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PubMed1973

Evidence for the direct action of colicin K on aerobic 32 P i uptake in Escherichia coli in vivo and in vitro.

Pentachlorophenol (PCP)-sensitive incorporation of (32)P-labeled orthophosphate ((32)P(i)) into nucleotides and nucleic acids by disrupted spheroplasts of Escherichia coli was inhibited by addition of colicin K. Incorporation by intact cells was also inhibited by a similar concentration of colicin K. Various colicin K-resistant mutants were isolated, and their ability to incorporate (32)P(i) was tested. When T6(r)-colK(r) mutants (T6 phage-resistant) and tol I mutants (T6-sensitive, colicin E-sensitive) were converted to disrupted spheroplasts, their (32)P(i)-incorporation became sensitive to colicin K. On the contrary, incorporation by disrupted spheroplasts from tol II mutants (T6-sensitive, colicin E-resistant) was fairly resistant to colicin K like that of intact cells. A modification of the cell surface of T6(r)-colK(r) mutants, caused by mutation to novobiocin-permeable, T4 phage-resistant cells, restored the sensitivity of the cells to colicin K. The modified T6(r)-colK(r) cells did not adsorb T6 phage or colicin K, indicating that the receptors for T6 phage or colicin K are not reactivated by this modification. Similar treatment of tol I mutants did not have this effect. These observations strongly suggest that colicin K can act on its target on the cell membrane if it can penetrate the cell surface to reach this target. The receptor for colicin K on the cell surface, which may be part of the T6 phage-receptor, may have some unknown function in relation to the action of colicin K in normal cells, but tends to become dispensable if the cells become permeable to colicin K.

Adsorption↗
PubMed1974

Orientation of membrane vesicles from Escherichia coli as detected by freeze-cleave electron microscopy.

The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K(+)-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na(+)-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K(+)-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that "dislocation" of membrane proteins occurs during osmotic lysis of spheroplasts.

Bacterial Proteins↗
PubMed1975

Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli.

In vitro deoxyribonucleic acid (DNA) synthesis systems based on an earlier system using pencillin have been developed which use osmotic lysis of lysozyme-formed spheroplasts of Escherichia coli cells embedded in an agarose matrix. An adenosine 5'-triphosphate (ATP)-dependent semiconservative mode, or replicative mode, of in vitro DNA synthesis is exhibited which is sensitivie to nalidixic acid. These systems require growth of the agar-embedded cells in a preincubation medium before spheroplast formation and osmotic lysis. Inhibitor studies suggest that one or more required macromolecular species are synthesized during this preincubation growth period. Osmotic shock fluid from E. coli contains macromolecular factors which preferentially stimulate the ATP- dependent semiconservative mode of in vitro DNA synthesis. In some cases, the ATP independent mode of synthesis is inhibited by shock fluid. Evidence is presented that the stimulating factors found in the osmotic shock fluid come from the E. coli periplasmic space. This stimulation is observed using either toluene-treated cells or lysed agar-embedded ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetate-lysozyme spheroplasts, and is thus independent of the in vitro DNA synthesis system used. Shock fluid obtained from a given E. coli dna mutant does not stimulate in vitro DNA synthesis by that mutant. However, in some cases, shock fluid from one class of dna mutants does stimulate ATP dependent in vitro DNA synthesis by another class of dna mutants, in a thermosensitive reacaction. Gently prepared cell extracts also stimulate ATP-dependent in vitro DNA synthesis, whereas cell extracts prepared by more severe procedures inhibit this in vitro synthesis. Severl stimulating DNA replication factors may be present in the osmotic shock fluid, including products of E. coli dna genes.

Adenosine Monophosphate↗
PubMed1976

Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols.

Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8.

Anaerobiosis↗
PubMed1977

Isolation and properties of two classes of low-density vesicles from Saccharomyces cerevisiae.

