Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.     

Multiple sites of action of cycloheximide in addition to inhibition of protein synthesis in Physarum polycephalum

The specificity of action of cycloheximide was tested using a cycloheximide resistant mutant of Physarum polycephalum. This resistance has previously been shown to reside with the ribosomes, making cytoplasmic protein synthesis refractile to the action of the drug. We show here that cycloheximide in the mutant strain causes specific alterations in metabolism without influencing the growth rate. These are: 1. lowered specific activity of glutamate dehydrogenase during starvation, 2. alteration of the molecular weight of glutamate dehydrogenase, 3. inhibition of uptake of amino acids from the medium into the internal pools. Possible explanations for these effects of cycloheximide outside of protein synthesis per se are considered. We conclude that cycloheximide may not be considered a specific inhibitor of protein synthesis, and that a causal relationship between protein synthesis and any biological process cannot be claimed unless such specificity is demonstrated in each case, preferably by use of mutants.     

Role of ribosomes in cycloheximide resistance of Neurospora mutants

Summary In Neurospora crassa, mutants resistant against cycloheximide appear with a marked time lag after mutation induction. We have suggested (Neuhäuser et al., 1970) that this lag indicates the time needed for the synthesis of altered ribosomes (phenotypic lag), that the drug in the wildtype acts upon the ribosomes, and that resistance is due to alterations in them.By measurements of poly-U directed poly-Phe synthesis on ribosomes of the wildtype and two different cycloheximide resistant mutants in a cell free system it is shown here that mutant ribosomes indeed differ from those of the wildtype. Poly-Phe synthesis on mutant ribosomes proceeds in the presence of the drug, whereas that on wildtype ribosomes is inhibited. This means that the earlier suggestions are correct.Abbreviation CHX cycloheximide     

Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum

Summary Cycloheximide-resistant mutants of Physarum polycephalum were induced in the haploid myxamoebae by the combined action of UV¹ and caffeine (Haugli and Dove, 1972) or by treatment with NMG². Eight independent mutants segregated in a Mendelian fashion (Table 1). Crosses between 6 of the mutants revealed 2 loci, actA and actB, for cycloheximide resistance (Table 2).All mutants are expressed in the plasmodium and are recessive in heterozygotes (Fig. 1 and 2). One mutation, conferring resistance to high levels of cycloheximide, was studied in heterokaryons and found to be incompletely recessive.An in vitro peptide synthesizing system was constructed from ribosomes from Physarum and supernatant factors from Saccharomyces cerevisiae. Cycloheximide strongly inhibited the activity of ribosomes derived from either wild type or mutants at the actB locus. In contrast, ribosomes from mutants at the actA locus were resistant to cycloheximide. Thus, the actA locus operates through the ribosomes.     

An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae.

Summary Mutants of Schizosaccharomyces pombe were isolated as resistant either to trichodermin or to anisomycin. Growth tests showed that the majority of mutants isolated were cross resistant to both drugs and also to cycloheximide. A limited genetic analysis showed that mutants at least four loci, tri3, tri4, ani1 and ani2, had this phenotype as was also the case for mutants at three cycloheximide resistant loci, cyh2, cyh3 and cyh4 reported previously (Ibrahim and Coddington, 1976). Allelism tests showed that the tri3, ani2 and cyh4 strains were allelic. A mutant at another trichodermin resistant locus, tri5, was cross resistant to anisomycin but sensitive to cycloheximide.Ribosomes from wild type and selected strains were analysed in a poly U directed cell free protein synthesising system. Three strains, cyh1-C7, ani1-F1 and tri-N15 (probably a tri5 allele) possessed ribosomes which were more resistant than the wild type to the drugs used in their isolation. In each case the site of the resistance was in the 60S subunit. Ribosomes from the cyh2, cyh3 and cyh4 strains were as sensitive to cycloheximide as those from wild type.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

