Antibiotic Cycloheximide Research Articles

Improvement of Ethanol Production by an Industrial Yeast Strain via Multiple Gene Deletions

The genetic engineering of yeasts used in commercial processes can be both time-consuming and laborious. This is because industrial yeasts possess largely uncharacterised genomes, which frequently carry at least two copies of any gene. Such strains are usually devoid of auxotrophic or other genetic markers and this requires the incorporation of positively selectable (and often heterologous) genes into plasmids or other transforming DNA molecules. In this paper, we demonstrate that multiple gene deletions may be readily performed in industrial yeasts. Using a specially designed loxPkanMX4 gene replacement cassette, we deleted the two PET191 alleles essential to respiration in the diploid, high alcohol-producing, wine yeast, K1. The two integrated deletion cassettes, which rendered the respiratory-deficient mutant, K1 Δpet191ab, resistant to the antibiotic geneticin were then excised from the genome following the expression of a cre recombinase gene harboured on the multi-copy plasmid YEP351-cre-cyh. This plasmid was maintained in the mutant under the selective pressure of the antibiotic cycloheximide and then removed when both genes had been successfully deleted. Batch fermentations were performed in homebrew style for strains K1 and K1Δpet191ab and revealed a 40% higher volumetric ethanol production rate and a 9% higher ethanol ceiling for the mutant. This demonstrates that, because of their respiratory deficiency, nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Furthermore, nuclear petites cannot metabolise the product of fermentation, ethanol, allowing higher ethanol titres to be achieved. We believe that the method of strain manipulation demonstrated here will be of interest to scientists in the alcoholic beverages industry, who wish to delete genes in production yeast strains, while simultaneously ensuring the removal of all foreign coding sequences.

Leptographium pini-densiflorae sp. nov. from Japanese red pine

ALeptographium species was isolated from deadPinus densiflora at six sites in Japan. The fungus is morphologically most similar toL. lundbergii but could be distinguished from that species by its short stipes, primary branches of conidiophores, and conidia with a rounded to sub-truncate base. In addition, the colony morphology, growth rate and tolerance to the antibiotic cycloheximide of theLeptographium species andL. lundbergii differed markedly. Here we describe the fungus as a new species,Leptographium pini-densiflorae.

Immunofluorescence Colony-staining (IFC) for Detection and Quantification of Ralstonia (Pseudomonas) solanacearum Biovar 2 (Race 3) in Soil and Verification of Positive Results by PCR and Dilution Plating

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of sucrose and the antibiotics cycloheximide and crystal violet to the non-selective trypticase soy broth agar resulted in increased colony size and staining intensity of R. solanacearum in IFC. Verification of IFC-results by picking cells from IFC-positive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with ‘spiked’ and naturally contaminated soils respectively. Several other bacterial species which cross-reacted with polyclonal antibodies in IFC also grew on SMSA and were difficult to distinguish from R. solanacearum, thereby necessitating confirmation of the results. Rapid verification of IFC-positive results directly by PCR-amplification with primers D2/B specific to division 2 of R. solanacearum had a success rate of 86% and 96% with ‘spiked’ and naturally contaminated soil samples, respectively. Primers D2/B reacted with all R. solanacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to detect consistently ca. 100 cfu g−1 of soil, a detection level similar to that found with direct plating on SMSA, but less laboriously, whereas detection level with a bioassay on tomato plants was only 104−105 cfu g−1 of soil.

Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance.

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10-20 microg/ml cycloheximide, at a maximum efficiency of 300 transformants/microg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events.

Evidence for the enhanced biodegradation of ethoprophos and carbofuran in soils from Greece and the UK

