Respiration-deficient Mutants Research Articles

Increase in gene expression by respiratory-deficient mutation.

Respiratory-deficient mutants (rho- cells) of Saccharomyces cerevisiae produced about 10 times as much human (h-) lysozyme as did wild-type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h-lysozyme gene. Introduction of intact mitochondria into the rho- cells resulted in a significant decrease in the production of h-lysozyme, indicating that the rho- mutation increased the expression of the h-lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h-lysozyme mRNA in the rho- cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h-lysozyme gene was also observed when a glyceraldehyde-3-phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho- mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho- mutants, pet mutants did not show higher gene expression compared with wild-type strains.

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase.

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to pet complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the MSM1 gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of MSM1 functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSM1 is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.

The induction of rho− mutants by UV or γ-rays is independent of the nuclear recombinational repair pathway in Saccharomyces cerevisiae

In order to discover whether the nuclear recombinational repair pathway also acts on lesions induced in mitochondrial DNA (mtDNA), the possible role of the RAD50, −51, −52, −55 and −56 genes on the induction of rho − mutants by radiations was studied. Such induction appeared to be independent of this pathway. Nevertheless, an efficient induction of respiration-deficient mutants was observed in γ-irradiated rad52 diploids. We demonstrate that these mutants do not result from a lack of mtDNA repair, but from chromosome losses induced by γ-rays. Such an impairment of the respiratory ability of diploids by chromosome losses was effectively observed in the aneuploid progeny of unirradiated RAD + cdc6 diploids incubated at the restrictive temperature.

Nuclear and mitochondrial revertants of a yeast mitochondrial tRNA mutant.

We isolated revertants capable of respiration from the respiratory deficient yeast mutant, FF1210-6C/170, which displays greatly decreased mitochondrial protein synthesis due to a single base substitution at the penultimate base of the tRNAAsp gene on mitochondrial (mt) DNA. Three classical types of revertant were identified: (1) same-site revertants; (2) intragenic revertants which restore the base pairing in the acceptor stem of the mitochondrial tRNAAsp; and (3) extragenic suppressors located in nuclear DNA. In addition a fourth type of revertant was identified in which the mutant tRNAAsp is amplified due to the maintenance of both the original mutant mtDNA and a modified form of the mutant mtDNA in which only a small region around the tRNAAsp gene is retained and amplified. The latter form resembles the mtDNA in vegetative petite (rho-) strains which normally segregates rapidly from the wild-type mtDNA. Each revertant type was characterized genetically and by both DNA sequence analysis of the mitochondrial tRNAAsp gene and analysis of the quantity and size of RNA containing the tRNAAsp sequence. These results indicate that the mitochondrial tRNAAsp of the mutant retains a low level of activity and that the presence of the terminal base pair in tRNAAsp is a determinant of both tRNAAsp function and the maintenance of wild-type levels of tRNAAsp.

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast.

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.

Two distinct mechanisms for deletion in mitochondrial DNA of Schizosaccharomyces pombe mutator strains : Slipped mispairing mediated by direct repeats and erroneous intron splicing

Mutator strains of the fission yeast Schizosaccharomyces pombe produce mitochondrial respiratory deficient mutants at a high rate, and roughly 20% of these mutants carry deletions in the range of 50 to 1500 base-pairs. To elucidate the mechanism of deletion we have sequenced ten deletion mutants in the mosaic gene encoding apocytochrome b ( cob) and three in the split gene coding for the first subunit of cytochrome c oxidase ( cox1). Of 13 deletions, ten are correlated with the presence of direct repeats, which could promote deletions by slipped mispairing during DNA replication. In some of these mutants, the termini are located in possible DNA secondary structures. In three independently isolated mutants with identical deletions in the cob gene, the 5′ deletion endpoint coincides with the 3′ splice point of the intron, whereas the 3′ endpoint of the deletion exhibits pronounced homology with the 5′ splice point of the intron. This result suggests that these deletions might be initiated by erroneous RNA splicing.

Fibrous component of yeast mitochondria

An intramitochondrial fibrous component (IMF) was consistently detected by electron microscopy in all eight strains of yeast examined, either respiratory competent or deficient, and including two species, Saccharomyces cerevisiae and S. uvarum. IMF was always found assembled into a layer 20 nm in thickness. In respiratory-deficient mutants, multiple IMF layers roll up concentrically to give rise to cylindrical inclusion bodies which attain several micrometers in length. In normal respiring yeasts, IMF occurs in close association with cristae to elaborate composite structures in which multiple parallel IMF layers are sandwiched between a pair of cristae. Evidence is presented to demonstrate the extramitochondrial origin of IMF.