A mixture of small (0.43-mum diameter) and large (0.62-mum diameter) low-density vesicles from spheroplasts of Saccharomyces cerevisiae was fractionated by rate centrifugation in a gradient of 0 to 8% (wt/vol) Ficoll to yield fractions rich (90 to 95%) in small or large vesicles. The large, but not small, vesicles swelled when diluted into mannitol solutions containing less than 0.4 M mannitol. The pH-electrophoretic mobility curve of the large vesicles showed that they are probably enclosed in a phospholipid-protein membrane. The dyes neutral red and toluidine blue, accumulated into large vesicles by intact cells and spheroplasts, were largely lost from large vesicles when these were separated from stained spheroplasts. Sudan black III stained small and large vesicles, both classes of vesicle retaining the stain on separation. Fractions rich in large vesicles contained proportionately more phospholipid and less free sterols, diacylglycerols, and free fatty acids compared with those enriched in small vesicles. The two classes of vesicles contained about the same proportions of esterified sterols and triacylglycerols. The free fatty acids in both small and large vesicles were free from unsaturated fatty-acyl residues; diacylglycerols and triacylglycerols contained appreciable proportions of unsaturated fatty-acyl residues. Small vesicles were richer in lipase activity, whereas the larger vesicles contained greater beta-glucanase and alpha-mannosidase activities. Phospholipase activity could not be detected in any of the fractions.

Acid Phosphatase↗
PubMed1983

Plasma membrane expansion terminates in Saccharomyces cerevisiae secretion-defective mutants while phospholipid synthesis continues.

Phospholipid synthesis activity and plasma membrane growth have been studied in the Saccharomyces cerevisiae temperature-sensitive, secretion-defective mutants isolated by Novick and Schekman (Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979; Novick et al., Cell 21:205-215, 1980). The mutants, sec1 through sec23, do not grow at 37 degrees C and exhibit lower rates of phospholipid synthesis than does the wild-type strain X2180. None of the mutants exhibits a decline in lipid synthesis rapid enough to explain secretion failure. Plasma membrane growth was assessed indirectly by examining the osmotic sensitivity of spheroplasts derived from cultures transferred from 24 to 37 degrees C. Spheroplasts from the normal-growing strain X2180 exhibited a small rapid increase in osmotic sensitivity and stabilized at a more sensitive state. Spheroplasts from the sec mutants exposed to the same temperature shift exhibited progressively increasing osmotic sensitivity. Cycloheximide treatment prevented progressive increases in osmotic fragility. These data are compatible with the hypothesis that plasma membrane expansion is restricted in the sec mutants. During incubation at 37 degrees C, the accumulation of intracellular materials within the no-longer expanding plasma membrane exerts osmotic stress on the membrane, increasing with time. The gene products defective in Novick and Schekman's sec mutants appear to be required for both extracellular protein secretion and plasma membrane growth in yeast cells.

Cell Membrane↗
PubMed1987

Efficient transfection of the archaebacterium Halobacterium halobium.

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage phi H DNA molecule routinely produced between 5 X 10(6) and 2 X 10(7) transfectants per microgram of DNA. Between 0.5 and 1% of spheroplasts were transfected per microgram of luminal diameter H DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.

Bacteriophages↗
PubMed1986

Saccharomyces cerevisiae nuclear fusion requires prior activation by alpha factor.

We have developed a protocol for efficient fusion of spheroplasts of the same mating type. Nuclear fusion in this whole-cell system is also efficient and closely parallels nuclear fusion in heterosexual mating of intact cells. In the spheroplast fusion system, nuclear fusion is dependent on both the KAR1 gene and prior exposure to alpha factor. The major products of nuclear fusion in the spheroplast fusion assay were true diploids that were homozygous at the mating-type locus. An additional 10% of the products were cells of ploidy greater than diploid. The dependence of nuclear fusion on alpha factor treatment could not be replaced by synchronization in G1 by mutations in CDC28 and CDC35 or by prior arrest in stationary phase. These data suggest that nuclear fusion is not a constitutive function of the nucleus, but rather is specifically induced by mating hormone.

Cell Nucleus↗
PubMedDate not supplied

[Localization of hydrogenase in the cells of the thermophilic hydrogen bacterium, Pseudomonas thermophila].