Degradation of carbofuran in a topsoil sample from a previously untreated field site in the UK was characterized by a short lag period followed by rapid degradation. Carbofuran added subsequently to the same soil degraded rapidly without the lag period. In a subsoil sample from the same site, the first application of carbofuran degraded only slowly but degradation rate increased with subsequent treatments and the third dose degraded as rapidly as in the pre-treated topsoil. In similar experiments with ethoprophos, degradation was quite slow and enhanced degradation of subsequent additions of ethoprophos was not observed. A single application of carbofuran in the field in the UK activated soils for rapid biodegradation of the insecticide for at least the subsequent four years. In contrast, in soils from Greece, enhanced degradation was evident six and 18 months after the last carbofuran treatment in the field, but not after three years. Fifty per cent of ethoprophos applied to soils from Greece previously treated with the nematicide was lost within approximately four days, compared with 38 days in a similar, but previously untreated, soil. Very rapid degradation of ethoprophos and carbofuran was observed in soil samples from Greece which had been treated annually with ethoprophos for the last 30 years but with no previous applications of carbofuran. Annual use of the thiocarbamate herbicide EPTC in the same field may have resulted in cross-activation for rapid biodegradation of carbofuran. Very slow degradation of both carbofuran and ethoprophos was observed in soil samples with a history of combined applications of the two pesticides, probably because of their low pH. Fumigation of soil with chloroform, or treatment with the antibacterial antibiotic chloramphenicol, inhibited ethoprophos degradation in a soil where rapid rates of loss had previously been induced, but the antifungal antibiotic cycloheximide had no effect on degradation rate. © 1999 Society of Chemical Industry

Rapid Consumption of Low Concentrations of Methyl Bromide by Soil Bacteria

A dynamic dilution system for producing low mixing ratios of methyl bromide (MeBr) and a sensitive analytical technique were used to study the uptake of MeBr by various soils. MeBr was removed within minutes from vials incubated with soils and ~10 parts per billion by volume of MeBr. Killed controls did not consume MeBr, and a mixture of the broad-spectrum antibiotics chloramphenicol and tetracycline inhibited MeBr uptake by 98%, indicating that all of the uptake of MeBr was biological and by bacteria. Temperature optima for MeBr uptake suggested a biological sink, yet soil moisture and temperature optima varied for different soils, implying that MeBr consumption activity by soil bacteria is diverse. The eucaryotic antibiotic cycloheximide had no effect on MeBr uptake, indicating that soil fungi were not involved in MeBr removal. MeBr consumption did not occur anaerobically. A dynamic flowthrough vial system was used to incubate soils at MeBr mixing ratios as low as those found in the remote atmosphere (5 to 15 parts per trillion by volume [pptv]). Soils consumed MeBr at all mixing ratios tested. Temperate forest and grassy lawn soils consumed MeBr most rapidly (rate constant [k] = 0.5 min-1), yet sandy temperate, boreal, and tropical forest soils also readily consumed MeBr. Amendments of CH4 up to 5% had no effect on MeBr uptake even at CH4:MeBr ratios of 10(7), and depth profiles of MeBr and CH4 consumption exhibited very different vertical rate optima, suggesting that methanotrophic bacteria, like those presently in culture, do not utilize MeBr when it is at atmospheric mixing ratios. Data acquired with gas flux chambers in the field demonstrated the very rapid in situ consumption of MeBr by soils. Uptake of MeBr at mixing ratios found in the remote atmosphere occurs via aerobic bacterial activity, displays first-order kinetics at mixing ratios from 5 pptv to ~1 part per million per volume, and is rapid enough to account for 25% of the global annual loss of atmospheric MeBr.

Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein.

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.

Leptographium elegans: a new species from Taiwan

Amongst collections of ophiostomatoid fungi occurring on woody hosts in Taiwan a new species Leptographium elegans , was isolated from freshly cut surfaces of Chamaecyparis formosensis which had been logged for milling. It is distinguished from other species by its short conidiophores and conidiogenous cells, as well as the presence of a Sporothrix synanamorph with prominently denticulate conidiogeneous cells. Although no evidence of a teleomorph has been found, its high tolerance of the antibiotic cycloheximide suggests that it is closely related to other species of Leptographium that share this characteristic and have Ophiostoma teleomorphs.

Evidence for the enhanced biodegradation of napropamide in soil

AbstractIn laboratory incubations, the time to 50% loss of napropamide was approximately 60; 21 and 8 days in soil treated for the first, second and third time respectively. In a survey of soils from commercial fields, there was evidence that enhanced biodegradation of the compound had been induced by normal field applications—in some soils by a single previous treatment. Confirmation of the observations of rapid rates of loss in pre‐treated soil was obtained in experiments with three formulations of napropamide. The rate of degradation in enhanced soils was unaffected by treatment of the soils with the antifungal antibiotic cycloheximide, but was inhibited by the antibacterial antibiotic chloramphenicol. Mixed bacterial cultures able to degrade the herbicide were obtained from three rapid‐degrading soils by enrichment culture. Isolates from two of them were able to degrade the herbicide in pure culture. These bacteria have, as yet, not been characterised.