Effect of methanol on the frequency of respiration-deficient mutation induced by ethidium bromide in yeast under growing and non-growing conditions.

The effect of methanol on the frequency of cytoplasmic respiration-deficient (RD) mutation induced by ethidium bromide was investigated in Saccharomyces cerevisiae. When growing cells were treated with ethidium bromide at concentrations higher than 1.0μg/ml, 95 to 100% of surviving cells were RD mutants as judged by the tetrazolium overlay method. The RD mutants induced by 1.0μg/ml ethidium bromide were drastically decreased by addition of 6-8% methanol in both growing and non-growing conditions. Methanol reduced uptake of ethidium bromide by yeast cells, but it did not modify the inhibitory effect of ethidium bromide on in vitro yeast mitochondrial deoxyribonucleic acid synthesis.

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli.

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.

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.

Growth and growth estimation of Saccharomyces cerevisiae in solid-state ethanol fermentation

The anaerobic growth of a respiration-deficient mutant of Saccharomyces cerevisiae on solid medium was estimated by the CO 2 evolution rate (CER). The cell growth and ethanol production were calculated by a growth-model associated with CER. The estimated cell growth agreed with the observed data. The calculation and the observed CER suggested that the maximum ethanol production and maximum cell groth are restricted by the initial moisture content of the solid medium.

Isolation and characterization of respiratory-deficient mutants of Escherichia coli K-12.

Several mutants of Escherichia coli K-12 defective in aerobic metabolism were isolated. One such mutant was found to be deficient in cytochromes, heme, and catalase. Aerobically grown cells did not consume oxygen and could grow only on fermentable carbon sources. Supplementation of the growth medium with delta-aminolevulonic acid, protoporphyrin IX, or hemin did not restore aerobic metabolism. The lack of heme and catalase in mutant cells grown on glucose was not due to catabolite repression, since the addition of exogenous cyclic AMP did not restore the normal phenotype. When grown aerobically on complex medium containing glucose, the mutant produced lactic acid as the principal fermentation product. This pleotropic mutation was attributed to an inability of the cells to synthesize heme, and preliminary data mapped the mutation to between 8 and 13 min on the E. coli genome.

Reduction of iron(III) byEscherichia coli K12: Lack of involvement of the respiratory chains

Extracts ofEscherichia coli, harvested from aerobic, anaerobic, or oxygen-limited cultures, reduced Fe(III) citrate with NADH or NADPH as reductants. The activity was predominantly soluble, not membrane bound. Cytochromes and quinones were shown, by the use of respiration-deficient mutants, not to be involved in electron transfer to Fe(III). Neither nitrate nor nitrite, alternative electron acceptors from the respiratory chain, directly inhibited Fe(III) reduction, but in anaerobic cultures containing nitrate and high Fe(III) concentrations, no Fe(II) accumulated; this was attributed to the reoxidation of Fe(II) by nitrite, the product of nitrate reduction. The pathway(s) of electron transport to the metal ion, the observed stimulation of Fe(III) reduction by ATP and cyanide, and the possible physiological significance of the reaction are discussed.

Assembly of the mitochondrial membrane system. MRP1 and MRP2, two yeast nuclear genes coding for mitochondrial ribosomal proteins.

Nuclear respiratory deficient mutants of Saccharomyces cerevisiae impaired in mitochondrial protein synthesis have been screened for lesions in ribosomal protein constituents. Two mutants, each representative of a separate pet complementation group, have been analyzed. One of the mutants, E795, was found to have altered mitochondrial ribosomes as evidenced by the absence of some ribosomal proteins. The second mutant studied, C167, appeared to have more grossly altered ribosomes that could not be isolated by standard preparative procedures. In addition to being defective in mitochondrial protein synthesis, the mutants exhibit an absence of "a" and "b" type cytochromes, are partially blocked in processing of intron bI4 of the apocytochrome b gene, have reduced levels of mitochondrial 15 S rRNA, and convert to rho- and rho 0 mutants at a high frequency. The wild type genes MRP1 and MRP2 were cloned by transformation of the pet mutations in E795 and C167, respectively, with a recombinant plasmid library of wild type yeast genomic DNA. MRP1 codes for a basic protein of 37 kDa with no significant homology to any known prokaryotic or eukaryotic ribosomal protein. MRP2 codes for a 14-kDa polypeptide homologous to protein S14 of the Escherichia coli small ribosomal subunit and to a chloroplast-encoded component of chloroplast ribosomes. The levels of MRP1 and MRP2 mRNAs were examined in glucose-repressed cells and in cells undergoing adaptation to aerobic metabolism of ethanol. The steady state concentrations of the mRNAs increased during the first 3 h of derepression, indicating that expression of these mitochondrial ribosomal protein genes is transcriptionally regulated by glucose in a fashion analogous to respiratory carriers such as cytochrome c.

