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PubMedDate not supplied

[Stability of Escherichia coli to the membranotropic antibiotic gramicidin S].

The work was aimed at studying the effect of gramicidin S on the intact cells, spheroplasts and membrane specimens of Escherichia coli K12S with the natural resistance to this antibiotic. The resistance was shown to be caused by the barrier properties of the cell wall: the spheroplasts were highly sensitive to the lytic action of gramicidin S. The differences in the sensitivity to gramicidin S of substrate oxidation carried by the membranes of E. coli and Micrococcus luteus, a sensitive organism, were not of crucial significance for the manifestation of the resistance. The resistance was not associated with the decrease of gramicidin S adsorption: the cells were capable of binding large quantities of the antibiotic and remaining viable. Gramicidin S appeared to be attached to the cell walls (most likely, the outer membranes) rather than the cytoplasmic membranes.

Cell Membrane↗
PubMed1991

Trimerization of an in vitro synthesized OmpF porin of Escherichia coli outer membrane.

The assembly of outer membrane proteins of Escherichia coli was examined using the OmpF porin as a model. Since this protein is made as a precursor, which is processed to a protein of Mr 37,000 before being assembled into trimers in the outer membrane, we synthesized a modified OmpF, which lacked 16 out of 22 amino acid residues from its signal sequence, in a coupled transcription-translation system. This modified protein resembled the unfolded, monomeric OmpF in its electrophoretic behavior, but much of the protein apparently existed in a more tightly folded conformation as it was recognized by a monoclonal antibody specific to a surface epitope of the native, trimeric OmpF porin. At least some conformers of this protein could be further incorporated into outer membrane or lipopolysaccharide bilayers, and assembled into trimers. The trimers formed were trypsin-resistant and heat-stable in sodium dodecyl sulfate up to 70 degrees C, thus showing the characteristics of the native trimeric protein. These results extend our earlier observation that OmpF monomer secreted by spheroplasts of E. coli can be trimerized in vitro (Sen, K., and Nikaido, H (1990) Proc. Natl. Acad. Sci. U.S. A 87, 743-747) and show that the trimerization can occur, albeit at a low efficiency, with porin monomers synthesized in vitro, presumably not contaminated by membrane fragments or other components of the cell envelope. However, comparison of trimerization efficiency of the nascent in vitro product with that of the same product already exposed to aqueous medium, as well as with that of the spheroplast-secreted product, leads us to the working hypothesis that the trimerization process in intact cells is accelerated either by accessory components or by the conformational changes accompanying the secretion through the cytoplasmic membrane and that the reactions observed in this study represent only part of the physiological process.

Amino Acid Sequence↗
PubMed1979

Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase.

Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts. When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein. When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases. These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane.

Apoenzymes↗
PubMed1979

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.

Bacteriophage phi X 174↗
PubMedDate not supplied

[The effect of cationic surface-active agents on the Escherichia coli ATPase].

The authors studied the effect of cationic surfactants (CS), such as alkyl(C8H17-C18H37)dimethylbenzylammonium (I), alkyl(C8H17-C16H33)benzyltrimethylammonium (II), alkyl(C8H17-C16H33)di-beta-hydroxyethylbenzylammonium (III) chlorides and chlorhydrate of glycine decyl ester (IV) on the ATPase activity of E. coli 1257 cell, spheroplasts, and isolated membranes. Changes in the ATPase activity of the E. coli cells and spheroplasts were found to depends on the concentration and the structure of the cationic surfactants. The removal of the cell wall increased the destroying effect of CS on the cytoplasmic membranes and enhanced the ATPase inhibition. The compounds with 16 and 18 carbon atom radical had the highest inhibitory effect. The action of cationic surfactants on the membrane is accompanied by changes in the protein and phospholipid composition and by significant solubilization of ATPase with pronounced inactivation of the enzyme. The kinetics of inhibition of E. coli membrane ATPase was studied to the presence of the homological series I and IV. The cationic surfactants under study inhibited the ATP hydrolysis catalysed by E. coli ATPase by a mixed type mechanism. Ki = 58.21.10(-4) M for IC10H21; 10.67.10(-4) M for IC12H25; 0.58.10(-4) M for IC16H33; 0.16.10(-4) M for IC18H37, and 5.93.10(-4) M for IV.

