A new type of mitochondrial mutation in Paramecium

Summary The mutant cl₁ of Paramecium previously described (Sainsard et al., 1974) differs from wild-type by a single recessive nuclear gene, cl ₁, and its mitochondria, M^cl, can be distinguished from wild-type mitochondria, M⁺, (Sainsard-Chanet, 1976). In order to determine the nature of the difference between M^cl and M⁺ mitochondria, the stability of the M^cl phenotype was studied. It was found that the M^cl character behaves like a mitochondrial mutation. Associated with a wild-type nucleus, M^cl mitochondria retain indefinitely their distinctive properties, i.e. compatibility with a cl ₁/cl ₁ nucleus and decrease of the cellular growth rate and cytochrome aa₃ content. Some properties of the cl₁ mutant which is in fact a double nuclear-mitochondrial mutant with the mitochondrial mutation partially suppressing the nuclear one are discussed.     

A regulatory system controlling the production of cytochrome aa3 in Neurospora crassa

Summary The mitochondria of the cyt-2-1, cya-3-16, cya-4-23 and 299-1 nuclear mutants and the [mi-3] and [exn-5] cytoplasmic mutants of Neurospora crassa are deficient in cytochrome aa ₃, while the cyb-1-1 and cyb-2-1 mutants have mitochondrial b-cytochrome dificiencies. However, the mitochondria from cyb-1-1 cyt-2-1, cyb-1-1 [mi-3] and cyb-2-1 [mi-3] double mutants contain 30% to 50% of the amount of cytochrome aa ₃ that is present in mitochondria from wild-type; i.e. cyb-1-1 and cyb-2-2 act as suppressors of the cytochrome aa ₃ deficiency phenotypes that are associated with the cyt-2-1 and [mi-3] mutations.The production of cytochrome aa ₃ can be induced in cyt-2-1 and [mi-3] by growing cells in medium containing antimycin A, an inhibitor of electron transport in the cytochrome bc ₁ segment of the mitochondrial electrontransport chain. Moreover, the growth of the [mi-3] mutant is strongly stimulated by low concentrations of antimycin A. The induction of cytochrome aa ₃ by antimycin treatments does not occur in [exn-5], cya-4-23 and 299-1 cells, but does take place in cya-3-16 cells.Although some of the seven constituent polypeptides of cytochrome aa ₃ are present the mitochondria of [mi-3], the holoenzyme complex is not formed in the mutant. In contrast, the mitochondria of cyb-1-1 [mi-3] and cyb-2-2 [mi-3] double mutants contain a fully assembled cytochrome oxidase complex as well as some unassembled subunit polypeptides.The observations are indicative of the existence of at least two regulatory systems controlling the production of cytochrome aa ₃. One of the circuits appears to control the basal or constitutive production of cytochrome oxidase, the other seems to coordinate the level of cytochrome aa ₃ with some function of the mitochondrial cytochrome bc ₁ complex, possibly electron transport.     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.     

Gene controlled selection of mitochondria in Paramecium.

Summary The slow growing mutant cl₁ of Paramecium, previously described (Sainsard, Claisse and Balmefrezol, 1974) differs from wild-type by a single recessive nuclear mutation and by a particular mitochondrial phenotype (M^cl) that gene cl ₁ distinguishes from the wild-type mitochondrial phenotype (M⁺). A further analysis of these nucleo-mitochondrial interactions was carried out by confronting the genes cl ₁ and cl ₁ ⁺ with mixed populations of M⁺ and M^cl mitochondria obtained after cytoplasmic exchange at conjugation. The following results were obtained: 1. M⁺ and M^cl mitochondria introduced respectively into mutant and wild-type cells do not multiply easily; 2. when a mixed population (M⁺+M^cl) is established, both mitochondrial types are maintained during the growth of the F₁ heterozygous cl ₁/cl ₁ ⁺ clones; 3. when the nuclear segregation occurs in F₂, the formation of homozygotes cl ₁/cl ₁ or cl ₁ ⁺ /cl ₁ ⁺ is soon followed by the segregation of the two mitochondrial types, M^cl or M⁺, reconstituting the two parental nucleo-mitochondrial associations.This paper is dedicated to Professor T.M. Sonneborn on the occasion of his 70th birthday     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

Growth,respiration and cytology of acetate-negative mutants ofCandida albicans

A number of acriflavine-induced mutants ofCandida albicans, characterized by their inability to grow on acetate as a source of energy, were screened for their cytochrome absorption spectra. Three mutants with different spectra, along with their parent, were selected for comparative studies, of their growth, respiratory activities and cellular structure. The spectrum of one of the mutants was the same as that of the wild-type, but the growth rate and yield of cells on glucose medium were only about 60% of the wild-type's; those of a second mutant deficient in cytochromes aa₃ were 50%, and those of a third mutant deficient in cytochromes aa₃ and b were less than 5% of those of the wild-type. The cytochrome-complete mutant and the wild-type showed respiratory activity both on glucose and ethanol well above the endogenous, the cytochrome aa₃-deficient mutant showed only endogenous respiration, and the cytochrome aa₃, b-deficient mutant no respiration at all. Electron microscopy of the wild-type cells revealed discrete, regular ovoidal, cristate mitochondria spaced near the periphery of the protoplasm; the cytochrome-complete mutant showed an abundance of large, cristate, but morphologically irregular mitochondria; the cytochrome aa₃-deficient mutant had fewer but still large, cristate, somewhat irregular mitochondria; and the cytochrome aa₃, b-deficient mutant only a few simple vesicles without discernible cristae.     