Effect of methanol and some metal compounds on the frequency of respiration-deficient mutation in yeast induced by manganese chloride.

Effect of methanol and some metal compounds on the frequency of respiration-deficient mutation in yeast induced by manganese chloride.

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis.

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae.

The respiratory deficient yeast nuclear mutant MK3 is defective in the synthesis of the mature transcripts of the mitochondrial COB and OX13 genes, which code for apocytochrome b and subunit I of cytochrome c oxidase, resp. Introns 3 and 4 of the COB transcript (bI3 and bI4) and intron 4 (aI4) of the OXI3 transcript can not be excised (Pillar et al. 1983a, b). When combined with mitochondrial genomes lacking introns bI1, bI2 and bI3, or lacking intron bI3 alone the mutant is respiratory competent. Thus, the non-excision of bI4 and aI4 turns out to be an indirect effect of the mutation. From a wild type yeast genebank a plasmid has been isolated with a 3.3 kb DNA insert, which complements the mutant. Subcloning experiments assigned the functional gene to a 1.6 kb HaeIII-Sau3A fragment. Hybridization experiments showed, that it is (i) a single copy gene, (ii) also present in strain D273-10B, containing the "short form" mitochondrial genome (lacking the COB introns bI1-bI3), and (iii) located on chromosome IX. The nuclear gene defective in mutant MK3, was named MRS1 (Mitochondrial RNA Splicing). The involvement of this nuclear gene in the excision of a single group I mitochondrial intron (bI3) of the COB transcript is discussed.

Reductive repression in Escherichia coli K-12 is mediated by oxygen radicals

Cyclic AMP (cAMP) content and the expression of cAMP-dependent phenotypes were positively correlated with respiration capacity in respiration-deficient mutants of Escherichia coli K-12 (“reductive repression,” R. Hertz, and J. Bar-Tana, (1982) Arch. Biochem. Biophys. 213, 193–199). Reductive repression in respiration-deficient mutants could not be accounted for by respective changes in either the energy charge of adenine nucleotides or the redox state of pyridine nucleotides but could be ascribed to an increased formation of oxygen radicals under conditions of limited respiration. Scavengers of superoxide radicals eliminated reductive repression in respiration-deficient mutants with a concomitant increase in cAMP content. Such scavengers also effected a partial escape from permanent glucose catabolite repression, thus indicating a possible role played by oxygen radicals in both repression modes.

Mechanism of action of Pseudomonas syringae phytotoxin, syringomycin. Interaction with the plasma membrane of wild-type and respiratory-deficient strains of Saccharomyces cerevisiae.

The effects of the phytotoxin, syringomycin, produced by Pseudomonas syringae pv. syringae, were examined on cells of a wild-type and a respiratory-deficient (rho0) mutant of Saccharomyces cerevisiae. The growth of both strains in liquid culture was inhibited by 0.5 micrograms syringomycin per ml and higher. Uptake rates of tetraphenylphosphonium and dimethyloxazolidine ions in cell suspensions of both strains increased when 1.5 micrograms per ml syringomycin was added. These responses were kinetically and quantitatively similar in the two strains and indicated increases in electrical potential (cell interior negative) and pH differences (cell interior alkaline) across the plasma membrane. Glucose (0.1 M) enhanced the effect on the electrical potential, was required for the pH changes, and increased the cellular ATP levels. These results show that the effects of syringomycin are energy-dependent and are due to alterations of plasma membrane and not to mitochondrial function.

Ethanol inhibition of growth, fermentation and starch hydrolysis in Schwanniomyces castellii

The inhibitory effect of ethanol on several parameters of starch fermentation was studied by comparing the strain Schwanniomyces castellii and the corresponding respiratory-deficient mutant. The effect of ethanol on cell viability and cell growth depends on the yeast metabolism. Inhibition was greater when the fermentative metabolism was preponderant. Amylases were shown to be inhibited at ethanol concentrations which are often obtained in fermentation. This inhibition conformed to non-competitive kinetics and was reversible. Enzyme biosynthesis was reduced in the presence of ethanol.