at the surface of the Arabidopsis seedling. Implications for the biomechanics in plant development

In diffuse growing cells the orientation of cellulose fibrils determines mechanical anisotropy in the cell wall and hence also the direction of plant and organ growth. This paper reports on the mean or net orientation of cellulose fibrils in the outer epidermal wall of the whole Arabidopsis plant. This outer epidermal wall is considered as the growth-limiting boundary between plant and environment. In the root a net transverse orientation of the cellulose fibrils occurs in the elongation zone, while net random and longitudinal orientations are found in subsequent older parts of the differentiation zone. The position and the size of the transverse zone is related with root growth rate. In the shoot the net orientation of cellulose fibrils is transverse in the elongating apical part of the hypocotyl, and longitudinal in the fully elongated basal part. Leaf primordia and very young leaves have a transverse orientation. Throughout further development the leaf epidermis builds a very complex pattern of cells with a random orientation and cells with a transverse or a longitudinal orientation of the cellulose fibrils. The patterns of net cellulose orientation correlate well with the cylindrical growth of roots and shoots and with the typical planar growth of the leaf blade. On both the shoot and the root surface very specific patterns of cellulose orientation occur at sites of specific cell differentiation: trichome-socket cells complexes on the shoot and root hairs on the root.     

Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives

In vivo cellulose ribbon assembly by the Gram-negative bacterium Acetobacter xylinum can be altered by incubation in carboxymethylcellulose (CMC), a negatively charged water-soluble cellulose derivative, and also by incubation in a variety of neutral, water-soluble cellulose derivatives. In the presence of all of these substituted celluloses, normal fasciation of microfibril bundles to form the typical twisting ribbon is prevented. Alteration of ribbon assembly is most extensive in the presence of CMC, which often induces synthesis of separate, intertwining bundles of microfibrils. Freeze- etch preparations of the bacterial outer membrane suggest that particles that are thought to be associated with cellulose synthesis or extrusion may be specifically organized to mediate synthesis of microfibril bundles. These data support the previous hypothesis that the cellulose ribbon of A. xylinum is formed by a hierarchical, cell- directed, self-assembly process. The relationship of these results to the regulation of cellulose microfibril size and wall extensibility in plant cell walls is discussed.     

Plants control the properties and actuation of their organs through the orientation of cellulose fibrils in their cell walls

Plants use the orientation of cellulose microfibrils to create cell walls with anisotropic properties related to specific functions. This enables organisms to control the shape and size of cells during growth, to adjust the mechanical performance of tissues, and to perform bending movements of organs. We review the key function of cellulose orientation in defining structural-functional relationships in cell walls from a biomechanics perspective, and illustrate this by examples mainly from our own work. First, primary cell-wall expansion largely depends on the organization of cellulose microfibrils in newly deposited tissue and model calculations allow an estimate of how their passive re-orientation may influence the growth of cells. Moreover, mechanical properties of secondary cell walls depend to a large extent on the orientation of cellulose fibrils and we discuss strategies whereby plants utilize this interrelationship for adaptation. Lastly, we address the question of how plants regulate complex organ movements by designing appropriate supramolecular architectures at the level of the cell wall. Several examples, from trees to grasses, show that the cellulose architecture in the cell wall may be used to direct the swelling or shrinking of cell walls and thereby generate internal growth stress or movement of organs.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : II. The Giant Ameba Pelomyxa carolinensis

Dividing nuclei from the giant ameba Pelomyxa carolinensis were fixed in osmium tetroxide solutions buffered with veronal acetate to pH 8.0. If divalent cations (0.002 M calcium, magnesium, or strontium as chlorides) were added to the fixation solution, fibrils that are 14 mµ in diameter and have a dense cortex are observed in the spindle. If the divalent ions were omitted, oriented particles of smaller size are present and fibrils are not obvious. The stages of mitosis were observed and spindle components compared. Fibrils fixed in the presence of calcium ions are not so well defined in early metaphase as later, but otherwise have the same diameter in the late metaphase, anaphase, and early telophase. Fibrils are surrounded by clouds of fine material except in early telophase, when they are formed into tight bundles lying in the cytoplasm unattached to nuclei. Metaphase and anaphase fibrils fixed without calcium ions are less well defined and are not observably different from each other. The observations are consistent with the concept that spindle fibrils are composed of polymerized, oriented protein molecules that are in equilibrium with and bathed in non-oriented molecules of the same protein. Partially formed spindle fibrils and ribosome-like particles were observed in the mixoplasm when the nuclear envelope had only small discontinuities. Remnants of the envelope are visible throughout division and are probably incorporated into the new envelope in the telophase. Ribosome-like particles are numerous in the metaphase and anaphase spindle but are not seen in the telophase nucleus, once the envelope is reestablished, or in the interphase nucleus.     

Cellulose synthesized by Acetobacter xylinum in the presence of multi-walled carbon nanotubes

The structure of bacterial cellulose is affected by the bacterial strain used, culture media and cultivation conditions. In this study, acid-treated multi-walled carbon nanotubes (MWNTs) were added into a static culture medium and their effect on bacterial cellulose structure was studied by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), CP/MAS (13)C NMR and X-ray diffractometry. The bacterial cellulose ribbons and the MWNTs interwound and formed a three-dimensional network architecture. Band-like assemblies with sharp bends and rigidity were also produced in the presence of MWNTs. The intermolecular hydrogen bonds in bacterial cellulose produced in the presence of MWNTs were weakened. The crystal structure, cellulose I(alpha) content, crystallinity index (CrI) and crystallite size all changed. The results may suggest that the acid-treated MWNTs containing hydroxyl groups interact with the sub-elementary bacterial cellulose fibrils, subsequently interfering with the aggregation and crystallization.     