The amiloride resistance gene, car1, of Schizosaccharomyces pombe

Amiloride, an inhibitor of various sodium transporters, is toxic to Schizosaccharomyces pombe at low concentration in minimal but not in rich media. Amiloride-resistant mutants were isolated and shown to represent a new locus (car1 for changed amiloride resistance) on chromosome I. The carl gene was cloned and sequenced. Sequence analysis revealed an open reading frame of 526 amino acids with a predicted molecular weight of 58 545 Da. It has 52% hydrophobic residues and belongs to the class of 12-transmembrane-domain transport proteins. Gene disruption of carl results in increased amiloride resistance. earl has sequence similarity to proteins from Candida associated with resistance to benomyl, methotrexate and cycloheximide. No single physiologically identifiable component of sodium transport appeared to be lost. We propose that earl serves an uptake function, perhaps as a symport with an unknown substrate and this carrier may transport amiloride into the cell. Further, we suggest that amiloride toxicity at low concentrations is not due to its effect on sodium transport but, rather, depends on intracellular interference with an unknown biosynthetic pathway.     

Tagging of genes involved in multidrug resistance in Aspergillus nidulans

We have used a plasmid containing the Neurospora crassa pyr4 gene to transform an Aspergillus nidulans pyrG89 mutant strain in the presence of BamHI, and isolated multidrug-sensitive mutants among the transformants. Using this approach, we hoped to identify genes whose products are important for drug resistance by analyzing gene disruptions that alter the drug sensitivity of the cell. About 1300 transformants isolated following transformation were screened for sensitivity to drugs or various stress agents with different and/or the same mechanism of action. Seventy-seven of these transformants showed sensitivity to at least one drug, while fourteen transformants showed a complex phenotype of sensitivity to different drugs. The pyr4 marker was shown to be tightly linked to the mutant phenotype in only 36% of the pleiotropic mutants analyzed in sexual crosses. Genetic crosses between our multidrug-sensitive transformants and cycloheximide-sensitive and imazalil-resistant mutants of A nidulans were performed to determine whether mutations were present at the same loci. We have shown that the gene imaD that confers resistance to imazalil may also be involved in cycloheximide and hygromycin sensitivity, since this mutation is allelic to scyB (mutant scy290). In addition, the cross between the transformant R223 and the imazalil-resistant mutant ima535 showed that both mutations are in the same complementation group, suggesting that the gene imaG could also be involved in cycloheximide and itraconazole sensitivity. Received: 30 August 1999 / Accepted: 22 February 2000     

Cloning and characterization of the histidine kinase gene<Emphasis Type="Italic"> Dic1</Emphasis> from<Emphasis Type="Italic"> Cochliobolus heterostrophus</Emphasis> that confers dicarboximide resistance and osmotic adaptation

A gene for a putative two-component histidine kinase, which is homologous to os-1 from Neurospora crassa, was cloned and sequenced from the plant-pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain, and six tandem repeats of 92-amino-acids at the N-terminal end that are found in histidine kinases from other filamentous fungi. Introduction of the histidine kinase gene complemented the deficiency of the C. heterostrophus dic1 mutant, suggesting that the Dic1 gene product is a histidine kinase. Dic1 mutants are resistant to dicarboximide and phenylpyrrole fungicides, and they are sensitive to osmotic stress. We previously classified dic1 alleles into three types, based on their phenotypes. To explain the phenotypic differences among the dic1 mutant alleles, we cloned and sequenced the mutant dic1 genes and compared their sequences with that of the wild-type strain. Null mutants for Dic1, and mutants with a deletion or point mutation in the N-terminal repeat region, were highly sensitive to osmotic stress and highly resistant to both fungicides. A single amino acid change within the kinase domain or the regulator domain altered the sensitivity to osmotic stress and conferred moderate resistance to the fungicides. These results suggest that this predicted protein, especially its repeat region, has an important function in osmotic adaptation and fungicide resistance.Communicated by C. A. M. J. J. van den Hondel     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-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 strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ 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 thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Transcription of bacteriophage Mu. I. Hybridization analysis of RNA made in vitro

Summary A mutation in the cyR1 gene of the fungus Podospora anserina confers resistance to cycloheximide and leads to an alteration of the 60S ribosomal protein L21 (Bégueret et al. 1977). Nine revertants of this mutant were isolated and the properties of these strains were analyzed. It was found that one revertant strain contains a new mutant form of L21. It is proposed that the cyR1 gene is the structural gene for protein L21 and that the alteration of this protein is responsible for the resistance to cycloheximide in vivo.     