Adenosine Triphosphatases↗
PubMed1985

[Transformation of the yeast Hansenula polymorpha by a hybrid plasmid containing the fragment of host DNA].

Spheroplasts of Hansenula polymorpha strain deficient in 2-isopropylmalate dehydrogenase have been shown to be transformed by the DNA of a hybrid plasmid pHRI, carrying the LEU2 gene from S. cerevisiae and 2.0 kilobase HindIII fragment of H. polymorpha genomic DNA. The frequency of transformants has reached 10(3) per 1 microgram of transforming DNA. Plasmid pHRI is maintained in transformants as an autonomous circular DNA molecule and is inherited by 1-2% fraction of cells from the population growing under the selective conditions. Transformation takes place under the same conditions that are required for spheroplast fusion. Thus, H. polymorpha becomes one more species of yeast susceptible to hybrid plasmid-mediated gene transfer in the process of DNA transformation.

DNA, Fungal↗
PubMed1985

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents.

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.

Adenosine Triphosphate↗
PubMed1985

[Importance of cellular homogenization for the posterior isolation of plasma membranes of the Candida utilis yeast].

Candida utilis plasma membranes were isolated from different homogenized, either whole cells or spheroplasts, by centrifugation and aggregation of mitochondrial and internal membranes at pH 4.0. As judged by electron microscopy, chemical analysis, disc electrophoresis and enzymatic assays, membrane preparations with different degrees of purity were obtained. The purest plasma membranes were those prepared by homogenization of spheroplasts in the Omnimixer Sorvall followed aggregation at mitochondrial isoelectric point as described above.

Candida↗
PubMed1977

[Morphologic characteristics of spheroid elements in a culture of bacterial L-forms according to scanning electron microscopy findings].

The authors studied stable L-cultures of Proteus valgaris, Bac. subtillis, Staphylococcus aureus, Streptococcus pyogenes of group A, and also unstable cultures of the L-forms of Proteus vulgaris and Proteus vulgaris culture at the stage of spheroplasts. Spheroid cells proved to appear at the stage of spheroplasts, prevailed at the log phase in stable and unstable L-cultures, but were less frequent at the stationary phase. Cross section of L-colonies showed that they were located at the surface. The size of spheroid elements was from 1 to 5 micron; their surface was smooth or slightly wrinkled with numerous protrusions and individual sockets. The spheroid cells were distributed in the colonies freely, in clusters, or were connected to one another by anastomosis. Several methods of reproduction of spheroid cells are described, including equal and unequal binary fission, budding, and formation of elementary bodies within the cell. Morphological connection of spheroid cells with large bodies, filamentous structures and structureless matrix of the L-colony apparently pointed to their origin from the corresponding elements of the L-cultures.

Bacillus subtilis↗
PubMed1970

A stable L-form of Haemophilus pleuropneumoniae.

A stable L-form of Haemophilus pleuropneumoniae (Shope) was isolated from primary pig kidney cell tissue cultures which had been inoculated 28 days previously with glycine induced spheroplasts of this organism.H. pleuropneumoniae was definitely cytopathic in primary pig kidney cell cultures, producing cell rounding, cytoplasmic vacuolation and nuclear enlargement with peripheral condensation of nuclear DNA. By contrast, the effect of spheroplasts was much less distinct, producing only loss of cytoplasmic granularity coincident with apparent loss of some cytoplasmic RNA, and slight nuclear enlargement. Both the organism and its L-form were shown to be related by cultural methods, antibiotic sensitivity tests, immunofluorescence and immunodiffusion. The L-form remained stable after 90 serial passages on agar and 45 in broth, each medium being capable of supporting the growth of both forms of the organism.

Animals↗
PubMedDate not supplied

[Location of RNA-dependent DNA polymerase within the endogenous template complex and discrimination of RNA- and DNA-dependent synthesis of DNA in Escherichia coli cells by alkylating dTTP gamma-amide].

