Effects of oligomycins on adenosine triphosphatase activity of mitochondria isolated from the yeasts Saccharomyces cerevisiae and Schwanniomyces castellii.

Functional mitochondria with respiratory control were isolated from the yeasts Saccharomyces cerevisiae and Schwanniomyces castellii. The presence of site I in Schw. castellii was indicated by higher ADP/O ratio than in S. cerevisiae where this site is absent. The ATPase Vmax was higher in S. cerevisiae than in Schw. castellii mitochondria. The latter was increased by the DR12 nuclear mutation. Nevertheless, the stimulation by heat and the inhibition profile of oligomycins on mitochondrial F1-F0 ATPase activities were similar in all three tested strains. In S. cerevisiae and Schw. castelli wild type or mutant mitochondria, the well-known inhibition of F1-F0 ATPase activity by low concentrations of oligomycins is abolished at high inhibitor concentrations near 60microg/ml suggesting uncoupling of F1 activity. At still higher oligomycin concentration the ATPase activity of both species and mutant is again strongly inhibited, suggesting an inhibitory effect on yeast F1 activity not detected so far.

Diarrhoeal toxin production at low temperature by selected strains of Bacillus cereus.

The growth of four Bacillus cereus strains producing diarrhoeal toxin at 32 degrees C (F4433/73 and 29.155, isolated on the occasion of foodborne outbreaks, and F4581/76L and F4581/76R, two variants of a clinical strain), a weakly toxigenic strain isolated in routine analysis of food (3505M) and an emetic isolate (F3502/73) was investigated at low temperature. Biomass was determined by protein assay. Generation times were: for strain F3502/73, which grew at > or = 12 degrees C, 8.71 h (at 12 degrees C); for other strains, which grew at > or = 10 degrees C, 10.2 to approximately 18.9 h (at 10 degrees C). Toxin production during growth was evaluated by a commercial kit (Oxoid) and by a toxicity test on Chinese hamster ovary cells. Strains F4433/73 and F4581/76, secreting high levels of diarrhoeal toxin during the exponential phase at 32 degrees C, produced high levels of toxicity at 10 degrees C until the stationary phase. Strain 29.155 had decreased toxin production at 10 degrees C. Toxicities for cellular extracts remained low when compared with culture filtrates. A correlation was found between the toxicity values given by the two detection methods tested, and the suitability of both methods for the detection of potential poisoning isolates is discussed.

Mitochondrial ATP synthase subunit 9 is not required for viability of the petite-negative yeast Kluyveromyces lactis.

Specific mutations in nuclear MGI genes encoding the alpha, beta and gamma subunits of the mitochondrial inner membrane F1-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K. lactis. In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal. These results imply that mtDNA encodes a gene that is essential. Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F0 component. The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly- 3. 9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein. Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes. These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1.

Production of diarrheal toxin by selected strains of Bacillus cereus.

The production of diarrheal toxin by six selected strains of Bacillus cereus was monitored during growth at 32 degrees C, a temperature described as near-optimal for growth and toxin production. Toxic activity was measured in culture filtrates and cellular extracts sampled at three different times during growth. Two alternative methods, a cytotoxicity test on Chinese hamster ovary (CHO) cells and a commercial immunological test (BCET-RPLA, Oxoid) were used. Toxin titres were in agreement with epidemiological characteristics and toxicity demonstrated by using other systems in other examinations. A comparison of intra- and extracellular toxicities measured at the exponential and stationary growth phases showed that the toxin was essentially secreted during the exponential phase. For several strains, secretion peaked during the period from the middle exponential phase to the beginning of the stationary phase. There was no important overall increase of the toxicity during full and late stationary phase. The level was stable or even lower, thus indicating that diarrheal toxin production during stationary phase was small, if any, and that the toxin was unstable under these conditions. Statistical analysis of toxicities showed that the cytotoxicity test was correlated with the immunological test (significant at a 1% level). For routine determinations, a toxicologic laboratory may use any of the two methods, depending on its facilities, the immunological test being relatively expensive.