A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents

Summary The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents, Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pl of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg⁺⁺ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50–100 mM. Nuclease 3 exhibits a temperature optimum of 42°C and is insensitive to N-ethylmaleimide. The enzyme is probably membrane-associated because it exhibits a strong tendency to aggregate and detergent is required for full solubilization.     

Identifying Escherichia coli genes involved in intrinsic multidrug resistance

Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.     

New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling

Summary We isolated new gyrA and gyrB mutations in Escherichia coli which have a graded effect on DNA supercoiling. The mutants, selected respectively for resistance to nalidixic acid and coumermycin, were sorted by means of a rapid in vivo assay of DNA gyrase activity (Aleixandre and Blanco 1987). Cells carrying a gyrB (Cou^r) mutation usually showed a decrease in DNA supercoiling, which would indicate a reduction in gyrase activity. In contrast, most of the gyrA (Nal^r) mutations had no significant effect on DNA supercoiling. Moreover, they conferred a high level of resistance to nalidixic acid and other quinolones, thus being similar to the gyrA(Nal^r) mutants currently used. We also detected rare gyrA mutants showing a reduction in DNA gyrase activity. These mutants were, in addition, resistant to only low concentrations of quinolones, which allowed us to use the phenotype of partial quinolone resistance as an indicator to score gyrA mutations affecting DNA supercoiling. When gyrB mutations were introduced into the gyrA mutants, these became more sensitive to quinolones and a decrease in supercoiling was observed. Moreover, the topA10 mutation sensitized gyrA(Nal^r) cells to quinolones. We conclude therefore that the GyrA-dependent quinolone resistance is diminished as a consequence of the reduction either in topoisomerase I or gyrase activities.     

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro

Summary We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.     

Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1

Summary Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl₂, The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.     

Cycloheximide-induced inhibition of chitin synthesis, and decay of apical vesicles and chitosomes in Phycomyces blakesleeanus

Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.     

Search for ribosomal mutants in Podospora anserina: Genetic analysis of mutants resistant to paromomycin

It has recently been shown that paromomycin, an antibiotic of the aminoglycoside family, is also active on eukaryotic cytoplasmic ribosomes. In the fungus Podospora anserina, genetic analysis of ten mutants resistant to high doses of paromomycin shows that this resistance is caused by mutations in two different nuclear genes. These mutants display pleiotropic phenotypes (cold sensitivity, mycelium and spore appearance and coloration, cross-resistance to other antibiotics). Double mutants are either lethal or very altered and unstable. Moreover, the cytochrome spectra of these mutants seem to indicate that cytoplasmic protein synthesis is affected. The mutants also display a slight suppressor effect. We can therefore assume that these mutations affect cytoplasmic ribosomes.This work was supported by a C.N.R.S. Grant (ATP Microbiologie No. 3052) and by a NATO Grant.     

Arabidopsis sensitivity to protein synthesis inhibitors depends on 26S proteasome activity

The 26S proteasome (26SP), the central protease of the ubiquitin-dependent proteolysis pathway, controls the regulated proteolysis of functional proteins and the removal of misfolded and damaged proteins. In Arabidopsis, cellular and stress response phenotypes of a number of mutants with partially impaired 26SP function have been reported. Here, we describe the responses of proteasome mutants to protein synthesis inhibitors. We show that the rpt2a-3, rpn10-1 and rpn12a-1 mutants are hypersensitive to the antibiotic hygromycin B, and tolerant to the translation inhibitor cycloheximide (CHX) and herbicide l-phosphinothricin (PPT). In addition to the novel mechanism for herbicide tolerance, our data suggests that the combination of hygromycin B, CHX and PPT growth-response assays could be used as a facile diagnostic tool to detect altered 26SP function in plant mutants and transgenic lines.