These data demonstrate that actinomycin D inhibits only 75-80% of DNA synthesis in cells of E. coli treated by lysozyme and ethyl enediaminetetraacetate. The residual synthesis is not the result of untemplated polymerization of dNTP. The DNA synthesis in spheroplasts does not correlate with replication of chromosomal DNA of E. coli catalyzed by DNA polymerase III sensitive to sulfhydryl blocking agents. N-ethylmaleimide does not inhibit this synthesis. No ATP stimulation of DNA synthesis is observed. The enzyme(s) responsible for DNA synthesis on endogenous template is (are) concentrated in interphase (D-fraction) as revealed by high speed centrifugation of spheroplasts lysate and they are absent in the chromosomal DNA fraction. dTTP 4-(N-2-chloroethyl-N-mehylamino) benzylamide suppresses completely the insensitive to actinomycin D action DNA synthesis and practically does not act on the sensitive one. It is suggested, that the DNA synthesis stable to the action of the antibiotic is catalyzed by RNA dependent DNA polymerase.

Alkylating Agents↗
PubMed1983

Inducible repair of thymine ring saturation damage in phi X174 DNA.

The susceptibility to inducible SOS repair of 5,6-dihydroxy-dihydrothymine (t') damage in single-stranded phi X174 DNA has been measured. Following exposure to osmium tetroxide, which introduces t' damage in DNA under the conditions used, biological survival of the DNA infected into spheroplasts of Escherichia coli which had received prior exposure to ultraviolet light was higher than in unexposed spheroplasts. From our results, we conclude that approximately 63% of the biological damage from t' products, which is one of the classes of damage present in DNA following ionizing radiation, is susceptible to repair by the inducible SOS repair system.

Bacteriophage phi X 174↗
PubMed1981

Release of alkaline phosphatase from cells of Xanthomonas oryzae by manipulation of surface permeability.

Xanthomonas oryzae was shown to contain a constitutive alkaline phosphatase (EC 3.1.3.1.). The enzyme was detectable in intact cells and releasable by osmotic shock or spheroplast formation indicating its periplasmic location in the cell. Sonication released about 85% of the total enzyme, and the releasable amount was increased to 97% when lysozyme was added to the sonicated cells prior to centrifugation. These changes suggest an association of the enzyme with peptidoglycan; the enzyme is not released unless the polymer is digested to small units. Adapted usual method of spheroplast formation released 85%, while modified osmotic shock procedure released about 75% of the enzyme. These procedures released decreased amounts of the enzyme following cell growth reflecting that some changes were taking place toward a tighter association between the enzyme and the cell envelope during aging of the culture. During osmotic shock, the major portion of the released enzyme distributed in supernatant of the first stage (S1) hypertonic solution. The enzyme was inhibited by EDTA, whereas the inhibition was partially removed by dialysis and completely reversed by addition of MgCl2. Data obtained also indicated that X. oryzae seems to have relatively high content of periplasmic protein.

Alkaline Phosphatase↗
PubMed1981

Synthesis and processing of in vitro and in vivo precursors of the vacuolar yeast enzyme carboxypeptidase Y.

The biosynthesis of carboxypeptidase Y, which is located in the lysosome-like vacuole of Saccharomyces cerevisiae, has been studied in vitro in a cell-free translation system from wheat germ and in vivo in intact spheroplasts. When a wheat germ system was programmed with yeast RNA, a translation product was immunoprecipitated by anti-carboxypeptidase Y antibodies, which had a slightly smaller molecular weight (Mr = 59,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the authentic glycoprotein (Mr = 60,000). In the presence of dog pancreatic microsomal membranes, an additional cross-reacting translation product of Mr = 68,000 was formed, which in contrast to the 59,000-dalton form was not susceptible to digestion by externally added proteinases, suggesting its segregation within the microsomal vesicles. The observed increase in molecular weight may be consistent with a core glycosylation of the translocated protein. During a pulse-chase labeling of spheroplasts, the antibody initially precipitated a form of carboxypeptidase Y, which co-migrated on sodium dodecyl sulfate gels with the 68,000-dalton in vitro translation product. Following a chase of 60 min, this early labeled immunoreactive protein was completely converted into the authentic enzyme (Mr = 60,000) and is therefore coincident with the in vivo precursor of carboxypeptidase Y previously described (Hasilik, A., and Tanner, W. (1978) Eur. J. Biochem. 85, 599-608). These data suggest that a vacuolar yeast enzyme is synthesized via a cotranslational segregation of its nascent polypeptide chain within the endoplasmic reticulum giving rise to a proenzyme, which is further processed in vivo into the vacuole-located mature enzyme.

