Adaptation of the yeast Yarrowia lipolytica to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents.

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes.

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Modification of proteins by active oxygen and their degradation

A detailed analysis of literary data concerning the oxidative modification of proteins by active oxygen species was carried out. It was shown that intermediate products of molecular oxygen reduction, e.g., superoxide anion radical, hydrogen peroxide and hydroxyl radical, can induce the inactivation of enzymes in vitro as a result of oxidative modification of certain amino acid residues necessary for the maintenance of native properties of the enzyme. In some cases modification of enzymes results in their degradation by proteolytic enzymes. Besides, some enzymes catalyzing the interconversions of active oxygen species (catalase superoxide dismutase, cytochrome P-450) are also inactivated in the course of catalysis under the oxidative action of active oxygen species. It was assumed that the oxidative modification of proteins appears to be one of the mechanisms which control their degradation in the cell. The hydroxyl radical oxidizing the amino acid residues located in the vicinity of the site of its synthesis is a direct modifying species. The superoxide anion radical and hydrogen peroxide are hydroxyl radical precursors and are responsible for the transport of oxidizing equivalents in the cell.     

Inducible resistance to oxidant stress in the protozoan Leishmania chagasi

Leishmania sp. protozoa are introduced into a mammalian skin by a sandfly vector, whereupon they encounter increased temperature and toxic oxidants generated during phagocytosis. We studied the effects of 37 degrees C "heat shock" or sublethal menadione, which generates superoxide and hydrogen peroxide, on Leishmania chagasi virulence. Both heat and menadione caused parasites to become more resistant to H(2)O(2)-mediated toxicity. Peroxide resistance was also induced as promastigotes developed in culture from logarithmic to their virulent stationary phase form. Peroxide resistance was not associated with an increase in reduced thiols (trypanothione and glutathione) or increased activity of ornithine decarboxylase, which is rate-limiting in trypanothione synthesis. Membrane lipophosphoglycan increased in size as parasites developed to stationary phase but not after environmental exposures. Instead, parasites underwent a heat shock response upon exposure to heat or sublethal menadione, detected by increased levels of HSP70. Transfection of promastigotes with L. chagasi HSP70 caused a heat-inducible increase in resistance to peroxide, implying it is involved in antioxidant defense. We conclude that leishmania have redundant mechanisms for resisting toxic oxidants. Some are induced during developmental change and others are induced in response to environmental stress.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H2O2, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pre-treatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H2O2, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

The topology of superoxide production by complex III and glycerol 3-phosphate dehydrogenase in Drosophila mitochondria

The topology of superoxide generation by sn-glycerol 3-phosphate dehydrogenase and complex III in intact Drosophila mitochondria was studied using aconitase inactivation to measure superoxide production in the matrix, and hydrogen peroxide formation in the presence of superoxide dismutase to measure superoxide production from both sides of the membrane. Aconitase inactivation was calibrated using the known rate of matrix superoxide production from complex I. Glycerol phosphate dehydrogenase generated superoxide about equally to each side of the membrane, whereas centre o of complex III in the presence of antimycin A generated superoxide about 30% on the cytosolic side and 70% on the matrix side.     

Regulations of the C2H4-forming system and the H2O2-scavenging system by heat treatment associated with self incompatibility in lily

The mechanism of self incompatibility in pistils of Lilium longiflorum Thunb. cv. Hinomoto, which is overcome by heat treatment, was analyzed. Immersing detached pistils in a distilled water bath held at 45°C for 5 min suppressed levels of ethylene and activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase at 6 h after self- and cross-incompatible pollination. However, the levels and activities showed no significant difference 48 h after pollination. Levels of ACC and activities of ACC synthase at 6 h after self-incompatible pollination were lower in pistils with heat treatment. Moreover, the heat treatment suppressed the activity of superoxide dismutase and enhanced the activity of catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In addition, the amount of hydrogen peroxide (H₂O₂) was reduced by heat treatment. In summary, heat treatment suppressed the ethylene-forming system and also enhanced the hydrogen peroxide-scavenging system in self-pollinated pistils associated with self incompatibility. A possible correlation between self incompatibility and stress in pistils after self-incompatible pollination is discussed based on the results obtained using heated pistils. Received: 12 April 2000 / Revision accepted: 19 September 2000     

Differential responses of stress proteins, antioxidant enzymes, and photosynthetic efficiency to physiological stresses in the Florida red tide dinoflagellate, Karenia brevis

