Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of Neurospora crassa and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-1 (wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction in the WCC mutants of stress factor signals that promote ROS (reactive oxygen species) formation.     

The role of thiol redox systems in the response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to far-UV irradiation

Escherichia coli mutants deficient in glutathione (gshA), glutaredoxin (grxA), thioredoxin (trxA), and thioredoxin reductase (trxB) synthesis were studied with respect to their resistance to far-UV (UV₂₅₄) exposure. The trxA, trxB, and grxA mutants subjected to a short-term UV exposure were found to be more resistant to UV irradiation than the parent cells. Under the same conditions, the trxA and trxB mutants demonstrated a high level of induction of the sulA gene, a component of the SOS regulon. The mutagenic effect of long-term UV exposure of all the mutants with redox deficiencies was more pronounced than in the case of the parent strain, and the trxA and trxB mutants were found to be the least viable microorganisms. Pretreatment of the cells with low concentrations of the thiol-oxidizing agent diamide enhanced the sulA gene expression; however, high concentrations of diamide inhibited sulA expression. The data obtained indicate that the thiol redox systems of E. coli are involved in its response to far-UV irradiation.     

The role of thiol redox systems in the peroxide stress response of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H₂O₂, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.     

Altered Species of Mitochondrial Transfer RNA associated with the <Emphasis Type="Italic">mi</Emphasis>-1 Cytoplasmic Mutation in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

THE mi-1 (poky) strain of Neurospora crassa is a relatively stable, respiration-deficient mutant, which exhibits cyto-plasmically-inherited reduction of growth rate and aberrations in the mitochondrial eytochrome system. In young cultures of mi-1, the cells accumulate up to sixteen times the amount of cytochrome c present in wild-type Neurospora and cytochromes b and a are not detectable spectroscopically in these same cells¹. In sexual crosses the mi-1 mutation is transmitted only through the cytoplasm of the protoperithecial parent and the pleiotropic mi-1 phenotype is caused by an alteration in a cytoplasmic gene², presumably in the mitochondrial DNA.     

A study of the nitrate and nitrite discharge from the mutant cells of <Emphasis Type="Italic">Neurospora crassa</Emphasis> lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Endogenous Salicylic Acid Levels and Signaling Positively Regulate <Emphasis Type="Italic">Arabidopsis</Emphasis> Response to Polyethylene Glycol-Simulated Drought Stress

Salicylic acid (SA) functions in the plant response to drought stress were assessed using SA-altering Arabidopsis mutants, including snc1 (with constitutively high levels of SA) and its nahG-transformed plants (named as snc1/nahG, with a comparable SA level to the wild type), sid2 and transgenic line nahG (both with SA deficiency), and npr1-1 (with SA signaling blockage). The drought stress was simulated by polyethylene glycol (PEG)-6000 treatment. Compared with wild-type (wt) plants, the snc1 plants displayed obvious easing of PEG-induced growth inhibition, leaf water loss, and photosynthesis-related impairment, whereas in nahG, sid2, and npr1-1 mutants the effect was more severe. PEG stress reduced stomatal conductance, to a higher extent in the snc1 line, whereas it was lower in nahG, sid2, and npr1-1 lines as compared with the wt. The snc1 plants accumulated higher levels of H₂O₂ than the other genotypes tested. PEG stress increased activities of superoxide dismutase and peroxidase, but decreased activities of catalase in all lines tested, to a greater extent in snc1 and less in sid2, nahG, and npr1-1 relative to wt. Proline was significantly increased, especially in snc1 line at 6 % and higher PEG stress. Noticeably, the performance of snc1 under PEG stress was dependent on SA levels, as the expression of nahG in snc1 plants did not only significantly reduce SA levels, but largely reversed the above-mentioned parameters, as well as eliminated the drought tolerance. Based on these data, it was concluded that endogenous SA levels and signaling provided a protective role in the Arabidopsis response to PEG-simulated drought.     

Molecular characterization of the <Emphasis Type="Italic">Aspergillus fumigatus NCS-1</Emphasis> homologue,NcsA

Here, we characterize the Aspergillus fumigatus homologue ncsA Neuronal Calcium Sensor. We showed that ncsA is not an essential gene and ?ncsA growth was decreased in the presence of EGTA and SDS. Furthermore, the ?ncsA mutant is more resistant to calcium chloride. NcsA:mRFP localizes to the cytoplasm and its cellular localization is not affected by the cellular response to either calcium chloride or EGTA. The ?ncsA mutant strain is more sensitive to voriconazole, itraconazole, and amphotericin. Polar growth in the ΔncsA mutant was also considerably more affected by lovastatin than in the wild type strain. The Spitzenkörper can be visualized in both strains and although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the ?ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the ΔncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.     