Spheroplasts that were osmotically stable in 0.2M Tris-HCl--0.02M EDTA were prepared from the autotrophically grown cells of Pseudomonas thermophila K-2. The spheroplasts possessed 90--95% of the hydrogenase activity of the whole cells. The half-life time of hydrogenase in the spheroplasts at 80 degrees C was 8.5 min. A spectrophotometric technique was developed for determining the membrane-bound hydrogenase in the presence of sulfhydryl compounds with methylene blue as electron acceptor. The maximal specific activity of hydrogenase in extracts prepared in the anaerobic conditions in the presence of dithiothreitol and Mg2+ and Mn2+ ions was 10 +/- 3 units per 1 mg of protein, which closely corresponded with the activity of hydrogenase in the whole cells. Almost all activity of hydrogenase assayed with methylene blue was localized in the membrane fraction. The activity of soluble NAD-specific hydrogenase was not detected. Large particles located in 60-70% sucrose had the highest hydrogenase activity upon fractionation in a continuous sucrose concentration gradient. The second, lower peak of the hydrogenase activity was detected in fractions of 40--50% sucrose. As was found by electron microscopy, the size of membrane vesicles with the hydrogenase activity varied within the range of 68--156 nm. The membrane preparations possessed the activity of NADH-dehydrogenase, NADH-oxidase and succinate dehydrogenase as well.

Centrifugation, Density Gradient↗
PubMed1989

[In vitro effect of itraconazole against various species of Candida].

The authors compared the in vitro antifungal activity of itraconazole to that of miconazole against various species of Candida. The MIC of 88 strains of Candida (C. albicans, C. guilliermondii, C. parapsilosis, C. glabrata) was determined by the agar dilution method using casitone medium at pH 5.5-5.6 for miconazole and pH 7.2-7.4 for itraconazole. C. albicans and C. glabrata were found to be less sensitive in vitro to itraconazole than to miconazole; the contrary was observed for C. parapsilosis. The sensitivity of C. guilliermondii to these antifungal agents was similar. The MIC 50, MIC 90 and G.MIC values for itraconazole with respect to 42 strains of C. albicans was 0.10, 1.4 and 0.138 micrograms ml-1, respectively; the MIC 90 and G.MIC values for miconazole were 0.5 and 0.021 micrograms ml-1, respectively. The effect of itraconazole on the ultrastructure of C. albicans yeast cells and spheroplasts was studied by scanning electron microscopy. This triazole molecule modified the cell wall of C. albicans, caused cell stretching and provoked defective separation between mother and daughter cells. Itraconazole altered the cytoplasmic membrane of the spheroplasts causing them to have a "spongy" appearance. Yeast cells treated with itraconazole appeared to liberate their spheroplast with difficulty.

Candida↗
PubMed1987

Electron microscopic analysis of in vitro interaction of Rickettsia prowazekii with guinea pig macrophages. I. Macrophages from nonimmune animals.

Monolayer cultures of peritoneal macrophages of intact guinea pigs were infected with Rickettsia prowazekii (strain Breinl) and examined by electron microscopy after 30 min, 4 and 24 hr post-infection (p.i.). Three parallel processes developed in infected macrophages: reproduction of rickettsiae in macrophage cytoplasm, destruction in phagolysosomes and production of spheroplast-like forms. Reproduction of rickettsiae yielded 2 cell types: those with dense and with light cytoplasm; they were located side by side in the microcolony and seemed to have a common capsule-like coat. Relatively small spheroplast-like forms of about 1 micron in size were regularly detected. Addition of immune serum to macrophages increased the number of rickettsiae, both of rod-shaped as well as of spheroplast-like ones located within phagosomes, but elicited no increase in the number of digested pathogen cells.

Animals↗
PubMedDate not supplied

[Study of the sequential stages of Proteus vulgaris L-transformation by scanning electron microscopy].