This study identifies stress proteins and antioxidant enzymes that may play a role in the survival strategies of the Florida red tide dinoflagellate, Karenia brevis. Heat shock protein 60 (Hsp 60), mitochondrial small heat shock protein (mitosHsp), chloroplastic small heat shock protein (chlsHsp), Mn superoxide dismutase (SOD), and Fe SOD were first identified by Western blotting. The induction of these proteins in laboratory cultures in response to elevated temperatures, hydrogen peroxide, lead, or elevated light intensities was next assessed. In parallel, F(V)/F(M), a measurement of photosynthetic efficiency and common proxy of cellular stress, was determined. Hsp 60, Fe SOD, and Mn SOD were induced following exposure to elevated temperatures, hydrogen peroxide, or lead. MitosHsp responded only to heat, whereas chlsHsp responded only to H(2)O(2)-induced stress. The expression of stress proteins and antioxidant enzymes appears to be a more sensitive indicator of heat or chemically induced stresses than F(V)/F(M). However, F(V)/F(M) decreased significantly in response to elevated light intensities that did not induce the expression of stress proteins. These results identify for the first time stress proteins and antioxidant enzymes in K. brevis, provide evidence for differential sensitivity of cellular organelles to various sources of stress, and confirm the presence of conserved stress responses observed across phyla in a dinoflagellate.     

Role of hydrogen peroxide in generation of a signal inducing heat tolerance of wheat seedlings

The effects of 1-min-long exposure to 42°C (hardening heating) on heat tolerance and dynamics of ROS (superoxide anion radical and hydrogen peroxide) generation were investigated in the wheat (Triticum aestivum L., cv. Elegiya) seedlings. During the initial 5–30 min after the onset of hyperthermia, ROS generation by roots and shoots was intensified, and superoxide dismutase (SOD) was activated. During the first hour after hardening heating, the seedling tolerance to injurious 10-min-long treatment with high temperature (46°C) decreased but subsequently it gradually rose, reaching maximum in 24 h. Transient accumulation of hydrogen peroxide induced by hardening was suppressed by seedling treatment with H₂O₂ scavenger dimethylthiourea, by inhibitors of NADPH-oxidase (imidazole) and DDC (sodium diethyldithiocarbamate). These compounds considerably reduced favorable effect of hardening on seedling heat tolerance. It was concluded that generation of a signal inducing the development of heat tolerance depended on NADPH-oxidase producing superoxide anion radical and SOD that transforms it into hydrogen peroxide (more stable ROS performing signaling functions).     

A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress.

Escherichia coli treated with nontoxic levels of the superoxide-generating redox-cycling agents menadione and paraquat showed dramatic changes in protein composition as monitored by two-dimensional gel analysis. The distribution of proteins synthesized after treatment with these agents overlapped significantly with that seen after hydrogen peroxide treatment, and it included all the proteins in the oxyR regulon. The redox-cycling agents also elicited the synthesis of at least 33 other proteins that were not seen with hydrogen peroxide, including three heat shock proteins, the Mn-containing superoxide dismutase, the DNA repair protein endonuclease IV, and glucose-6-phosphate dehydrogenase. At least some of these redox-inducible proteins appear to be part of a specific response to intracellular superoxide. E. coli is thus equipped with a network of inducible responses against oxidative damage, controlled in multiple regulatory pathways.     

Evidence for suitability of glutathione peroxidase as a protective enzyme: studies of oxidative damage, renaturation, and proteolysis

The stability of glutathione peroxidase was assessed in vitro via oxidative inactivation by peroxides and a peroxidizing fatty acid and by renaturation and proteolysis. The stability of glutathione peroxidase to methyl ethyl ketone peroxide, H2O2, linoleic acid hydroperoxide, and peroxidizing methyl linolenate was compared with the stability of several other enzymes. Sulfhydryl enzymes were the most labile to all four treatments. Some of the enzymes tested were very stable to methyl ethyl ketone peroxide but very labile to linoleic acid hydroperoxide treatment. Glutathione peroxidase in the absence of glutathione was relatively slowly inactivated by each treatment. Linoleic acid hydroperoxide damage to glutathione peroxidase was characterized by release of a nonstoichiometric amount of selenite from the protein. Glutathione peroxidase samples lost all of their activity when (i) acidified to pH 2, (ii) heated 5 min at 100 degrees C, and (iii) treated with 6 M guanidinium hydrochloride or 8.5 M urea and heated 5 min at 100 degrees C. When the pH 2 sample was neutralized or the guanidinium hydrochloride-treated sample was diluted 101-fold, about 80% of the original activity was recovered in 30 min. The samples treated with urea and heat recovered no activity when diluted 101-fold. No loss of glutathione peroxidase occurred during treatment for 24 h within trypsin or thermolysin. Based on these results, glutathione peroxidase appears to be a relatively stable enzyme, and thus is is well-suited to perform its role in peroxide detoxification and prevention of oxidative deterioration of cells.     

