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PubMed1999

Transformation of Candida albicans by electroporation.

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.

Blotting, Southern↗
PubMed1995

A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.

Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys. The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form. A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified. The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20. Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence. p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins. Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro. These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane.

Amino Acid Sequence↗
PubMed1982

[Arthrobacter globiformis - a new yeast-lysing bacterium].

A bacterium capable to lyse viable yeast cells was isolated from compost enriched with baker's yeast cells and was identified as Arthrobacter globiformis. The yeast lytic enzyme complex produced by shaking culture was precipitated with ammonium sulfate, dialyzed and lyophilized. The crude residue contained beta-glucanase and alpha-mannanase, yet not chitinase. Optimale carbon sources in the culture medium for a high enzyme synthesis were 0.5% beta-glucan for the production of beta-glucanase, resp., 3% alpha-mannan for the production of alpha-mannanase. Addition of 10% whole died baker's yeast cells to the culture medium effected similar results. The crude enzyme residue released among other things spheroplasts from cells of the yeast Pichia membranaefaciens, Metschnikowia pulcherrima, Hansenula anomala and Candida guilliermondii "H". However, it did not or only weakly lyse viable cells of the yeasts Rhodotorula rubra, Rhodosporidium toruloides and Sporobolomyces roseus. The mutants of Candida guilliermondii "H" with modified glucan, resp., mannan concentrations in the cell walls did not indicate differences in their susceptibility to lysis.

Arthrobacter↗
PubMed1980

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria.

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH2-cytochrome c reductase activity with a turnover number of 15.7 sec(-1) and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c1 per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (35S)-methionine in the absence or presence of inhibitors of cytoplasmic or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using anti-serum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (35S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.

Cytochrome b Group↗
PubMed1992

RNA delivery in Saccharomyces cerevisiae using electroporation.

An efficient delivery method for introducing in vitro synthesized RNA into yeast has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introducing those RNAs that cannot be made in vivo, such as poly(A)- mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation.

Animals↗
PubMed1999

Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain.

The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.

Amino Acid Sequence↗
PubMed1994

pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.

Filamentous phage pIV is an outer membrane protein required for phage assembly and secretion. Chemical cross-linking and sedimentation experiments have been used to demonstrate that pIV from f1-infected Escherichia coli exists as a homo-multimer, probably composed of 10 to 12 subunits. pIV secreted from spheroplasts remains soluble and does not form multimers. Synthesis of pIV from distantly related filamentous phages or from a bacterial homolog that participates in a specialized form of extra-cellular protein secretion in the same cell with pIVf1 resulted in the formation of mixed multimers. This suggests that the homologous proteins themselves form homo-multimers. These structures could form gated channels that conduct assembling phage or specific substrate proteins across the outer membrane to the extracellular milieu.

Bacterial Proteins↗
PubMed1997

Improved membrane isolation in the purification of beta 2-adrenoceptors from transgenic Escherichia coli.

beta 2-Adrenoceptors (beta 2-AR) have been purified from many mammalian tissues. Unfortunately, other beta-AR subtypes expressed in the same cells are usually copurified, contaminating the preparation and interfering with subsequent investigations such as receptor characterization, ligand binding studies, immunoprecipitation, or development of anti-receptor antibodies. The advent of molecular biology techniques has facilitated the expression of beta 2-AR in cells in which no other similar molecules are present; thus, receptor purification has been simplified. beta 2-AR expressed in Escherichia coli provides a convenient source of receptor without the need for specialized culture facilities required for eukaryotic cells. The greater complexity of the gram-negative cell wall structure, however, complicates the purification of membrane-bound receptor from this source. In this report, we describe a reliable method for the partial purification of membrane-bound beta 2-AR from transgenic E. coli. Spheroplast formation followed by cell disruption and a carbonate wash procedure provided beta 2-AR bound to bacterial inner membrane in high yield.

Carbonates↗
PubMed1992

Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri.

Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N2O reductase and nitrite reductase (cytochrome cd1) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N2O reductase in a mutant strain deficient in the chromophore of N2O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.

