Mutants of the Killer Plasmid of SACCHAROMYCES CEREVISIAE Dependent on Chromosomal Diploidy for Expression and Maintenance

Mutants of the killer plasmid of Saccharomyecs cerevisiae have been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.—Haploids carrying these mutant plasmids (called d for diploid-dependent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K⁺R⁺) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.—Sporulation of the phenotypically K⁺R⁺ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.     

Isolation and characterization of temperature-sensitive mak mutants of Saccharomyces cerevisiae.

The K1 killer plasmid of Saccharomyces cerevisiae is a 1.5-megadalton linear double-stranded ribonucleic acid molecule. Using simplified screening and complementation procedures, we have isolated mutants in three chromosomal genes that are temperature sensitive for killer plasmid maintenance or replication. One of these genes, mak28-1, was located on chromosome X. Two of the temperature-sensitive mutants rapidly lost the wild-type killer plasmid of A364A during spore germination and outgrowth at nonpermissive temperatures, but during vegetative growth, they only lowered the plasmid copy number. These two mutants did not lose two other wild-type K1 killer plasmids, indicating a heterogeneity of the killer plasmids in laboratory yeast strains.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

Defective Interference in the Killer System of Saccharomyces cerevisiae

The K₁ killer virus (or plasmid) of Saccharomyces cerevisiae is a noninfectious double-stranded RNA genome found intracellularly packaged in an icosahedral capsid. This genome codes for a protein toxin and for resistance to that toxin. Defective interfering virus mutants are deletion derivatives of the killer virus double-stranded RNA genome; such mutants are called suppressive. Unlike strains carrying the wild-type genome, strains with these deletion derivatives are neither toxin producers nor toxin resistant. If both the suppressive and the wildtype virus are introduced into the same cell, most progeny become toxin-sensitive nonkillers (J. M. Somers, Genetics 74:571-579, 1973). Diploids formed by the mating of a killer with a suppressive strain were grown in liquid culture, and RNA was extracted from samples taken up to 41 generations after the mating. The ratio of killer RNA to suppressive RNA decreased with increasing generations; by 41 generations the killer RNA was barely detectable. The copy numbers of the suppressive genome and its parental killer were virtually the same in isogenic strains, as were the growth rates of diploid strains containing either virus alone. Therefore, suppressiveness, not being due to segregation or overgrowth by faster growing segregants, is likely due to preferential replication or maintenance of the suppressive genome. Three suppressive viruses, all derivatives of the same killer virus (T. K. Sweeney et al., Genetics 84:27-42, 1976), did not coexist stably. The evidence strongly indicates that the largest genome of the three slowly suppressed both of the smaller genomes, showing that larger genomes can suppress smaller ones and that suppression can occur between two suppressives. Of 48 isolates of strains carrying the suppressive viruses, 5 had newly detectable RNA species, all larger than the original suppressive genomes. At least seven genes necessary for maintenance of the wild-type killer virus (MAK genes) were needed by a suppressive mutant. No effect of ski mutations (affecting regulation of killer virus double-stranded RNA replication) on suppressiveness was observed.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

Parental effects and phenotypic characterization of mutations that affect early development in Caenorhabditis elegans

Genetic tests for parental effects were performed on 24 temperature-sensitive embryonic-lethal mutants of the nematode Caenorhabditis elegans. For 21 of these mutants, maternal expression of the wild-type allele is sufficient for embryonic survival, regardless of the embryo's genotype. For 11 of these 21 mutants, maternal expression of the wild-type allele is necessary for embryonic survival (strict maternals). For the remaining 10, either maternal or embryonic expression is sufficient for survival (partial maternals). One mutant shows a paternal effect; that is, a wild-type extragenic sperm function appears to rescue homozygous mutant embryos. Similar parental-effect tests were performed on 11 larval-lethal mutants. In 4 mutants, 1 of which blocks as late as the second larval stage after hatching, maternal contributions still can rescue mutant larvae. The remaining 3 embryonic lethals and 8 larval lethals show no parental effects; that is, zygotic expression of the wild-type allele is necessary and sufficient for embryonic survival. Temperatureshift experiments on embryonic-lethal embryos showed that all but 1 of the strict maternal mutants are temperature sensitive only before gastrulation. One of the partial maternal mutants is temperature sensitive prior to gastrulation, suggesting that some zygotic genes can function early in embryogenesis. At the nonpermissive temperature, 7 of the strict maternal mutants either show cleavage abnormalities in early divisions or stop cleavage at less than 100 cells, or both.     

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Leucine-rich repeat kinase 2 mutants I2020T and G2019S exhibit altered kinase inhibitor sensitivity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of late-onset autosomal dominant Parkinson’s disease (PD). Some disease-associated mutations directly affect LRRK2 kinase activity and inhibition of LRRK2 is viewed as a potential therapeutic treatment for PD. We demonstrate by both binding and enzymatic assays that alterations in the kinase activity of the PD-associated mutants I2020T and G2019S are due in part to altered ATP affinity. In binding assays, G2019S and I2020T have approximately 2-fold lower and 6-fold higher ATP affinity, respectively, than wild-type LRRK2. Furthermore, using an in vitro kinase activity assay, we demonstrate that at ATP concentrations close to cellular levels (1 mM) I2020T is approximately 10-fold more resistant to ATP-competitive kinase inhibitors than wild-type whereas G2019S is 1.6-fold more sensitive. These results predict that LRRK2 status may impact kinase inhibitor potencies in vivo or in cellular models.     

Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli.

The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.     

Inhibition of Rhizobium etli Polysaccharide Mutants by Phaseolus vulgaris Root Compounds

Crude bean root extracts of Phaseolus vulgaris were tested for inhibition of the growth of several polysaccharide mutants of Rhizobium etli biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher levels in roots and nodules than in stems or leaves. Inoculation with either wild-type or polysaccharide-deficient R. etli did not appear to affect the level of activity. Sequential extractions of the crude root material with petroleum ether, ethyl acetate, methanol, and water partitioned inhibitory activity into each solvent except methanol. The major inhibitors in the petroleum ether and ethyl acetate extracts were purified by C₁₈ high-performance liquid chromatography. These compounds all migrated very similarly in both liquid and thin-layer chromatography but were distinguished by their mass spectra. Absorbance spectra and fluorescence properties suggested that they were coumestans, one of which had the mass spectrum and nuclear magnetic resonances of coumestrol. These results are discussed with regard to the hypothesis that one role of rhizobial polysaccharides is to protect against plant toxins encountered during nodule development.     

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15–25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8–M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5–12 %) and SO₂ (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.     

The genetics and biochemistry of urease in Ustilago violacea

Two complementing loci in different linkage groups of the basidiomycete Ustilago violacea are involved in urease activity: a structural one (ure-1) and a second inferred to involve a permease (ure-2) locus. Two types of complementing mutations occur in the structural locus: null activity (ure-1a) and obviously reduced activity (ure-1b). The ure-2 mutants lacked urease activity in vivo on the phenol red-urea test medium, but gave extracts with wild-type activity. Extracts from wild-type strains gave one site of urease activity after polyacrylamide gel electrophoresis. A number of ure-1b mutants and active revertants from ure-1a mutants yielded electrophoretically variant urease sites. The results are discussed in terms of enzyme polymorphism in haploid eukaryotes by one (missense) or two (null, then missense) mutations.     

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.     

Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function.

M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only on M plasmid. Mutants with neutral (nonkiller [K-], immune [R+]) or suicide (killer [K+], sensitive [R-] phenotypes were examined. All mutants became K- R- sensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor-product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.     

Killer double-stranded ribonucleic acid synthesis in cell division cycle mutants of Saccharomyces cerevisiae.

The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdc14, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc21, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature.     

The occurrence of killer,sensitive, and neutral yeasts in Brazilian Riesling Italico grape must and the effect of neutral strains on killing behaviour

 The occurrence of killer toxins amongst yeasts in Brazilian Riesling Italico grape must was investigated by using the sensitive strain EMBRAPA-26B as a reference strain at 18°C and 28°C. From a total of 85 previously isolated yeasts, 21 strains showed ability to kill the sensitive strain on unbuffered grape must/agar (MA-MB) and 0.1 M citrate/phosphate-buffered yeast extract/peptone/dextrose/agar (YEPD-MB) media both supplemented with 30 mg/l methylene blue. The killer activity of only four yeasts depended on the incubation temperature rather than the medium used. At 28°C, the strains 11B and 53B were not able to show killer action. On the other hand, strains 49B and 84B did not kill the sensitive yeast at 18°C. The killer strain EMBRAPA-91B and a commercial wine killer yeast K-1 were employed to examine the sensitivity of the isolated yeasts on YEPD-MB and MA-MB at 18°C. The sensitivity and neutral characteristics of yeasts were shown to be dependent on the medium and the killer strain. Interactions, including K^- R^-, K^- R⁺ and K⁺ R⁺ strains, simultaneously, have revealed that some K^-R⁺ strains appear to protect the K^- R^- strain against the killer toxin. Sensitive dead cells, although to a less extent, also exhibited similar protection. Kinetic studies have shown that the maximum specific growth rates were higher for the 20B YEPD-MB-sensitive strain (μ_max=0.517 h^-1) than for both the 91B (μ_max=0.428 h^-1) and K-1 (μ_max= 0.466 h^-1) killer strains. The protective capacity of neutral or sensitive cells that contaminate a fermentation, as well as the higher maximum specific growth rate of sensitive yeasts, besides other factors, may preclude the dominance of a killer strain. This protective capacity may also reduce the risk of a sensitive inoculum being killed by wild-type killer yeasts in open non-sterile fermentation. Received: 3 November 1995/Received revision: 11 March 1996/Accepted: 15 April 1996     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

Serum resistance in bvg-regulated mutants of Bordetella pertussis

Serum resistance, or resistance to killing by antibody dependent pathway of complement, in Bordetella pertussis is bvg-regulated and the Bordetella resistance to killing (brk) locus mediates much of the resistance. Here we examined whether other bvg-regulated proteins contribute to serum resistance. We found that neither pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin, dermonecrotic toxin, tracheal colonization factor, nor Vag8 mutants were sensitive to serum killing compared to the wild-type. Filamentous hemagglutinin has been reported to bind C4 binding protein, an inhibitor of complement, but this activity does not appear to contribute to serum resistance, as evidenced by the resistant phenotype of FHA mutants. Clinical isolates were serum resistant and wild-type strains possessing an additional copy of the brk locus were 2–5-fold more resistant to serum killing.