Ionic strength effects on cytochrome aa3 kinetics

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa₃ can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa₃ increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa₃ complex limits the ferrocytochrome c association to the low affinity site.2. At low ionic strength, the reduction of cytochrome c-aa₃ complex by ferrocytochrome c₁ proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c₁ and aa₃.3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa₃ complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c.4. Dissociation rates of both high and low affinity sites on cytochrome aa₃ were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 μM for the high and low affinity site, respectively.     

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa₃ and to the (porphyrin) cytochrome c-cytochromeaa₃ complex.2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2 · 10⁸ M^?1 · s^?1 and about 10⁷ M^?1 · s^?1 (I = 8.8 mM, pH 7.0, 10° C), respectively.3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa₃ reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa₃. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa₃ has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa₃ has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.     

Haem O and a putative cytochrome bo in a mutant of Bacillus cereus impaired in the synthesis of haem A

In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically. The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected. In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O. The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation. External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant. The presence of membrane-bound cytochrome caa ₃ -OII and aa ₃ -II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit). Western blotting also revealed that in contrast to the wild-type – strain PYM1 contained the membrane-bound subunits caa ₃ -COI and aa ₃ -I, but in low amounts. The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo. Received: 21 June 1996 / Accepted: 9 October 1996     

Abnormal-abdomen beiDrosophila hydei

Several strains ofDrosophila hydei exhibiting an abnormal-abdomen (aa) phenotype were investigated. They proved to be mutant at one and the same major gene, located on chromosome 2 (homologous to 3R ofD. melanogaster).This mutant gene,aa, causes skeletal abnormalities of the adult abdomen. Sternites and, less frequently, tergites are imperfectly sclerotized. They may be dissolved into separate areas, or may be missing altogether. Often they are normal in shape, while the number of hairs is reduced. Compound sclerites originating from parts of adjoining segments were never observed. Small hypoderm spherules with internal hairs occur frequently under the ventral skin. In aa larvae, segmentation is normal, in contrast to the situation in abnormal-abdomen mutants ofD. melanogaster. Even where theaa mutant expression is extremely strong, puparia are perfectly normal. Theaa gene thus appears to act exclusively during metamorphosis. This was confirmed by temperature experiments. The expression of theaa gene is much reduced at low temperature, but low temperature is effective during metamorphosis only.The seven aa strains compared, showed considerable differences in penetrance and expressivity as well as in the amount of dominance in crosses. Reciprocal crosses yield in the F₁ values either intermediate, or lower or higher, compared to those of the parent strains. The F₁ values show a nonlinear correlation between penetrance and expressivity.On the basis of chromosomal location and phenotypic effect, it is unlikely thataa ofD. hydei is homologous to any of the abnormal-abdomen mutants ofD. melanogaster described so far. However, a new dominant autosomal mutant was found inD. simulans, which seems to be a homologue.     

Efficiency of insecticidal crystal protein production in a Bacillus thuringiensis mutant with derepressed expression of the terminal oxidase aa 3 during sporulation

A Bacillus thuringiensis respiratory mutant (AB1 strain) that shows premature sporulation and insecticidal crystal protein (ICP) production was isolated. The mutant strain harbours the cryIC and cryID insecticidal genes and could be important for the production of ICP highly toxic to Spodoptera sp. The mutant was selected by its increased capacity to oxidize. N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). In this strain, cytochrome aa ₃ expression is not repressed during the sporulation phase, in contrast with the wild-type strain. The growth, spore production, dissolved O₂, O₂ consumption, CO₂ evolution rate and ICP production were recorded as a function of time. The AB1 mutant strain has a similar growth yield to the wild-type strain, but begins sporulation at least 4 h earlier. The AB1 strain consumes 74.5% more O₂ than the wild-type strain, during the fermentation process. The mutation on strain AB1 has an important positive effect on ICP production. This procedure shows that ICP production could be increased during fermentation by increasing the respiration capacity of Bacillus thuringiensis. Correspondence to: A. Bravo     

Cytochrome mutants of bradyrhizobium induced by transposon tn5

Transposon Tn5 was used to mutate Bradyrhizobium japonicum USDA 61N. From over 5000 clones containing Tn5, 12 were selected and purified using a chemical reaction to identify oxidase-deficient clones. Four classes of mutants were identified based on the alterations in cytochromes. Most of the mutants had alterations in more than one cytochrome. Southern hybridization analysis of restricted genomic DNA of a representative strain of each class demonstrated that each mutant had a single Tn5 insert. Thus a single Tn5 insert produced pleiotropic effects on cytochromes. One class, which was totally deficient in cytochromes aa₃ and c, produced ineffective nodules on soybeans. Most of the strains representing the other classes produced effective nodules but exceptions were observed in each class. Bacteroids of the wild-type strain contained cytochrome aa₃. Bacteroids from one class of mutants were totally devoid of cytochrome aa₃. Several of these strains produced effective symbioses indicating that cytochrome aa₃ is not required for an effective symbiosis in this DNA homology group II strain which normally has this terminal oxidase in bacteroids.     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Oxidative deficiencies and some ultrastructural features of acridine-induced male strains of Allomyces arbusculus

Summary A high proportion of small, frequently TTC^- and highly (some 100%) masculinized gametophytic colonies have been produced from meiospores of Allomyces arbusculus plated on acridine-containing media.Endogenous O₂-consumption as well as succinate dehydrogenase and cytochrome oxidase activities are reduced in the acridine-treated masculinized strains studied. Such sharp deficiencies in the oxidative metabolism are paralleled in an acridine-male strain by structural defects such as highly reduced number of cristae in the mitochondria.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.