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At least 865 records · Page 48Linked to original sources
PubMed2002

Electrorelease of Escherichia coli nucleoids.

Bacterial chromosome is assembled and folded into one or several nucleoids, depending on the metabolic status of the cell. Development of reliable nucleoid isolation protocols has always been an objective for researchers. A rapid and reproducible procedure for isolation of E. coli nucleoids is described here, while the cell envelope is maintained. Membrane dispersions and vesicles were prepared by lysozyme-EDTA treatment with subsequent rupture of the spheroplasts by electric field. Under these conditions the yield of electroreleased nucleoids was around 90%. The extent of DNA-envelope contacts was determined by light microscopy employing phase contrast and fluorescence modes.

Cell Fractionation↗
PubMed1998

Two internal pools of soluble polyphosphate in the cyanobacterium Synechocystis sp. strain PCC 6308: an in vivo 31P NMR spectroscopic study.

Two intracellular pools of soluble polyphosphate were identified by in vivo 31P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate. The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. 31P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment. Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value. These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA. Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells.

Cyanobacteria↗
PubMed1999

Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris.

We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichia pastoris expression system. A cDNA fragment encoding the amino-terminal domain of mouse SHH was inserted downstream of the Saccharomyces cerevisiae alpha-mating factor secretion signal. The DNA fragment was introduced into the host genome by the spheroplast transformation method. Transformants were selected based on their resistance to G418: His+ transformants which showed resistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His+ clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of mouse SHH into culture fluid. The yield of secreted SHH was estimated to be 50 micrograms/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH.

Animals↗
PubMed2005

Accumulation of polyphosphates and expression of high molecular weight exopolyphosphatase in the yeast Saccharomyces cerevisiae.

The effect of cultivation time and concentration of inorganic phosphate (P(i)) in the culture medium on the accumulation of polyphosphates (polyP) and the activity of two cytosolic exopolyphosphatases of the yeast Saccharomyces cerevisiae was studied: an exopolyphosphatase of 40 kD encoded by PPX1 and a high molecular weight exopolyphosphatase encoded by another gene. Depletion of polyP in the cells on P(i) starvation is a signal factor for the accumulation of polyP after the subsequent addition of 5-20 mM P(i) and glucose to the cells or spheroplasts. A high activity of both exopolyphosphatases does not prevent the accumulation of polyP. The expression of the high molecular weight exopolyphosphatase is due to the acceleration of metabolism in cells that have reached the stage of growth deceleration on the addition of P(i) and glucose or complete culture medium. This process may occur independently from the accumulation of polyP. The activity of exopolyphosphatase PPX1 depends less on the mentioned factors, decreasing 10-fold only under conditions of phosphate surplus at the stationary growth stage.

Acid Anhydride Hydrolases↗
PubMed1987

Methods for DNA extraction from Candida albicans.

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.

Candida albicans↗
PubMed1989

Preparation of RNA from unspheroplasted yeast cells (Saccharomyces cerevisiae).

High-quality RNA can be prepared from up to 100-ml culture volumes of unspheroplasted yeast cells (Saccharomyces cerevisiae) via homogenization in high-temperature phenol:chloroform mixtures. The yield of RNA from this preparative method is equivalent to those of other methods requiring preliminary spheroplasting of cells. Quality and quantity of recovered RNA are independent of yeast strain and cell growth medium used, and the method works equally well on cells in either log phase growth or in stationary phase. Mitochondrial RNAs recovered as part of whole cell RNA mixtures may be slightly degraded. Analyses of individual transcripts in the recovered RNA mixtures suggest that there is no selection for or against any specific single transcript or any group of transcripts when RNA is prepared by this method.

Blotting, Northern↗
PubMed1977

Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum.

Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.

Bacterial Chromatophores↗
PubMed1985

Kinetics of the c-cytochromes in chromatophores from Rhodopseudomonas sphaeroides as a function of the concentration of cytochrome c2. Influence of this concentration on the oscillation of the secondary acceptor of the reaction centers QB.

