R. Malcolm Brown

Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli, and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants.

Structural investigations of microbial cellulose produced in stationary and agitated culture

Structural characteristics of microbial cellulose synthesized by two different methods have been compared using FT-IR and X-ray diffraction techniques. Cellulose synthesized by Acetobacter xylinum NQ-5 strain from agitated culture conditions is characterized by a lower Iϑ mass fraction than cellulose that was produced statically. Such a decrease was in good correlation with smaller crystallite sizes of microfibrils produced in agitated culture. Formation of characteristic cellulose spheres during agitation has been investigated by various electron and light microscopic methods. On this basis, a hypothetical mechanism of sphere formation and cell arrangement in the agitated culture has been proposed. During agitation, cells are stacked together in organized groups around the outer surface of the cellulose sphere.

Simultaneous glutaraldehyde-osmium tetroxide fixation with postosmication - An improved fixation procedure for electron microscopy of plant and animal cells

SummaryA fixation procedure for electron microscopy is described which includes a simultaneous glutaraldehyde-OsO4 fixation followed by postosmication. This procedure was found to have considerable advantages in preserving structures of plant and animal cells.

Towards electronic paper displays made from microbial cellulose

Cellulose (in the form of printed paper) has always been the prime medium for displaying information in our society and is far better than the various existing display technologies. This is because of its high reflectivity, contrast, low cost and flexibility. There is a major initiative to push for a dynamic display technology that emulates paper (popularly known as “electronic paper”). We have successfully demonstrated the proof of the concept of developing a dynamic display on cellulose. To the best of our knowledge, this is the first significant effort to achieve an electronic display using bacterial cellulose. First, bacterial cellulose is synthesized in a culture of Acetobacter xylinum in standard glucose-rich medium. The bacterial cellulose membrane thus formed (not pulp) is dimensionally stable, has a paper-like appearance and has a unique microfibrillar nanostructure. The technique then involves first making the cellulose an electrically conducting (or semi-conducting) sheet by depositing ions around the microfibrils to provide conducting pathways and then immobilizing electrochromic dyes within the microstructure. The whole system is then cased between transparent electrodes, and upon application of switching potentials (2–5 V) a reversible color change can be demonstrated down to a standard pixel-sized area (ca. 100 μm2). Using a standard back-plane or in-plane drive circuit, a high-resolution dynamic display device using cellulose as substrate can be constructed. The major advantages of such a device are its high paper-like reflectivity, flexibility, contrast and biodegradability. The device has the potential to be extended to various applications, such as e-book tablets, e-newspapers, dynamic wall papers, rewritable maps and learning tools.

The Biosynthesis of Cellulose

Abstract Cellulose is one of the major commercial products of Sweden and constitutes the most abundant of the natural polymer systems. Thus, it is of interest to review the molecular design and architecture of cellulose with particular reference to the controls of its biosynthesis. The bioassembly process is highly ordered and structured, reflecting the intricate series of events which must occur to generate a thermodynamically metastable crystalline submicroscopic, ribbonlike structure. The plant cell wall is an extremely complex composite of many different polymers. Cellulose is the “reinforcing rod” component of the wall. True architectural design demands a polymer which can withstand great flexing and torsional strain. Using comparative Hydrophobic Cluster Analysis of a bacterial cellulose synthase and other glycosyl transferases, the multidomain architecture of glycosyl transferases has been analyzed. All polymerization reactions which are processive require at least three catalytic sites located on ...

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.

About the structure of cellulose: debating the Lindman hypothesis

The hypothesis advanced in this issue of CELLULOSE [Springer] by Bjorn Lindman, which asserts that the solubility or insolubility characteristics of cellulose are significantly based upon amphiphilic and hydrophobic molecular interactions, is debated by cellulose scientists with a wide range of experiences representing a variety of scientific disciplines. The hypothesis is based on the consideration of some fundamental polymer physicochemical principles and some widely recognized inconsistencies in behavior. The assertion that little-recognized (or under-estimated) hydrophobic interactions have been the reason for a tardy development of cellulose solvents provides the platform for a debate in the hope that new scientific endeavors are stimulated on this important topic.

Cellulose biosynthesis : A model for understanding the assembly of biopolymers

This study provides an updated review of the current status on cellulose biosynthesis. The centerpiece of this work is the presentation of a new model of cellulose biogenesis. This model and its parts are presented to better understand the mechanisms of polymerization and crystallization leading to biopolymer formation. The new information has been derived largely from sequence analysis, biochemistry and ultrastructural data relating to cellulose, Natures most abundant macromolecule.

Airborne Algae: Their Abundance and Heterogeneity

The literature on the occurrence of airborne algae is reviewed briefly. Airborne algae were isolated into culture in both quantity and diversity. Qualitative experiments and culture techniques are discussed, as are quantitative sampling techniques and preliminary correlations of the occurrence of algae, fungi, and pollen in the air. The data disclose an important pathway for the dispersion of soil algae and support an observation that algae may be important as causal agents in inhalant allergies.

Cellulose I microfibril assembly : computational molecular mechanics energy analysis favours bonding by van der Waals forces as the initial step in crystallization

In vitro and abiotic synthesis of cellulose I have indicated that glucan sheet formation is most likely to be the first stage in crystallization of this allomorph of cellulose. Bonding schemes for the different glucan sheets found in crystals of cellulose Iα and Iβ were analysed energetically with the molecular mechanics program, MM3. Using high and low dielectric constants, favouring van der Waals forces and hydrogen bonding, respectively, van der Waals-associated mini-sheets had lower energies than hydrogen bonded mini-sheets. Furthermore, the first glucan mini-sheet most likely to form is the one with the lowest energy. In the case of cellulose Iβ, this mini-sheet was the one along the (110) plane; in the case of cellulose Iα, it was the one along the (010) plane. Incorporating these results into known experimental evidence, we theorize the requirement of at least three sequential steps for native cellulose I crystallization: (1) formation of mini-sheets by van der Waals forces, (2) association of these sheets by hydrogen bonding into mini-crystals, and (3) the convergence of mini-crystals to form the crystalline microfibril.