Carboxypeptidases↗
PubMed1982

[Morphological alterations in Escherichia coli and Serratia marcescens induced by cefotetan (author's transl)].

The effects of cefotetan (CTT, YM09330) and cefmetazole (CMZ) on the morphological changes of Escherichia coli NY-17 and Serratia marcescens IID 620 were examined with scanning electron microscope. 1) CTT and caused E. Coli NY-17 filamentous forms at 0.024 microgram/ml but CMZ showed same morphological changes at 0.2 microgram/ml which is about 10 fold higher concentration than that of CTT. Spheroplast like structures and cell lysis were observed at lower concentrations than those of CMZ. 2) In case of Serratia marcescens IID 620, filamentous forms, spheroplast like structures and cell lysis were also observed at lower concentrations than those of CMZ. The action of CTT against the both strains was bactericidal.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Carboxysomes of the thermophilic hydrogen bacterium Pseudomonas thermophila].

The technique of electron microscopy was used to detect the presence of paracrystal hexagonal inclusions in the cells and spheroplasts of the thermophilic hydrogen bacterium Pseudomonas thermophila. The connection of the inclusions with DNA threads can easily be seen on photomicrographs of the spheroplasts. The cells were disintegrated by freezing and thawing and the resultant homogenates were centrifuged in a sucrose density gradient; up to 80% of the activity of ribulose-1,5-diphosphate carboxylase (RDP carboxylase, EC 4.1.1.39) was found in the fraction of particles containing hexagonal paracrystal inclusions (carboxysomes). Their granular content and the tendency to diffuse were seen at a magnification x100,000. The percentage of the insoluble enzyme was higher in cells in the stationary growth phase than in growing cells. Only 25% of the enzyme activity was detected in the particles after the cells treated with lysozyme had been subjected to osmotic shock. A possible role of carboxysomes in cells as a compartment storing RDP carboxylase is discussed.

Carboxy-Lyases↗
PubMed1994

Cell wall deficiency in "slime" strains of Neurospora crassa: osmotic inhibition of cell wall synthesis and beta-D-glucan synthase activity.

1. The RCP-3 S/H mutant of Neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form of an fz, sg, os-1 ("slime"-like) segregant. The mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (Pietro et al. (1990). Journal of General Microbiology, 136: 121-129). The carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkali-soluble galactosaminoglycan polymer. Furthermore the mutant cell wall had a somewhat lower content of beta-glucan relative to that of chitin. 2. Increasing concentrations of sorbitol in the culture medium of the mutant inhibited by 10-fold the formation of cell wall relative to total biomass. The cell wall of the mutant cultured in the presence of sorbitol lacked mannose- and galactose-containing polymers, and also showed progressively lower amounts of beta-glucan relative to chitin. 3. The activity of membrane-bound (1-3)-beta-D-glucan synthase from the mutant grown in the absence of sorbitol shared several properties with the wild type enzyme (i.e., Km app., Vmax, stability at 30 degrees C, activation by GTP gamma S, and dissociability by treatment with NaCl and Tergitol NP-40 into a membrane-bound catalytic center and a GTP-binding activating protein). On the other hand, the enzyme from the mutant but not that from the wild type was inactivated by about 15% by treatment with NaCl and detergent. 4. At high concentrations of sorbitol (1.0 M) the RCP-3 S/H mutant exclusively produced spheroplasts devoid of (1-3)-beta-D-glucan synthase activity. The defect was at the level of the membrane-bound catalytic center. The activity of the GTP-binding activating factor was apparently normal in these cells. 5. These results suggest that the definitive loss of cell wall in the N. crassa "slime" RCP-3 S/H mutant was due to a defect in (1-3)-beta-D-glucan synthase activity which was exaggerated in the presence of high osmolyte concentrations.

Cell Wall↗
PubMed1999

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells.

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.

Fungal Proteins↗