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R Lamed

Publications and source records attributed to R Lamed.

At least 19 recordsLinked to original sources
PubMed1992

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.

Amino Acid Sequence↗
PubMed1992

Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.

The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens.

Amidohydrolases↗
PubMed1992

The cellulose paradox: pollutant par excellence and/or a reclaimable natural resource?

The various aspects of cellulose as a pollutant are considered in view of its lack of toxicity on the one hand and its recalcitrant durable nature on the other. The microbial degradation of cellulosics is discussed, and the contrast between its success in handling natural cellulosic wastes versus its failure to cope with man-made refuse is described. Research carried out in the past decade has demonstrated that cellulolytic organisms are provided with cell surface multifunctional multienzyme conglomerates, called cellulosomes, which are capable of solubilizing solid cellulosic substrates. The intriguing properties of such complexes include their cohesive nature, their many enzymatic components, and a characteristic glycosylated cellulose-binding, 'scaffolding' component. The latter appears to serve as a substrate-targeting carrier, which delivers the other (hydrolytic) components to the cellulose. Progress in establishing efficient model systems for in vitro solubilization of purified cellulose or natural cellulosic substrates has been achieved using purified cellulosome preparations, fortified with beta-glucosidase and pectinase. The latter enzymes were required in order to alleviate the phenomenon of product inhibition which reduces the efficiency of the free cellulosome. Such combined enzyme systems are proposed as examples of future tailor-made cellulolytic systems for the degradation of natural cellulosics.

Biodegradation, Environmental↗
PubMed1991

Primary structure of O-linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains.

The cell-free forms of the multiple cellulase-containing protein complex (cellulosome), isolated from the cellulolytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5-7% (by mass), consisting of O-linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast-atom-bombardment mass spectrometry (FAB-MS). In addition to the previously described major oligosaccharide, (formula; see text) [Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027-1035], the following partial structures of this compound could be established: (formula; see text). Cell-free and cell-associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.

Bacterial Proteins↗
PubMed1991

Anomalous dissociative behavior of the major glycosylated component of the cellulosome of Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is a highly cohesive multienzyme complex that is capable of completely solubilizing insoluble cellulose. One of the major cellulosomal components, the glycosylated S1 subunit, is believed to play an important structural role and normally migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 210,000. It is shown here that by simply altering the conditions (pH or ionic strength) of the environment prior to electrophoresis, a different migratory profile for S1 emerges, yielding a collection of bands, all of which migrate faster than the parent band. The original electrophoretic behavior of S1 can be reproduced on restoration of the original pH and ionic strength. These results may bear important significance for the physiological role of the S1 subunit in facilitating the observed synergistic action of the other (cellulolytic) components of the cellulosome.

Biotechnology↗
PubMed1991

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.

Animals↗
PubMed1990

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.

Candida↗
PubMed1990

Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry.

A method is presented for encapsulating single microbial cells in small spheres suitable for analysis and sorting by flow cytometry. The entrapped cells are able to multiply and form colonies contained within their respective microspheres. The system is based on ejecting the cells suspended in a gellable liquid through an orifice vibrating at ultrasonic frequencies, thus shearing the cell-containing jet into uniform droplets. When low-melting-temperature agarose was used, the droplets could be gelled into solid spheres during flight by appropriately directed colling air streams. This gelling was accompanied by significant dehydration, resulting in a twofold decrease in bead diameter and a corresponding increase in agarose concentration. Nevertheless, the microbeads obtained were highly uniform and had diameters which could be precisely controlled in the range of 10 to 40 microns. A variety of bacterial and yeast species were entrapped in agarose beads by using this system. In all cases the cells were able to develop into microcolonies containing as many as several hundred cells. This system enables one to apply the powerful method of flow cytometry to the analysis and sorting of whole microbial colonies. Potential applications of this technology in various areas of microbiology are considered.

Bacteria↗
PubMed1990

Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes.

