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R Lamed

Publications and source records attributed to R Lamed.

At least 55 records · Page 3Linked to original sources
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

Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A.

A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60 degrees C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75 degrees C and pH 5.0, respectively. Both the enzyme activities were stable up to 70 degrees C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70 degrees C and 45 min at 75 degrees C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by beta- and gamma-cyclodextrins but not by alpha-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in alpha1,6 linkages and produced multiple saccharides from cleavage of alpha-1,4 linkages in starch. The K(m)s for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.

Journal Article↗
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↗
PubMed1983

Rapid identification of Streptococcus pyogenes by flow cytometry.

Flow cytometry combined with immunofluorescence of Streptococcus pyogenes was used to assay bacteria suspended in buffer solution and in saliva derived from throat swabs of healthy volunteers. The method allowed the enumeration of as few as 5 X 10(3) and 5 X 10(4) CFU per milliliter of buffer and saliva respectively. Controls including Streptococcus salivarius instead of Streptococcus pyogenes or buffer instead of specific antibodies confirmed the specificity of the detection of Streptococcus pyogenes in the samples. The results suggest that flow cytometry may serve as a basis for an automated reliable method for the diagnosis of streptococcal infections.

Culture Media↗
PubMed1983

Tetra- and hexahydro derivatives of aldosterone and 18-hydroxycorticosterone by chemical and microbial reductions.

Preparative methods were developed for reduction with NaBH4 at 0 of 3 beta, 5 alpha- and 3 alpha, 5 beta-tetrahydroaldosterone (1) and (12) to their respective 20 alpha-ol derivatives 2a and 13a. Corroboration of structures was obtained by periodate oxidations to the lactols 3b and 14b and thence, by further oxidation, to the lactones 4 and 15 respectively; these lactones were also independently obtained from 1 and 12. Reduction with NaBH4 at 80 degrees C converted 1 and 12 into 18-hydroxy-3 beta, 5 alpha, 20- and 18-hydroxy-3 alpha, 5 beta, 20-hexahydrocorticosterone 6a and 17a respectively, which were mixtures of epimers at C-20. Compound 17a could also be prepared by reduction of the lactone 21 with sodium aluminum bis-(methoxyethoxy) hydride. Again, periodate oxidations of 6a and 17a gave the lactols 7b and 22b and thence, by Jones oxidation, the diketolactones 8 and 23, which were also prepared from 18-hydroxy-11-dehydrocorticosterone (10) and 18-hydroxycorticosterone (24) respectively. Improved conditions for reduction with Clostridium paraputrificum permitted convenient conversion of aldosterone (11), the corresponding 18 leads to 11 lactone 18a and 18-hydroxycorticosterone (24) into their 3 alpha, 5 beta-tetrahydro derivatives.

18-Hydroxycorticosterone↗
PubMed1983

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.

Agglutination↗
PubMed1983

Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.

The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding. It appears that the CBF is not only responsible for the adherence of the cells to cellulose but also constitutes a major part of the cellulolytic apparatus of this organism.

Cellulase↗
PubMed1981

Thermostable, ammonium-activated malic enzyme of Clostridium thermocellum.

"Malic" enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) was purified from Clostridium thermocellum by DEAE-cellulose, agarose-NADP and Sephadex G-200 column chromatography. The 117-fold purified "malic" enzyme displayed a maximum activity of 135 units/mg at 40 degrees C and represented 0.8% of the total cell protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of the protein suggested 90% purity and an approximate tetrameric subunit molecular weight of 40 000. The enzyme absolutely required both bivalent and monovalent cations for catalysis. Mn2+ and NH4+ were the most effective cationic activators examined. Increasing NH4+ concentration increased both enzyme activity and affinity toward L-malate. The apparent Km for L-malate was 3 X 10(-4) M at 0.4 mM NH4Cl. Enzyme activity increased linearly when temperature was raised between 22-60 degrees C and a Q10 of 2.1 was calculated from an Arrhenius plot. The enzyme was stable at heating at 60 degrees C but was denatured at higher temperatures. The enzyme half-life was 10 min at 72 degrees C. The enzyme displayed a broad pH optimum (7.2-87.2 for Tris-HCl buffer) but was inactivated by p-chloromercuribenzoate. The high thermal stability, low apparent molecular weight and NH4+ activation are properties not common to all previously described "malic" enzymes.

Ammonium Sulfate↗
PubMed1981

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.

Acetone↗
PubMed1980

Glucose fermentation pathway of Thermoanaerobium brockii.

Thermoanaerobium brockii was shown to catabolize glucose via the Embden-Meyerhof-Parnas pathway into ethanol, acetic acid, H(2)-CO(2), and lactic acid. Radioactive tracer studies, employing specifically labeled [(14)C]glucose, demonstrated significant fermentation of (14)CO(2) from C-3 and C-4 of the substrate exclusively. All extracts contained sufficient levels of activity (expressed in micromoles per minute per milligram of protein at 40 degrees C) to assign a catabolic role for the following enzymes: glucokinase, 0.40; fructose-1,6-diphosphate aldolase, 0.23; glyceraldehyde-3-phosphate dehydrogenase, 1.73; pyruvate kinase, 0.36; lactate dehydrogenase (fructose-1,6-diphosphate activated), 0.55; pyruvate dehydrogenase (coenzyme A acetylating), 0.53; hydrogenase, 3.3; phosphotransacetylase, 0.55; acetaldehyde dehydrogenase (coenzyme A acetylating), 0.15; ethanol dehydrogenase, 1.57; and acetate kinase, 1.50. All pyridine nucleotide-linked oxidoreductases examined were specific for nicotinamide adenine dinucleotide, except ethanol dehydrogenase which displayed both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked activities. Fermentation product balances and cell growth yields supported the glucose catabolic pathway described. Representative balanced end product yields (in moles per mole of glucose fermented) were: ethanol, 0.94; l-lactate, 0.84; acetate, 0.20; CO(2), 1.31; and H(2), 0.50. Growth yields of 16.4 g of cells per mole of glucose were demonstrated. Both growth and end product yields varied significantly in accordance with the specific medium composition and incubation time.

Bacteria↗
PubMed1980

Ethanol production by thermophilic bacteria: relationship between fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and Thermoanaerobium brockii.

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H(2) and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H(2) addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate-activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K(m), V(max), and Q(10) values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H(2) production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.

Acetate Kinase↗