Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Superoxide radicals and antiradical defence of propionic acid bacteria

Superoxide dismutase activity was demonstrated for 6 strains of 3 propionibacteria species. Rather high level of superoxide dismutase activity found in propionibacteria was in accordance with high level of catalase activity reported for propionibacteria previously. Both these activities were shown to have cytozolic localization. For the first time peroxidase activity was detected in gel-fractionated crude cell extracts of propionibacteria. The ability to produce superoxide radicals in NADH-dependent oxidation system was revealed for three strains of the bacteria. The level of superoxide production by the membrane particles of the propionic acid bacteria correlated with the levels of superoxide dismutase and catalase activities and was the lowest for Propionibacterium shermanii. The ability to perform monovalent oxygen reduction during succinate oxidation was not revealed. The intact cells of P. globosum, P. vannielii, P. shermanii apparently did not excrete superoxide radicals into culture fluid during respiration.     

Biosynthesis of oxygen-detoxifying enzymes in Bdellovibrio stolpii.

Axenically grown Bdellovibrio stolpii (i.e., grown independently of the host) was examined for superoxide dismutase, catalase, and peroxidase activities. Kinetics of enzyme synthesis were determined for aerobically grown cultures and for cultures exposed to 100% oxygen. Enzymatic activities varied with the age of the culture. Normally grown cultures exhibited maximum activity during the first 10 h of growth and again as the stationary phase was approached, beginning at about 48 h. Polyacrylamide gel electropherograms of cell-free extracts revealed that B. stolpii contained one major band (1) and two minor bands (II, III) of superoxide dismutase activity. Each of these enzymes was inactivated by H2O2, indicating that they were iron-containing enzymes. Manganese-containing superoxide dismutase was not detected in B. stolpii. Increased oxygenation did not appreciably stimulate enzyme synthesis, for only superoxide dismutase was induced, reaching maximum activity at 10 h and then rapidly falling to normal levels. Superoxide dismutase appears to be the main enzymatic defense against oxygen toxicity in B. stolpii. Induction of superoxide dismutase with 100% oxygen was manifested as an increase in the intensities of the two minor bands of activity, suggesting that isozyme I is constitutive, whereas isozymes II and III are inducible. The induction of isozymes II and III by 100% oxygen was prevented by an inhibitor of protein biosynthesis, chloramphenicol.     

Variability of antioxidant enzyme activity and isoenzyme profile in needles of Serbian spruce (Picea omorika (Panč.) Purkinye)

Variations were studied of the activity and isoenzyme patterns of soluble peroxidase, catalase, catechol oxidase and superoxide dismutase, in needles of the Balkan endemic conifer Serbian spruce, Picea omorika (Pan?.) Purkinye. The samples were collected from the natural habitat of the species, Mt. Tara. Seasonal changes were found to affect enzymatic activities and isoenzyme profiles. Total protein content was significantly lower in the summer than in other seasons. Several isoforms of peroxidase, catechol oxidase and superoxide dismutase (SOD), as well as two catalase isoenzymes were detected. The number of peroxidase isoenzymes was greatest during the vegetative season. Catalase and catechol oxidase peaked in summer and spring, respectively. Total SOD and Mn-SOD activities were significantly higher in the winter samples than the summer ones.     

Changes in activity of antioxidative enzymes in wheat (Triticum aestivum) seedlings under cold acclimation