Bacterial Proteins↗
PubMed1994

In vivo analysis of chromatin following nystatin-mediated import of active enzymes into Saccharomyces cerevisiae.

In vivo DNA-protein interactions are usually studied at the molecular level using DNA-degrading agents of low molecular weight. In order to be useful, macromolecular probes of chromatin structure, such as enzymes must first cross the cell membrane. In this paper we describe the introduction and evaluation of macromolecules with enzymatic activity into yeast spheroplasts treated with the polyene antibiotic nystatin. We report the low resolution analysis of chromatin structure in the promoter region of the Saccharomyces cerevisiae gene encoding DNA topoisomerase I by this technique using micrococcal nuclease and restriction enzymes.

Base Sequence↗
PubMed1989

High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.

DNA, Fungal↗
PubMed1981

Liposome-mediated genetic transformation of Neurospora crassa.

Transformation of Neurospora crassa spheroplasts is reported for three different genes, using uncloned Neurospora DNA, both naked and encapsulated in synthetic phosphatidylserine liposomes. Whereas transformation by naked DNA is DNase-sensitive, that by liposomes is not. Per unit of transforming DNA, liposome transformation is significantly more efficient than that with naked DNA, ranging from 19x for the am gene to 41x for pyr-3. Levels of activity of pyr-3 and am transformants, and segregation data on pyr-3 transformation are given.

DNA, Fungal↗
PubMed1989

Thiolutin inhibits utilization of glucose and other carbon sources in cells of Escherichia coli.

Thiolutin was found to inhibit the utilization of glucose and other growth substrates in Escherichia coli. The inhibition was detected by a sharp drop of the respiration rate after addition of the antibiotic. The actual function affected was allocated to the cytoplasmic membrane of the bacterial cells by the following evidence: --spheroplasts were affected like intact cells, --individual reactions of either the electron transport chain or the glycolytic pathway were not inhibited, --glucose consumption in the culture stopped and the cells accumulated guanosine tetraphosphate as under starvation conditions, --activation of the cell's apo-glucose dehydrogenase restored respiration via bypassing the glucose phosphotransferase system. It was concluded that the transport of certain substrates across the membrane was inhibited.

Anti-Bacterial Agents↗
PubMed1975

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides.

A cell envelope fraction had been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm3) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm3) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O2 pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm3). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.

Aerobiosis↗
PubMed1975

Interaction between Bdellovibrio bacteriovorus and the cytoplasmic membrane of Escherichia coli B.

Adsorption of Bdellovibrio bacteriovours (Bdv) on the surface of Escherichia coli is accompanied by a sharp decrease in the initial rate of entry of alpha-methylglucoside-C-14 and thiomethlgalactopyranoside-C-14 into the host cell. Interaction between the parasite and E. coli leads to the rapid departure of previously accumulated labeled glucosides and beta-galactosides from the bacteria. Meanwhile the ATPcontent in E. coli falls sharply. Adsorption Bdv E.coli spheroplasts was established as a fact. The possible mechanisms of interaction between Bvd and the host cell at the cytoplasmic membrane level are discussed.

Adenosine Triphosphate↗
PubMedDate not supplied

Genetic organization of Acetobacter for acetic acid fermentation.

Plasmid vectors for the acetic acid-producing strains of Acetobacter and Gluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochrome c, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene of A. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of an A. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of the Acetobacter strains was developed and applied to improve their properties.

Acetates↗
PubMed1995

Killer-toxin-resistant kre12 mutants of Saccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor.

The Saccharomyces cerevisiae killer toxin K1 is a secreted alpha/beta-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to beta-1,6-D-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of the KRE genes whose products are involved in synthesis and/or assembly of cell wall beta-D-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strains indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), rendering kre12 mutants incapable of binding significant amounts of toxin to the membrane. Since kre12 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from a kre12 mutant and from an isogenic Kre12(+) strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in the kre12 mutant. A model for K1 killer toxin action is presented in which the gene product of KRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.

Cell Wall↗