Characterization of alternative respiratory pathways in the yeast Schwanniomyces castellii by the study of mutants deficient in cytochromes a+a3 and/or b.

After a general review of the proposed mechanisms and physiological roles of the alternative respiratory pathways found in various organisms, the studies are focussed on the amylolytic yeast Schwaniomyces castellii. In addition to the cytochrome chain, the wild type presents two alternative pathways insensitive to antimycin A. One is salicylhydroxamic acid (SHAM)-sensitive and azide-insensitive; the other is SHAM-insensitive and sensitive to high azide concentration. Conditions for mutagenesis and screening are described, which allow isolation of mutants deficient in cytochromes a+a3 and/or b in this yeast previously classified as petite negative. The relative proportions of the alternative respiratory pathways are compared in the wild type and mutant strains following inhibition by SHAM and azide at optimal concentration as determined by iso-inhibition curves. The growth of the cytochrome deficient mutants on citrate, a non-fermentable carbon source, and the ability of the wild type to grow on citrate+antimycin A, after a lag of about 10 h, indicate an involvement of the alternative pathway(s) in energy production. Rotenone sensitivity of respiration and ATP level confirm the presence of a functional phosphorylation site 1. The role of each alternative respiratory pathway in energy production is discussed.

Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase. Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships.

We have established the nucleotide sequence of the wild-type and that of a trans-acting mutant located in the third (bi3) intron of the Saccharomyces cerevisiae mitochondrial cytochrome b gene. The intron, 1691 base-pairs long, has an open reading frame 1045 base-pairs long, in phase with the preceding exon and the mutation replaces the evolutionarily conserved Gly codon of the second consensus motif by an Asp codon and blocks the formation of mature cytochrome b mRNA. Splicing intermediates of 5300 and 3900 bases with unexcised bi3 intron and a characteristic novel polypeptide (p50), the size of which corresponds to the chimeric protein encoded by upstream exons and the bi3 intronic open reading frame (ORF), accumulate in this and other bi3 splicing-deficient mutants. We conclude that the protein encoded by the bi3 ORF is a specific mRNA maturase involved in the splicing of the cytochrome b mRNA. The open reading frame of the third intron is remarkably similar to that of the unique intron of the cytochrome b gene (cob A) of Aspergillus nidulans. Both are located in exactly the same position and possibly derive from a recent common ancestor by a horizontal transfer. We have established the nucleotide sequence of an exonic mutant located in the B3 exon. This missense mutation changes the Phe codon 151 into a Cys codon and leads to the absence of functional cytochrome b but does not affect splicing. Finally, we have studied the splicing pathway leading to the synthesis of cytochrome b mRNA by analysing, in a comprehensive manner, the 22 splicing intermediates of several mutants located in bi3.

Impairment of insulin assimilation and beta-fructosidase activity due to a petite mutation in Kluyveromyces marxianus.

A respiratory deficient mutant of Kluyveromyces fragilis was isolated using ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a + a3 and deficiency in cytochrome b. This petite mutant has brought about modifications in the excretion pattern of beta-fructosidase active on saccharose and inulin. The mutant practically no longer excretes the enzyme, and is incapable of growth and fermentation in the presence of inulin. The study of the activities of different enzyme extracts (culture medium, whole and disrupted cells) on inulin and saccharose suggests the existence of an unique enzyme system capable of taking several forms, and also shows the influence of the growth substrate on the I/S activity ratio.

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth, respiration and protein biosynthesis.

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O2 consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.

Isolation of a respiratory-deficient Kluyveromyces fragilis mutant for the production of ethanol from Jerusalem artichoke.

A respiratory-deficient, mutant of Kluyveromyces fragilis was isolated using a ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a + a3 and by an improvement of its inulinase activity. Under anaerobic conditions this mutant was always better than the wild strain for ethanol production especially from Jerusalem artichoke extracts containing large amounts of high polyfructosans ("early" extracts).

Protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes.

Yeast mitochondria isolated from two different wild type strains (gal+ and gal-), whether grown on galactose or glucose, synthesize all mitochondrial polypeptides with similar efficiencies and in proportions approximating those detected in vivo. Mitochondria isolated from mit- mutants synthesize in vitro a mutant pattern of mitochondrial proteins, indistinguishable from the in vivo products. The mutant pattern is restored to the wild type one in mitochondria isolated from pseudorevertant strains carrying an additional nuclear (nam3-1 and R705) or mitochondrial (mim3-1) informational suppressor gene. Suppression is expressed in isolated mitochondria without the obligatory presence of cytosol at the level of both respiratory control and specific polypeptide synthesis. Translation in isolated mitochondria is sensitive to paromomycin. The antibiotic differentiates between translation in mitochondria from wild type strains and that in nam-type gene carrying strains. This strongly suggests that nam-type mutations affect the mitoribosome, enhancing ambiguity of translation, thus allowing for the pseudoreversion of mit- phenotypes.

Isolation and characterization of a mutant of Schwanniomyces castellii with altered respiration.

We have tried to isolate respiratory deficient mutants of the amylolytic yeast Schwanniomyces castellii CBS 2863 after mutagenesis with acriflavine. One of the mutants called DR 12 has been studied in more detail. Pasteur effect present in the wild-type is lost in the mutant, on the contrast an obvious Crabtree effect was observed: fermentation was almost as active in aerobiosis as in anaerobiosis. Moreover, the rate of anaerobic fermentation of the mutant was almost twice that of the wild type. This mutant was cytochrome b-deficient while the amount of the other cytochromes was larger than in the wild-type. Moreover, the level of these remaining cytochromes in the mutant was higher on non-repressive medium than on glucose medium. However, the fact that the mutant DR 12 retained a cyanide-sensitive respiration and that it was able to grow on ethanol as a non-fermentable substrate is noteworthy.

Phenotypic suppression and nuclear accommodation of the mit- oxi1-V25 mutation in isolated yeast mitochondria.

Phenotypic suppression by the antibiotic, paromomycin, of the mitochondrial oxi1- -V25 mutation, a mutation which arrests by premature ochre codon the synthesis of the cox II subunit, was studied in isolated yeast mitochondria competent in translation. This antibiotic is known to suppress the mutation in vivo (Dujardin et al. 1984) and allowed in vitro, at concentrations of 20-1100 micrograms per ml. the synthesis of the cox II subunit. This strongly suggests that phenotypic suppression of mit- mutations is due to the direct action of paromomycin on mitochondrial ribosomes. The effect of paromomycin bears a resemblance to the function of the omnipotent nuclear suppressor mutation R705. The nuclear suppression was expressed in isolated mitochondria; suppressor mutation influenced the structure of the mitoribosome. Therefore, it appears that mitoribosomes are indeed the common target in the phenotypical and genetic nuclear suppression of the oxi1-V25 mutation.

Tubulin evolution: ciliate-specific epitopes are conserved in the ciliary tubulin of Metazoa.

In spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised against Paramecium and Tetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.

Antibodies against synthetic oligopeptides allow identification of the mRNA-maturase encoded by the second intron of the yeast cob-box gene.

Genetic and biochemical evidence has strongly suggested that several introns located in yeast mitochondrial genes specifying apocytochrome b or cytochrome oxidase encode trans-acting proteins (termed mRNA-maturases) responsible for splicing the cognate intron and maturation of the mRNA. We have chemically synthesized three oligopeptides, predicted from the DNA sequence of the open reading frame (ORF) present in the second intron of the cob-box gene, and raised antibodies against them. These antibodies have allowed us to identify a protein of 42 kd as the product translated from the ORF of the wild-type intron. In two splicing-deficient mutants this protein is replaced by shorter polypeptides whose lengths and antigenic properties are in full agreement with the positions of TAA codons established by the DNA sequence of the intron's ORF.