Carbohydrate Spectrum of Extremophilic Yeasts <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under pH Stress

Alterations in the concentrations of cell cytosol carbohydrates of polyextremophilic yeasts Yarrowia lipolytica under stresses of diverse nature were observed. Under pH stress, mannitol was the main storage carbohydrate (up to 89% of the total cytosol carbohydrates), while arabitol, glucose, and inositol were present in insignificant amounts (3 to 6%). Experiments with inhibition of de novo mannitol synthesis by bis(p-nitrophenyl) disulfide revealed that the cytoprotective effect of mannitol was most noticeable in the cells grown under acidic conditions (pH 4.0), while the role of catalase and superoxide dismutase, the enzymes of the first line of antioxidant protection, increased under alkaline conditions (pH 9.0). The constitutively high mannitol content in Y. lipolytica cells was hypothesized to be a part of the core mechanism of stress resistance in this yeast species.     

Effect of oxylipins on <Emphasis Type="Italic">Neurospora crassa</Emphasis> growth and differentiation

The effect of the natural oxylipins 3(R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE) and 18-hydroxy-(9Z,12Z)-octadecadienoic acids (18-HODE) on the growth and hypha aggregation, as well as on some light-depending processes, such as carotenoid biosynthesis, protoperithecia formation (sexual cycle), and conidiation (asexual cycle), of the ascomycete Neurospora crassa was studied. Hypha aggregation and growth slowdown were induced by 3-HETE, 18-HODE, and linoleic acid. At concentrations from 5 to 50 μM, these compounds had no significant effect on the light-induced carotenogenesis. At the same time, these 3-HETE and 18-HODE concentrations, unlike linoleic acid, induced the formation of protoperithecia in the dark. At the concentration of 5 μM, an additive effect of oxylipins and light was revealed. The studied oxylipins had different effects on the asexual reproduction of N. crassa: 3-HETE induced conidiation in the dark, whereas 18-HODE induced conidiation in the light. The possible involvement of oxylipins in the regulation of the processes of sexual and asexual reproduction of N. crassa is discussed.     

Repair of cisplatin-DNA adducts in mutants for genes controlling spontaneous and induced mutagenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast

Sensitivity to the lethal action of the anticancer substance cisplatin was studied in the yeast mutants him1, hsm2, hsm3, and hsm6, deficient for repair of spontaneous and induced mutations. The him1 and hsm3 mutants were as resistant to the agent under study as the wild-type strain. The survival of the double mutant rad2 hsm3 was higher than that of the single mutant rad2. The hsm2 and hsm6 mutants were more cisplatin-sensitive than the wild type. Cisplatin was shown to have high mutagenic and recombinogenic effects on yeast cells.     

<Emphasis Type="Italic">Neurospora tetrasperma</Emphasis> crosses heterozygous for hybrid translocation strains produce rare eight-spored asci-bearing heterokaryotic ascospores

During ascogenesis in Neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel T ^Nt strains by introgressing four Neurospora crassa insertional translocations (EB4, IBj5, UK14-1, and B362i) into N. tetrasperma. We now show that crosses of all the T ^Nt strains with single-mating-type derivatives of the standard N. tetrasperma pseudohomothallic strain 85 (viz. T ^Nt a × 85A or T ^Nt A × 85a) can produce rare eight-spored asci that contain heterokaryotic ascospores, or ascospores with other unexpected genotypes. Our results suggest that these rare asci result from the interposition of additional mitoses between the post-meiotic mitosis and the partitioning of nuclei into ascospores, leading to the formation of supernumerary nuclei that then generate the heterokaryotic ascospores. The rare asci probably represent a background level of ascus dysgenesis wherein the partitioning of ascospores becomes uncoupled from the post-meiotic mitosis. Ordinarily, the severest effect of such dysgenesis, the production of mating-type heterokaryons, would be suppressed by the N. crassa tol (tolerant) gene, thus explaining why such dysgenesis remained undetected thus far.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Exploiting the <Emphasis Type="Italic">ZIP4</Emphasis> homologue within the wheat <Emphasis Type="Italic">Ph1</Emphasis> locus has identified two lines exhibiting homoeologous crossover in wheat-wild relative hybrids

Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.