Avoiding high-valent iron intermediates: superoxide reductase and rubrerythrin

The Fenton or Fenton-type reaction between aqueous ferrous ion and hydrogen peroxide generates a highly oxidizing species, most often formulated as hydroxyl radical or ferryl ([Fe(IV)O](2+)). Intracellular Fenton-type chemistry can be lethal if not controlled. Nature has, therefore, evolved enzymes to scavenge superoxide and hydrogen peroxide, the reduced dioxygen species that initiate intracellular Fenton-type chemistry. Two such enzymes found predominantly in air-sensitive bacteria and archaea, superoxide reductase (SOR) and rubrerythrin (Rbr), functioning as a peroxidase (hydrogen peroxide reductase), contain non-heme iron. The iron coordination spheres in these enzymes contain five or six protein ligands from His and Glu residues, and, in the case of SOR, a Cys residue. SOR contains a mononuclear active site that is designed to protonate and rapidly expel peroxide generated as a product of the enzymatic reaction. The ferrous SOR reacts adventitiously but relatively slowly (several seconds to a few minutes) with exogenous hydrogen peroxide, presumably in a Fenton-type reaction. The diferrous active site of Rbr reacts more rapidly with hydrogen peroxide but can divert Fenton-type reactions towards the two-electron reduction of hydrogen peroxide to water. Proximal aromatic residues may function as radical sinks for Fenton-generated oxidants. Fenton-initiated damage to these iron active sites may become apparent only under extremely oxidizing intracellular conditions.     

Interaction of a small heat shock protein with light-harvesting cyanobacterial phycocyanins under stress conditions

Phycobiliproteins such as phycocyanins are the most abundant proteins found in cyanobacteria which are assembled to form the phycobilisome. Here, we showed that a small heat shock protein, HspA, interacts directly with phycocyanins from the cyanobacterium Synechococcus sp. strain PCC 7942 in vitro and suppresses inactivation of their light-harvesting functions due to heat denaturation in the presence of hydrogen peroxide. Under the denaturing conditions, phycobilisomes were de-assembled to lighter complexes and then aggregated. HspA associated with phycocyanins in the dissociated complexes, and suppressed the aggregation. The specific interaction between a small heat shock protein and phycocyanins was further supported by the fact that HspA and alpha-crystallin protected isolated phycocyanins from denaturation, while HtpG and lysozyme did not. The maximum protection was observed at a molar ratio of four HspA monomer per one phycocyanin (alpha beta) monomer.     

Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress

In Escherichia coli, the coordinate action of two antioxidant enzymes, superoxide dismutase and hydroperoxidase (catalase), protect the cell from the deleterious effects of oxyradicals generated during normal aerobic respiration. To evaluate the relative importance of these two classes of enzymes, strains of E. coli deficient in superoxide dismutase and (or) hydroperoxidase were constructed by generalized transduction and their physiological responses to oxygen and oxidant stress examined. Superoxide dismutase was found to be more important than hydroperoxidase in preventing oxygen-dependent growth inhibition and mutagenesis, and in reducing sensitivity to redox-active compounds known to generate the superoxide anion. However, both types of enzymes were required for an effective defense against chemical oxidants that generate superoxide radicals and hydrogen peroxide.     

Affinity inactivation of bovine Cu,Zn superoxide dismutase by hydroperoxide anion, HO2-

Bovine liver Cu,Zn superoxide dismutase (SOD) is inactivated by hydrogen peroxide at alkaline pH, and full inactivation correlates with the loss of 1.1 histidine/subunit. At each pH utilized, saturation of the rate of inactivation is observed. This process is characterized by a half-saturation constant for peroxide and a maximum pseudo-first-order rate constant for inactivation. At 25 degrees C, the former decreases from 15.7 to 3.2 mM as the pH is increased from 9.0 to 11.5, while the latter increases from 0.83 to 2.43 per min over the same pH range. We have previously (Arch. Biochem. Biophys. 224, 579 (1983] proposed that the true affinity reagent for the inactivation of yeast SOD is the hydroperoxide anion, and we now believe the same is true for bovine SOD. However, a subtle difference between the two enzymes exists, for while the maximum pseudo-first-order rate constant for inactivation of bovine SOD increases with increasing pH, the same parameter for the yeast enzyme is pH-independent.