Activated oxygen species such as superoxide radicals, singlet oxygen, hydrogen peroxide and hydroxyl radicals can be produced in plants exposed to low, non-freezing, non-injurious temperatures. To prevent or alleviate oxidative injury, plants have evolved several mechanisms which include scavenging by natural antioxidants and enzymatic antioxidant systems such as superoxide dismutases, catalase and peroxidases. Although overproduction of hydrogen peroxide and increased tolerance to oxidative stress can be induced in wheat by low-temperature treatments, data concerning changes in the enzymatic antioxidant systems are almost absent. With the aim to provide this information, antioxidant enzyme (superoxide dismutases, catalase and peroxidases) activities were analysed in leaves and roots of Triticum aestivum cvs Brasilia (frost resistant in field) and Eridano (less frost resistant in field) seedlings grown at day/night temperatures of 24/22°C (control treatment) and 12/5°C (low-temperature treatment). Our data showed that superoxide dismutase activities were unaffected by low-temperature treatment both in leaves and roots. Catalase activity in leaves and roots was decreased in 12/5°C-grown seedlings, but Brasilia maintained higher catalase activity than Eridano. Differences were also observed in guaiacol peroxidase activities between control and acclimated seedlings: Higher guaiacol peroxidase activities were found in the leaves of 12/5°C-grown seedlings while in roots these activities were lower. Moreover, Brasilia guaiacol peroxidase activities were higher than Eridano. Superoxide dismutase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by low-temperature treatment. Changes in the activities of antioxidant enzymes induced by cold acclimation support the hypothesis that a frost-resistant wheat cultivar, in comparison with a less frost-resistant one, maintains a better defence against activated oxygen species during low-temperature treatment.     

Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats

The role of oxidative stress in pilocarpine-induced status epilepticus was investigated by measuring lipid peroxidation level, nitrite content, GSH concentration, and superoxide dismutase and catalase activities in the hippocampus of Wistar rats. The control group was subcutaneously injected with 0.9% saline. The experimental group received pilocarpine (400 mg.kg(-1), subcutaneous). Both groups were killed 24 h after treatment. After the induction of status epilepticus, there were significant increases (77% and 51%, respectively) in lipid peroxidation and nitrite concentration, but a 55% decrease in GSH content. Catalase activity was augmented 88%, but superoxide dismutase activity remained unaltered. These results show evidence of neuronal damage in the hippocampus due to a decrease in GSH concentration and an increase in lipid peroxidation and nitrite content. GSH and catalase activity are involved in mechanisms responsible for eliminating oxygen free radicals during the establishment of status epilepticus in the hippocampus. In contrast, no correlations between superoxide dismutase and catalase activities were observed. Our results suggest that GSH and catalase activity play an antioxidant role in the hippocampus during status epilepticus.     

Response characteristics of Scirpus trioueter and its rhizosphere to pyrene contaminated soils at different growth stages

Scirpus triqueter (Triangular club-rush), a typical wetland species, is used to study the response characteristics to pyrene. A pot experiment was conducted to investigate the growth parameters (height, diameter, shoot number, total volume, underground biomass, above-ground biomass and total biomass), and enzymes (catalase and superoxide dismutase) of S. triqueter. The characteristics of soil enzymes (catalase and polyphenol oxidase) and microorganisms (bacteria and fungi) were also assessed after pyrene treatment. Elevated pyrene concentration (80 mgkg(-1)) in the soil reduced the shoot number and biomass significantly, especially at the early growth stage. In root tissue, the enzyme catalase was activated at 80 mgkg(-1) of pyrene. Compared to roots, shoots had higher enzyme activities. Catalase activities in the rhizosphere increased throughout the growth period of S. triqueter. Polyphenol oxidase activities in the rhizosphere were higher than those in the bulk soil and unplanted soil. The populations of bacteria (total bacteria, pyrene-tolerant bacteria, and actinomyces) and fungi decreased under the stress of high pyrene concentration, while that of pyrene-tolerant bacteria increased with the increasing pyrene concentration. The presence of pyrene did not benefit the growth of S. triqueter. S. triqueter and soil enzymes varied within the growth stages. The presence of S. triqueter could improve the activity of soil enzymes and facilitate the propagation of microorganisms which could help eliminate pyrene contamination.     

Effects of growth conditions on superoxide dismutase and catalase activities inSaccharomyces cerevisiae var.ellipsoideus

The superoxide dismutase (SOD) activity inSaccharomyces cerevisiae var.ellipsoideus increased when the cells were exposed to hyperbaric oxygen tension. Ethanol-grown cells, having a more oxidative metabolism, showed higher SOD activities than did glucosegrown cells. In a glucose-limited chemostat the SOD activity increased with the specific oxygen consumption rate. The increase in SOD activity may be explained by a higher intracellular flow of superoxide radicals at higher respiration rates. The catalase activity decreased with increasing growth rates in a glucose-limited chemostat, and the activity was lower in glucosethan in ethanol-grown cells.     