Consecutive stages of L-transformation caused by penicillin in Proteus vulgaris were studied, from spheroplasts and unstable, easily reversing L-forms to stable L-forms which did not reverse. To maintain natural topography of the culture on the growth medium, millipore filters were placed on the surface of the medium and then were fixed with the culture. All the main elements of L-cultures were found at the stage of spheroplast formation, before the first passage onto the medium for cultivating L-forms, i.e. spherical cells having a diameter from 3.5 to 1 mcm, elementary bodies 1.0-0.6 mcm large, filamentous structures with a diameter to 4 mcm, large bodies having dimensions of 20 mcm and more, and "cell-free" masses. The composition of the structural elements and their reproduction are supposed to be almost the same in the bacterial culture and L-forms at the stage of spheroplast formation.

L Forms↗
PubMed1982

[Production of pesticins on synthetic nutrient media].

It was found that 96.2 per cent of the plague microbe cultures studied had a capacity to produce pesticin I on solid and liquid minimum synthetic nutrient media providing growth of the cultures. No pesticin III was detected in the spheroplasts of plague microbes, when they were grown on the synthetic nutrient medium, while on the complete nutrient medium the inhibition zones were observed which was indicative of pesticin III synthesis. The average value characterizing the titer of pesticin I produced by the rod-shaped forms of the plague microbe on the complete and synthetic nutrient media was 320. An increase in the titer of the spheroplast pesticin was shown, while the pesticin titer of the rod-shaped forms was low (1 : 20--1 : 320). As a result the average titer of the spheroplast pesticin was higher than that of pesticin of the rod-like forms. On the complete nutrient medium it was 1280 and on the synthetic medium it was 640.

Bacteriocins↗
PubMed1976

[Mechanism of the stimulating effect of protamines on the infectivity of DNA phages].

The interaction of styrin (protamine from sturgeon milt) with E. coli spheroplasts was investigated with the aid of the fluorescent probe--5-dimethylamino-1-naphthalenesulfonyl-chloride (DNS). Not less than 40% of the total (15 mug ml-1) DNA-protamine concentration was bound to spheroplasts. A part of the protein was found in the isolated membranes and inside the cell. There was a correlation between the binding of protein with spheroplasts and its biological action. The dansylation somehow decreased the protein stimulating activity in transfection.

Animals↗
PubMed1994

Separation of inner and outer membrane vesicles from Escherichia coli in self-generating Percoll gradients.

A rapid and simple method for separating and isolating the inner and outer membranes of Escherichia coli is described. Membrane vesicles were prepared either by passing the bacteria through a French press or by conversion of the cells to spheroplasts by the lysozyme-EDTA treatment and disruption of the spheroplasts by sonication. The membrane vesicles were collected by ultracentrifugation and suspended in a Percoll-containing buffer. The membranes were separated by centrifugation of the membrane-Percoll mixture in a fixed angle rotor at 27,000gmax for 30 min in a preparative centrifuge. One low-density and one high-density band was obtained, corresponding to the inner and outer membranes, respectively. For the membranes prepared by French pressing 69 and 3.3% of the total activity in the gradient of the inner membrane marker NADH-oxidase was found in the low-density and the high-density bands, respectively. For the outer membrane marker 2-keto-3-deoxyoctonate (KDO), 69 and 7.3% of the total amount of KDO in the gradient was found in the high-density and the low-density bands, respectively. For the membranes prepared by sonication of spheroplasts the same figures were 39 and 6.5% for NADH-oxidase and 52 and 9.0% for KDO. The total time of preparation of membrane vesicles, from harvesting the bacteria to the separation of the inner and outer membrane vesicles, is about 6 h. A good separation of the inner and outer membranes was still obtained when samples corresponding to about 10 mg of membrane protein were added to a 33-ml gradient.

Cell Membrane↗
PubMed1982

Depurination of DNA as a possible mutagenic pathway for cells.

