Specialized cell surface structures in cellulolytic bacteria.

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.     

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Blot analyses of glycoconjugates: enzyme-hydrazide--a novel reagent for the detection of aldehydes

A procedure for the general staining of glycoproteins and other glycoconjugates on protein blots has been developed. Aldehydes are formed on the sugars of glycoconjugates by periodate oxidation which then react with hydrazide groups of enzyme-hydrazides, a novel reagent designed for aldehyde detection. The bound enzyme-hydrazide is demonstrated histochemically. The new assay is advantageous over periodic-acid Schiff staining of gels as its reagents and signals are stable and the process is simple and expedient, and provides greater sensitivity.     

Capsule of Escherichia coli K29: ultrastructural preservation and immunoelectron microscopy.

The polysaccharide capsule of Escherichia coli K29 fully surrounds the microorganism and thus occupies an extracellular space ca. 20 times larger in volume than that of the decapsulated cell. Since more than 95% of the capsule consists of water, dehydration for electron microscopy causes the material to collapse. We describe here a method for embedding the capsule in an uncollapsed form. Dehydration of gelatin-enrobed, glutaraldehyde-fixed cells was performed in dimethyl formamide. The cells were embedded in Lowicryl K4M with the "progressive lowering of temperature" method and UV polymerization. In ultrathin sections, the capsule can be identified by its low electron contrast. It occupies a layer 3/4 micron thick thick and shows fibrous strands embedded in a fine granular matrix. The thin strands extend radially from the cell wall and transverse the capsule. The entire capsule domain, as well as the outer membrane, binds specific anticapsular antibody, whereas the periplasmic space and most of the inner membrane lack capsule-specific immunostain.     

Adherence of Clostridium thermocellum to cellulose

The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant. This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2. The CBF antigen could be removed from cell extracts by adsorption to cellulose. A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity. During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS. However, the amounts decreased in stationary-phase cells. Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid. Increased levels of CBF were observed when cells were grown on cellulose. In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence. The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species.