Unusual Growth Phase and Oxygen Tension Regulation of Oxidative Stress Protection Enzymes, Catalase and Superoxide Dismutase, in the Phytopathogen Xanthomonas oryzae pv. oryzae

The enzymes catalase and superoxide dismutase play major roles in protecting phytopathogenic bacteria from oxidative stress. In Xanthomonas species, these enzymes are regulated by both growth phase and oxygen tension. The highest enzyme levels were detected within 1 h of growth. Continued growth resulted in a decline of both enzyme activities. High oxygen tension was an inducing signal for both enzyme activities. An 80,000-Da monofunctional catalase and a manganese superoxide dismutase were the major forms of the enzymes detected at different stages of growth. The unusual regulatory patterns are common among several Xanthomonas strains tested and may be advantageous to Xanthomonas species during the initial stage of plant-microorganism interactions.     

Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Superoxide dismutase and catalase activities were studied in Azotobacter vinelandii grown diazotrophically at different ambient oxygen concentrations in continuous culture. Activities were expressed either as specific activity or activity per cell. Specific superoxide dismutase activity increased by a factor of 1.6 with increasing oxygen concentration from about 1% to 90% air saturation of the growth medium whereas specific catalase activity increased only slightly, if at all. Since cell volumes increased in parallel to increases in the oxygen concentration cellular superoxide dismutase activities increased by a factor of 4.3 while cellular catalase activities increased by a factor of 3.3. Under all conditions only the Fe-containing form of superoxide dismutase was detected. The possible function of these enzymes in the protection nitrogenase from oxygen damage is discussed.Abbreviation SOD superoxide dismutase     

Antioxidant enzymes responses to cadmium in radish tissues

To investigate the antioxidant responses of radish (Raphanus sativus L.) to cadmium (Cd) treatment, seedlings of a tolerant variety were grown in increasing concentrations of CdCl(2), ranging from 0.25-1 mM, for up to 72 h in a hydroponic system. Analysis of Cd uptake indicated that most of the Cd accumulated in the roots, but some was also translocated and accumulated in the leaves, especially at the higher concentrations of Cd used in the experiments. Roots and leaves were analysed for catalase, glutathione reductase and superoxide dismutase activities. Catalase and glutathione reductase activities increased considerably in the roots and leaves after 24 h exposure to the metal, indicating a direct correlation with Cd accumulation. The analysis of native PAGE enzyme activity staining, revealed several superoxide dismutase isoenzymes in leaves, with the two predominant isoenzymes exhibiting increases in activity in response to Cd treatment. The results suggest that in radish, the activity of antioxidant enzymes responds to Cd treatment. The main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of Cd-binding proteins.     

Production and some properties of catalase and superoxide dismutase from the anaerobe Bacteroides distasonis.

The catalase level of Bacteroides distasonis (ATCC 8503, type strain) varied with the amount of hemin supplied to the medium when the cells were grown in either a prereduced medium containing 0.5% peptone, 0.5% yeast extract, and 1% glucose or in a prereduced, defined heme-deficient medium. The effect of hemin on catalase production could not be duplicated by ferrous sulfate or ferrous ammonium citrate. Catalase activity reached peak values in late log phase, whereas superoxide dismutase specific activity remained constant throughout the culture growth cycle. The catalase was a nondialyzable, cyanide and azide-sensitive, heat-labile protein that coeluted with bovine erythrocyte catalase from Sepharose 6 B. Analysis of polyacrylamide gels stained for catalase activity and for heme showed a correspondence between the single catalytic activity band and one of three heme-protein bands. These data suggest a heme-protein of approximately 250,000 molecular weight. The superoxide dismutase was a cyanide-insensitive protein of approximately 40,000 molecular weight that migrated electrophoretically on acrylamide gels as a single band of activity.     