The possible consequences of depurination for both spontaneous and induced mutagenesis were investigated using in vitro and in vivo assays. Depurination of synthetic polynucleotide templates such as poly [d(A-T)] or poly [d(G-C)] leads to increased misincorporation of noncomplementary nucleotides when these templates are copied by prokaryotic and eukaryotic DNA polymerases. The ability of Escherichia coli DNA polymerase I to copy over apurinic sites was demonstrated using single-stranded circular DNA of bacteriophage 0X174 as a template and starting DNA synthesis at a fixed point. Analysis of the newly synthesized 0X174 restriction fragments on neutral and alkaline sucrose gradients shows that synthesis proceeded past apurinic sites. When using depurinated 0X174 DNA containing the am3 amber mutation as a template for copying by E. coli DNA polymerase I, an increased reversion to wild type is observed after transfection into E. coli spheroplasts. The enhancement in reversion frequency is proportional to the extent of depurination, suggesting that depurination is also mutagenic during copying natural DNA in vitro. When noncopied depurinated 0X174 am3 DNA is transfected in E. coli spheroplasts, no increase in reversion frequency is observed above background level. However, when the spheroplasts are derived from bacteria in which the SOS response had been induced by UV irradiation, a substantial increase is observed for depurinated molecules, whereas no increase is observed for nondepurinated templates, suggesting in vivo mutagenesis at depurinated sites. In each of the different assay systems investigated, the increase in misincorporation or reversion frequency is a linear function of the number of sites and is abolished by treatment of the depurinated templates with alkali, which rapidly induces strand breakage at apurinic sites.

Apurinic Acid↗
PubMed1979

Regulation of mitochondrial ribosomal RNA synthesis in yeast. I. In search of a relaxation of stringency.

Studies were undertaken to determine if mitochondrial rRNA synthesis in yeast is regulated by general cellular stringent control mechanism. Those variables affecting the relaxation of a cycloheximide-induced stringent response as a result of medium-shift-down or tyrosine limitation include: 1) the stage of cell growth, 2) carbon source, 3) strain differences and, 4) integrity of the cell wall. The extent of phenotypic relaxation decreased or was eliminated entirely in a strain dependent manner as cells entered stationary phase of growth or by growth of cells on galactose or in osmotically stabilized spheroplast cultures. Cytoplasmic and mitochondrial RNA species were extracted from regrowing spheroplast cultures subjected to different experimental regimens and analyzed by electrophoresis on 2.5% polyacrylamide gels. Relative rates of synthesis were determined in pulse experiments and normalized by double-label procedures to longterm label material. Tyrosine starvation was found to inhibit synthesis of the large and small rRNA species of both cytoplasmic and mitochondrial rRNAs to about 5-20% of the control values. Chloramphenicol inhibits mitochondrial and cytoplasmic rRNA synthesis to 60-80% of control; however, chloramphenicol addition does not relax the stringent inhibition of either class of rRNAs. Cycloheximide addition results in 70-80% inhibition of synthesis of both cellular speceis of rRNAs. As noted above, cycloheximide does not relax the stringent response of cytoplasmic rRNA synthesis in spheroplasts, and also does not relax the stringent inhibition of mitochondrial rRNA synthesis. From these studies, we conclude that both cytoplasmic and mitochondrial rRNA synthesis share common control mechanisms related to regulation of protein synthesis by shift-down or amino acid limitation.

Cycloheximide↗
PubMed1995

The frequency of integrative transformation at phase-specific genes of Candida albicans correlates with their transcriptional state.

The phase transition between the white and opaque phenotypes in the switching system of Candida albicans strain WO-1 is accompanied by the differential expression of the white-specific gene WH11 and the opaque-specific gene PEP1. The frequency of integrative transformation at the white-specific gene locus WH11 is between 4.5 and 7.0 times more frequent in white than in opaque spheroplasts, and the frequency of disruptive transformation at the opaque-specific gene locus PEP1 is 30.5 times more frequent in opaque spheroplasts than in white spheroplasts. In contrast, the frequencies of integrative transformation at the constitutively expressed loci ADE2 and EF1 alpha 2 are similar in the white and opaque phases. Therefore, the frequency of integration of linear plasmid DNA containing sequences of phase-specific genes correlates with the transcriptional state of the targeted locus.

Aspartic Acid Endopeptidases↗