Xylanase activity of Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, was characterized. The activity was localized both in the cellulosome (the principal multienzyme, cellulose-solubilizing protein complex) and in noncellulosomal fractions. Each of these fractions contained at least four major polypeptide bands which contributed to the xylanolytic activity. In both cases, pH and temperature optima, product pattern, and other features of the xylanase activity were almost identical. The main difference was in the average molecular weights of the respective polypeptides which appeared responsible for the activity. In the noncellulosomal fraction, xylanases with Mrs ranging from 30,000 to 65,000 were detected. Distinct from these were the cellulosomal xylanases, which exhibited much larger Mrs (up to 170,000). The cellulosome-associated xylanases corresponded to known cellulosomal subunits, some of which also exhibited endoglucanase activity, and others which coincided with subunits which appeared to express exoglucanaselike activity. In contrast, the noncellulosomal xylanases hydrolyzed xylan exclusively. beta-Glucosidase and beta-xylosidase activities were shown to be the action of different enzymes; both were associated exclusively with the cell and were not components of the cellulosome. Despite the lack of growth on and utilization of xylan or its degradation products, C. thermocellum produces a highly developed xylanolytic apparatus which is interlinked with its cellulase system.

Bacterial Adhesion↗
PubMed1989

Novel O-linked carbohydrate chains in the cellulase complex (cellulosome) of Clostridium thermocellum. 3-O-Methyl-N-acetylglucosamine as a constituent of a glycoprotein.

Alkaline borohydride treatment of the cellulosome of Clostridium thermocellum yielded two major oligosaccharide-alditols, namely D-Galp-beta(1----4)-D-GalOH and (formula; see text) The compounds, isolated via gel permeation chromatography and high performance liquid chromatography, were analyzed by monosaccharide analysis, methylation analysis, gas-liquid chromatography/mass spectrometry, fast atom bombardment/mass spectrometry, and one- and two-dimensional 500-MHz (COSY, HOHAHA, ROESY) 1H NMR spectroscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with blotting technology indicated that the tetrasaccharide is mainly associated with one of the cellulosome subunits.

Acetylglucosamine↗
PubMed1987

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.

Bacteria↗
PubMed1986

Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose.

The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy. When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures. However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate). In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate. The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.

Bacterial Proteins↗
PubMed1985

Organization and distribution of the cellulosome in Clostridium thermocellum.

The properties of the cellulosome (the cellulose-binding, multicellulase-containing protein complex) in Clostridium thermocellum were examined by comparing the cellulase systems derived from the wild type and an adherence-defective mutant. The growth conditions--specifically, growth either on cellulose (Avicel) or on cellobiose as insoluble or soluble carbon sources, respectively--were found to be critical to the distribution of the cellulosome in the mutant system: the cellobiose-grown mutant (in contrast to the wild type) lacked the cellulosome on its surface and produced only minor quantities of the extracellular cellulosome accompanied by other relatively low-molecular-weight cellulases. The polypeptide composition of the respective purified cellulosome was dependent on the nature of the carbon source and was similar for both wild-type and mutant cells. Ultrastructural analysis revealed the presence of novel polycellulosomal protuberances on the cell surface of the cellobiose-grown wild type which were absent in the mutant.

Cell Membrane↗
PubMed1976

Comparative studies on amino and thiol groups in myosins from different sources.

Myosins from rabbit white and red skeletal, rabbit heart, fish skeletal and chicken gizzard muscles, as well as from human platelets were subjected to trinitrophenylation by trinitrobenzene sulfonate and alkylation by N-ethylmeleimide which affected their amino and thiol groups, respectively. The blocking of amino groups was carried out in the presence or in the absence of Mg-ADP and was followed both spectrophotometrically and enzymatically. Essential amino groups, whose modification throughly changes the enzymic characteristics of myosin, were found in heart and in all skeletal muscle myosins but were absent in myosins from chicken gizzard muscle and from human platelets. The reaction of these amino groups was highly retarded in the presence of Mg-ADP. Alkylation of thiols led to loss of the K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in all myosins. However, the rate of loss of activity varied from one myosin to another and, for a given myosin, was affected by the presence of nucleotides and by the value of the ionic strength. The change in Ca(2+)-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) on alkylation was influenced by the presence of Mg - ADP during the reaction. In the absence of this nucleotide, the Ca(2+)-ATPase activity increased and reached a plateau as a consequence of modification. The extent of activation largely depended on the origin of the myosin. When alkylation was carried out in the presence of Mg-ADP, the Ca(2+)-ATPase activity as a function of time exhibited a maximum but the descending part of the curve was absent in myosins from heart and gizzard muscles.

Adenosine Triphosphatases↗
PubMed1976

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K+- and Ca2+-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.

Adenosine Triphosphatases↗