The oxidation kinetics of Cyt c1 and c2 have been measured in normal chromatophores and in chromatophores fused with liposomes in order to increase the internal volume. The kinetics of Cyt c1 oxidation were found to be dependent on Cyt c2 concentration. The initial rate of Cyt c1 oxidation decreased after fusion by a factor of about two, indicating a process dependent on diffusion. The results do not allow a clear distinction between a diffusion of Cyt c2 along the inner membrane surface or through the inner volume of the vesicle; two- and three-dimensional models are discussed. In contrast to Cyt c1, the kinetics of oxidation of Cyt c2 were not influenced by changes in concentration. It is concluded that reduced Cyt c2 is preferentially bound to the reaction centers. A binary pattern as a function of flash number from the dark-adapted state was measured in the turn-over of the two-electron gate of the reaction center. In chromatophores with more than 0.5 cytochrome c2 molecules per reaction center, this binary pattern titrated out with a midpoint around 340 mV on reduction of the suspension. In experiments with chromatophores with a low Cyt c2 content, or with spheroplast-derived vesicles which had lost Cyt c2, the binary oscillation in the two-electron gate could be observed at much lower potentials. The results suggest that the binding of reduced cytochrome c2 modifies the behavior of the two-electron gate. A model in which reaction center dimers are stabilized by Cyt c2 is proposed to explain the effect.

Bacterial Chromatophores↗
PubMed1979

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae.

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.

Adenosine↗
PubMed1979

Small angle x-ray scattering from the inner and outer membranes from Escherichia coli.

Small angle X-ray data from purified forms of inner or cytoplasmic and outer membranes from Escherichia coli have been obtained and appear to be qualitatively similar. Transitory changes are apparent in the circularly averaged X-ray profiles from inner membranes. Such results could be due to the loss or denaturation of peripheral membrane proteins. Some partially dried forms of outer membrane are partly ordered and produce diffraction patterns which support an underlying bilayer structure. An extra light membrane fraction which results from membrane preparations utilizing a French pressure cell for spheroplast disruption has been characterized and shown to be similar to inner membrane. The purified membranes produce small angle X-ray diffraction patterns which are much different from those of lipid dispersions and the differences are attributable to the high protein content of the intact membranes. While the small angle X-ray region may be useful for characterizing the membrane preparations, the paucity of detail in the diffraction pattern suggest that it will be of little value in describing the complex underlying membrane structure.

Cell Membrane↗
PubMed1976

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.

Amidohydrolases↗
PubMed1975

13C-enriched phosphatidylethanolamines from Escherichia coli.

Escherichia coli have been grown using [1-13C]acetate and [2-13C]acetate as the sole carbon source. The 13C NMR spectra of the whole cells and spheroplasts can be readily obtained but give limited information. The 13C NMR spectra of t-e isolated 13C-enriched phosphatidylethanolamines are assigned and analysed to give the biosynthetic pathway for acetate incorporation. This method provides a ready source of 13C-enriched phospholipids.

Acetates↗
PubMed1977

Inactivation of infectivity of phiX174 DNA by menadione and reduced menadione.

Interaction of menadione and reduced menadione with phage phiX174 DNA was investigated. A concentration of 2-10(-4) M menadione inactivated 60% of the infectivity of phiX174 DNA to spheroplasts of Escherichia coli, while reduced menadione inactivated 97% of the infectivity of phiX174 DNA at the same concentration. Alkaline sucrose gradient centrifugation revealed 2-10(-5) M reduced menadione caused approximately 24% of phiX174 DNA to produce strand break under the condition of 80% lethanlity. DNA strand break was not observed even at 4 - 10(-4) M menadione. These results indicated that there were different mechanisms for inactivation of phiX174 DNA between menadione and reduced menadione.

Coliphages↗
PubMed1977

Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.

Coliphages↗
PubMed1978

Heterologous transfection with bacteriophage phiX174 DNA: and improved system.

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage phiX174 DNA was established. By mild washing of the speroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of phiX174 progeny phage production and not on those of phiX174 dougle-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage phiX174 DNA can be detected and the number of molecules can be determined with accuracy.

Bacteria↗
PubMed1975

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Amino Acids↗
PubMed1983

Simultaneous isolation of the yeast cytosol and well-preserved mitochondria with negligible contamination by vacuolar proteinases.

Disruption of yeast spheroplasts by DEAE-dextran in isoosmotic conditions allows isolation of relatively undamaged subcellular fractions from yeast. The preservation of mitochondria and vacuoles permits the simultaneous isolation of the cytosol with negligible contamination by vacuolar proteinases and therefore, virtually eliminates proteolytic artefacts.

Cell Fractionation↗
PubMed1996

Formation of disulfide bonded dimer of mutated heat-labile enterotoxin in vivo.

One of the two cysteines in the B subunit of heat-labile enterotoxin has been changed to a serine by site-directed mutagenesis so that the internal disulfide bond cannot form. The mutant protein, like the wild-type protein synthesised in the presence of the reducing agent dithiothreitol, does not form pentamers in the periplasm but binds to available membranes. Binding to membranes is disrupted by chaotropic agents but not by salt. More than half the molecules of mutant protein form disulfide-bonded dimers when exported to the periplasm but no dimer is detected when the protein is exported to the medium by spheroplasts.

Bacterial Toxins↗