A new species of Phialocephala (Hyphomycetes)

A new species in the Hyphomycete genus Phialocephala is described. The new species Phialocephala virens produces an intense greyish green to turquoise colour on certain media and has smaller conidia, shorter phialides, and shorter conidiophores than previously described Phialocephala species. Its tolerance to low levels of the antibiotic cycloheximide suggests the fungus may have affinities with species of Ophiostoma. Cultures of the fungus cause a bright green stain when inoculated onto sapwood of some conifers. Key words: Phialocephala virens, new species, Hyphomycetes, sapstain.

The effect of some platinum compounds on the activity of the CTP synthetase of Ehrlich ascites tumor cells: In vitro and in vivo studies

The present work investigates the effect of cis-DDP (DDP, diamminedichloroplatinum(II)), trans-DDP, SPC (spermine platinum(II) complex), and K 2PtCl 4 on the activity of the CTP synthetase in the cytosol of Ehrlich ascites tumor cells. To study their in vitro effect, the platinum compounds were supplemented to the incubation mixture for the enzyme assay. A concentration dependent inhibition of the CTP synthetase was found which was strongest in the case of trans-DDP. When ascites cells collected from mice, pretreated in vivo with platinum compounds, were used, the enzyme assay showed that the inhibition is strongest in the case of cis-DDP and K 2PtCl 4 (about 90% inhibition). This distinct inhibitory effect of the platinum compounds in the present experiments may be explained with the metabolic conversions of the compounds in the organism to their more active forms and/or with the inhibition of the protein biosynthesis under their influence because the lifetime of the CTP synthetase is short. This last assertion is proved in this work by control experiments with the antibiotic cycloheximide, which is an inhibitor of the protein biosynthesis.

Cycloheximide genotoxicity in in vitro and in vivo test systems

The aim of this study was to investigate if there was any genotoxic effect produced by the antibiotic cycloheximide, widely used as a fungicide in agriculture as well as in everyday laboratory practice. The battery of test systems included the bacterium Salmonella typhimurium (strains TA98 and TAl00), the yeast Saccharomyces cerevisiae (D7), Allium cepa somatic cells and mouse bone marrow cells. This combination of test systems enabled us to establish possible effects caused by cycloheximide at different levels of the genome and to indicate a possible mechanism of action. The results obtained in experiments showed that cycloheximide did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. In S. cerevisiae cycloheximide had only toxic effects but no increase of mitotic gene conversion was noticed under the conditions of the experiment. However, in A. cepa somatic cells as well as in mouse bone marrow cells cycloheximide showed its activity causing different genetic damages, e.g., chromosome breaks, mitotic disturbances and nuclear abnormalities.

Central effects of cycloheximide alone and of its combination with T-2 toxin

The antibiotic cycloheximide inhibits protein synthesis in eukaryotic cells. This overall effect is similar to that of T-2 toxin, but the mechanisms of intoxication by these two inhibitors are different. This is shown here by intracerebral injections of mixtures of T-2 toxin and cycloheximide, leading to potentiation of their toxic effects. The histopathology of cerebral intoxication by the two compounds is similar, but after cycloheximide the lesions appear earlier, and repair is faster.

Different solution and solid‐state conformations of the antibiotic cycloheximide

AbstractThe 1H NMR spectra of the antibiotic cycloheximide in CDCl3 and CD3OD have been assigned. Analyses of coupling constants and difference NOE spectra showed different conformations in these two solvents, due to changeover between intra‐ and inter‐molecular hydrogen bonding. The twist boat cyclohexanone found in the solid state was not detected in solution. The results are compared with the solution and solid‐state conformations of the antitumour agent sesbanimide A.

Uncoupling of translocation across microsomal membranes from biosynthesis of influenza virus hemagglutinin.

This communication presents our recent studies on the biosynthesis of influenza virus hemagglutinin (HA) in a mammalian-cell-free system and its translocation across microsomal membranes. RNAs coding for wild-type (full-length) and mutant (truncated) forms of HA were generated by in vitro transcription by using bacteriophage T7 DNA-dependent RNA polymerase. These RNAs were translated in a rabbit reticulocyte system that was supplemented with dog pancreas membranes, either before translation was initiated or after it had been artificially terminated with the antibiotic cycloheximide. All forms of HA could be cotranslationally translocated. However, only truncated molecules (83% of full length) could translocate after protein synthesis had been terminated. Posttranslational translocation was dependent on the presence of a functional N-terminal signal sequence and occurred only in the presence of ribosomes. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed based on the signal hypothesis.