THE DINOFLAGELLATE PELLICULAR WALL LAYER AND ITS OCCURRENCE IN THE DIVISION PYRRHOPHYTA1

Forty-five species of dinoflagellates were surveyed for the presence of a pellicular layer in the amphiesma or cell covering. Such a layer was found in 15 of the 20 genera studied. Half the pellicles tested were resistant to acetolysis and may contain a sporopollenin-like material similar to that of some dinoflagellate cyst walk. Most organisms which formed pellicles were capable of reinforcing this layer with cellulose. Pellicles of Heterocapsa niei (Loeblich) Morrill & Loeblich and Scrippsiella trochoidea (Stein) Loeblich were studied with the electron microscope. Evidence is presented indicating that dividing cells of S. trochoidea from new walls while still enclosed in the parental pellicular layer.     

Electrically conductive bacterial cellulose by incorporation of carbon nanotubes

Electrically conducting polymeric membranes were prepared by incorporating multiwalled carbon nanotubes (MWCNTs) into bacterial cellulose pellicles produced by Gluconacetobacter xylinum. The MWCNTs were dispersed in a surfactant (cationic cetyl trimethylammonium bromide) solution, and cellulose pellicles were dipped into the solution for 6, 12, and 24 h. The surfactants were then extracted in pure water and dried. Electron microscopy showed that the individual MWCNTs were strongly adhered to the surface and the inside of the cellulose pellicle. The conductivity of the MWCNTs-incorporated cellulose pellicle, as measured by a four-probe at room temperature, was 1.4 x 10(-1) S/cm, based on the total cross-sectional area (approximately 9.6 wt % of MWCNTs). This suggests that the MWCNTs were incorporated uniformly and densely into the pellicles.     

The Effect of Homologous Rabbit Antiserum on the Growth of Leishmania tarentolae—a Fine Structure Study

SYNOPSIS. The fine structure of Leishmania tarentolae growing in the presence of homologous rabbit antiserum was investigated. Immediately after agglutination of the promastigotes, a dense band between pellicles of adjacent cells could be seen, and a surface coat was rendered visible. The cells did not fuse. At dilutions of antiserum from 1:100–1:1200 the promastigotes continued to grow, forming large masses. By 5 days these consisted of individual cells adhering to one another. In cross-section, any 2 cells were separated by 2 membranes with associated subpellicular fibrils and a dense band between the 2 membranes. The term syncytium generally refers to a multinucleate cell originating by fusion of several cells and has been used to mean a multinucleate cell arising from nuclear division unaccompanied by cytokinesis. Therefore, it is unlikely that the masses formed in the presence of homologous antiserum are syncytia.     

Characterization of fibrous compound of Golgi vesicles in tapetal cells ofDelphinium

Summary It was shown that Golgi structures abundantly appearing in tapetal cells ofDelphinium Ajacis L. developing anthers, prior to meiocytes meiosis, show a fine fibrous material within their vesicles. At the time of the formation of tapetal cell wall this fibrous component, released by an exocytotic process, is incorporated into the cell wall. The membrane of dictyosomes derived vesicles participates in the development of plasma membrane. Fibrous material appears to be morphologically similar to the fibrils of tapetal cell wall; this cell wall gives a positive reaction for cellulose and pectins, as visible in the light microscope. Moreover, the fibrous and pectinase resistant compound of dictyosomes derived vesicles and the fibrils of cell wall disappear partly after cellulase digestion which proves their cellulosic character. On the other hand pectinase treatment as well as ruthenium red staining suggest associated with cellulose pectins within Golgi vesicles.     

Alternative Environmental Roles for Cellulose Produced by Acetobacter xylinum

The cellulose-producing bacterium Acetobacter xylinum has been considered a strict aerobe, and it has been suggested that the function of cellulose is to hold cells in an aerobic environment. In this study, we showed that A. xylinum is capable of growing microaerophilically. Cellulose pellicles provided significant protection to A. xylinum cells from the killing effects of UV light. In experiments measuring colonization by A. xylinum, molds, and other bacteria on pieces of apple, cellulose pellicles enhanced colonization of A. xylinum on the substrate and provided protection from competitors which use the same substrate as a source of nutrients. Cellulose pellicles produced by A. xylinum may have multiple functions in the growth and survival of the organism in nature.     

The Arabidopsis COBRA Protein Facilitates Cellulose Crystallization at the Plasma Membrane

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a K_D of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.”     

Effects of CMC addition on bacterial cellulose production in a biofilm reactor and its paper sheets analysis

Bacterial cellulose (BC) can be grown into any desired shape such as pellicles, pellets, and spherelike balls, depending on the cultivation method, additives, and cell population. In this study, Acetobacter xylinum (ATCC 700178) was grown in the production medium with different concentrations of carboxylmethylcellulose (CMC) and were evaluated for BC production by using a PCS biofilm reactor. The results demonstrated that BC production was enhanced to its maximum (～13 g/L) when 1.5% of CMC was applied, which was 1.7-fold higher than the result obtained from control culture. The major type of the produced BC was also switched from BC pellicle to small pellets. The ratio of BC pellets in suspension increased from 0 to 93%. Fourier transform infrared (FTIR) spectroscopy demonstrated that CMC was incorporated into BC during fermentation and resulted in the decreased crystallinity and crystal size. The X-ray diffraction (XRD) patterns indicated that CMC-BC exhibited both lower crystallinity (80%) and crystal size (4.2 nm) when compared with control samples (86% and 5.3 nm). The harvested BC was subjected to paper formation and its mechanical strength was determined. Dynamic mechanical analysis (DMA) results demonstrated that BC paper sheets exhibited higher tensile strength and Young's modulus when compared with regular paper.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.