Tubulin evolution: an electrophoretic and immunological analysis.

This paper summarizes a survey of the electrophoretic behavior of the tubulins of 23 species (mostly protists) as well as their reactivity towards 4 anti-tubulin antibodies (raised against two ciliate tubulins and two vertebrate ones). Some generalizations concerning the relative migration rates of alpha VS beta tubulin could be made, in particular the alpha/beta inversion, first described in Physarum was extended to several ciliates. Antivertebrate tubulin antibodies displayed a very broad spectrum of reactions, reacting with virtually all the species tested. They appear to correspond to auto-antibodies no exclusively directed against species specific determinants. In contrast, the two anti-ciliate tubulin antibodies displayed a narrow species specificity reacting only with a limited subset of protists. They were shown to be specific for a small number of immunological determinants present on ciliate tubulins. This allowed a rough evaluation of evolutionary relatedness between the various groups of protists analyzed. The results are discussed within the framework of a number of published phyllogenies and shown to be in striking agreement with some of the schemes.

Recombinational analysis of oxi2 mutants and preliminary analysis of their translation products in S. cerevisiae.

Genetic and biochemical studies were performed with mutants allocated to the mitochondrial oxi2 gene.Recombinational analysis of 19 oxi2 mutants was performed using α and a mutant strains derived from the same genetic background. The frequencies of wild-type recombinants in oxi2 (-) × oxi2 (-) crosses varied from 0.002 to 17%. The map of oxi2 mutations constructed on the basis of these frequencies shows many internal inconsistencies. In the course of rho (-) deletion mapping five classes of oxi2 mutations were distinguished. The results of deletion analysis are in agreement with those of recombinational mapping.The analysis of mitochondrial translation products by SDS-polyacrylamide electrophoresis of 20 oxi2 mutants shows that 17 of them are connected with conspicuous changes of 22 kd polypeptide band corresponding to subunit III of cytochrome oxidase. At least four of them carried instead of subunit III clearly visible significantly shorter polypeptides (12.8 to 20.1 kd). These were, most likely, shorter fragments of subunit III resulting from chain termination mutations. Colinearity was observed between the lenght of new polypeptides and the positions of the respective mutations on the recombinational map. These data confirm hat oxi2 encodes subunit III of cytochrome oxidase and suggest that translation of the oxi2 gene is in the direction from V303 to V273.

Mutations within an intron and its flanking sites: patterns of novel polypeptides generated by mutants in one segment of the cob-box region of yeast mitochondrial DNA.

We have undertaken a systematic examination of the polypeptides accumulating in thirteen (out of 23) mutants in the intron cluster box7 and its flanking clusters box2 and box9 of the cob-box (cytochrome b) region of the mitochondrial genome of Saccharomyces cerevisiae. We have subjected these polypeptides to fingerprint analysis, both sequential and in parallel, with two proteases in order to disclose sequence homologies and differences between the different novel polypeptides themselves, and between them and the wild type product of the gene, i.e. apocytochrome b. One of our aims has been to establish the existence of possible correlations between the nature of the novel polypeptides and the fine structure genetic map of that segment of the mitochondrial genome. Our results show that all box7 mutants accumulate the following set of polypeptides not seen in wild type cells: a) a characteristic set of "large" polypeptides consisting of three species: p56, p42 and p35 or p34.5; b) a polypeptides p23; c) a much shorter fragment (of which the apparent molecular weight varies from 12.5 to 13, according to the mutation) with the exception of two mutants; d) in addition, the majority accumulate in varying amounts a polypeptide p30 closely related to but not identical with apocytochrome b. Moreover only two box7 mutants accumulate a polypeptide in the range of mobilities corresponding to 25-27 Kd (referred to as class p26) while such a polypeptide is seen in all box9 and box2 mutants examined with one exception in box2.