Effects of microbial inhibitors on the degradation rates of metamitron, metazachlor and metribuzin in soil

AbstractRates of carbon dioxide evolution and degradation rates of metamitron, metazachlor and metribuzin were measured in two soils in the presence of three microbial inhibitors. The nonselective microbial inhibitor sodium azide reduced both carbon dioxide evolution and the rate of loss of all three herbicides in both soils, although the reduction in degradation rate of metamitron was small. The antibacterial antibiotic novobiocin enhanced carbon dioxide evolution from both soils but had variable effects on the rates of herbicide degradation. It inhibited degradation of metazachlor and metribuzin, and in one of the soils its effects on metazachlor degradation were similar to those of sodium azide. Novobiocin inhibited degradation of metamitron to a small extent in one soil only. The antifungal antibiotic cycloheximide also enhanced carbon dioxide evolution from both soils. In general, its effects on herbicide degradation were similar to those of novobiocin, although the extent of inhibition was usually less pronounced. The results are discussed in terms of the relative involvement of microorganisms in degradation of the three herbicides.

4-Nitropyrocatechol ethers as possible photoaffinity labels : Photochemical reactions of 4-nitropyrocatechol ethers of biologically active compounds

The photochemical reactions of O- and N-(2-methoxy-4-nitro)phenoxyalkyl derivatives of estrone and of the antibiotic cycloheximide with methylamine afford clean substitutions of the methoxy group. From these experiments it is inferred that 2-methoxy-4-nitrophenyl ethers can be good photoaffinity labels.

Acquired cycloheximide resistance inNeurospora crassa andSclerotium rolfsii

Acquired resistance to the antibiotic cycloheximide developed byNeurospora crassa andSclerotium rolfsii was studied. Both the fungi gained certain level of tolerance to the antibiotic just after a single exposure and by serial transfers could adapt to the several-fold initial inhibitory dosage. Cycloheximide-resistance in both the cases was not a stable characteristic and was lost gradually on serial transfers in fungicide-free media. The resistant strains of both the fungi showed poor growth and decreased sporulation or sclerotia formation in fungicide-free media.Sclerotium rolfsii developed cross-resistance to Difolatan but not to Polyoxin-D, Hinosan and Bayleton. The cycloheximide-resistance inSclerotium rolfsii was probably due to the conversion of cycloheximide into isocycloheximide which is a less toxic structural analog, as revealed by thin layer chromatographic studies of culture filtrates of resistant and sensitive strains. The resistant strain ofSclerotium rolfsii retained its pathogenicity to tomato, mustard and chilli seedlings. However, the loss of sclerotia forming capacity and the instability of the acquired resistance trait may prove to be of ecological disadvantage to the resistant strain.

The nucleophilic aromatic photosubstitution in photoaffinity labelling. A model study of a cycloheximide derivative.

The photoreactions of several 4-nitrocatechol ethers with the relatively hard nucleophile methylamine occurs at the meta position respect to the nitro group. A derivative of the antibiotic cycloheximide carrying a 4-nitrocatechol ether moiety reacts with methylamine affording an in vitro model for photoaffinity labelling.

Dual action of cycloheximide on memory formation in day-old chicks

Amnesia for a single trial discriminated passive avoidance learning task in day-old chicks, resulting from the antibiotic cycloheximide, is shown to begin after 30 min following learning when cycloheximide is given immediately after or before learning. However, amnesia does not begin until after 50 min following learning when cycloheximide is given 5 min or later after learning. The results are interpreted in the context of a 3 stage model of memory formation. It is suggested that the second or intermediate stage involves two phases: phase A which lasts up to 30 min following learning, is energy dependent, and is susceptible to inhibition by 2,4-dinitrophenol; phase B which occurs after phase A, lasts up to 50 min following learning, is energy independent, and is not susceptible to inhibition by 2,4-dinitrophenol. It is concluded that cycloheximide given immediately after or before learning inhibits expression of phase B of intermediate memory as well as formation of long-term memory, while cycloheximide given 5 min or later after learning only inhibits formation of long-term memory. Another antibiotic, anisomycin, is shown to have no effect on intermediate memory, independent of time of administration.