Antioxidative defense to lead stress in subcellular compartments of pea root cells

Lead, similar to other heavy metals and abiotic factors, causes many unfavorable changes at the subcellular and molecular levels in plant cells. An increased level of superoxide anion in Pisum sativum root cells treated with 1 mM Pb(NO3)2 evidenced oxidative stress conditions. We found increased activities of enzymatic components of the antioxidative system (catalase and superoxide dismutase) in the cytosol, mitochondrial and peroxisomal fractions isolated from root cells of Pisum sativum grown in modified Hoagland medium in the presence of lead ions (0.5 or 1 mM). Two isoenzyme forms of superoxide dismutase (Cu,Zn-SOD and Mn-SOD) found in different subcellular compartments of pea roots were more active in Pb-treated plants than in control. Increased amount of alternative oxidase accompanied by an increased activity of this enzyme was found in mitochondria isolated from lead-treated roots. These results show that plants storing excessive amounts of lead in roots defend themselves against the harmful oxidative stress caused by this heavy metal.     

Effect of cold stress in the rhizosphere on the activity of antioxidant enzymes in the tissues of Pinus sylvestris

The activities of lipase, peroxidase, IAA-oxidase, superoxide dismutase, and catalase have been comparatively studied in the needles, inner bark of stem and roots of 10-year-old self-sawn Pinus sylvestris trees in Central Siberia under natural conditions and in experiment imitating the effect of permafrost. It is shown that a decrease in the rhizosphere temperature for self-sawn Pinus sylvestris causes not only a change in the morphogenesis of the sprouts of the current year and reduction of the annual ring but also a shift of the natural dynamics of antioxidant enzyme activity to a later time. Before soil thawing, the activity of antioxidant enzymes on the experimental plot weakened thus implying the enhancement of the oxidative stress, while the growth of buds and sprouts is hindered during this period because of the high activity of IAA-oxidase. An active part in the elimination of the oxidative stress consequences belongs to the conjugated pair of antioxidant enzymes superoxide dismutase—catalase.     

Catalase activity and isozyme pattern of the metalloenzyme system, superoxide dismutase, as a function of leaf development during growth of Pisum sativum L. plants

The catalase activity and the isozyme pattern of the metalloenzyme system superoxide dismutase (SOD) have been determined in pea ( Pisum sativum L., cv, Lincoln) leaves of different ages (apical, middle and lower), during several stages of plant development. Pea seedlings were grown in full nutrient solution in a phytotron. Catalase activity was determined polarographically, and superoxide dismutase isozymes (Mn-SOD, Cu, Zn-SOD I and Cu, Zn-SOD II) were separated by acrylamide gel electrophoresis and their relative amounts quantified by densitonietry. The results indicate that the relative amounts of SOD isozymes are slightly different in leaves of different ages during plant growth and, interestingly, each molecular form of SOD shows a clearly distinct pattern during plant development. These changes in the relative percentages of SOD isozymes could be due to the induction of the distinct molecular forms of SOD by the metals Mn, Cu and Zn, translocated to the different leaves as a result of plant development. The relative percentage of the Mn-SOD isozyme showed a similar pattern to that of catalase activity, suggesting a possible link between these two metalloenzymes at subcellular level, both cooperating to remove the toxic effects of O^-₂ and H₂O₂.
An additional conclusion is that before a certain metalloenzyme can be used as a marker to assess the plant micronutrient status, it is essential to have a detalled study of its activity pattern in leaves of different age during plant development.     

Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H₂O₂) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H₂O₂ was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 μM) of H₂O₂. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.     

Time course of catalase and superoxide dismutase during acclimatization and growth of micropropagated Calathea and Spathiphyllum plants

Catalase and superoxide dismutase (SOD) activities were followed in leaves during ex vitro acclimatization and growth of micropropagated Spathiphyllum floribundum Schott Petite and Calathea louisae Gagnep Maui Queen. During acclimatization of both plants catalase activity increased, reaching a maximum 4 weeks after transplantation, while total superoxide dismutase activity increased with plant growth reaching a maximum in the 24th week. Variations in the pattern of catalase and SOD isoforms were observed; a second Mn-SOD band appeared in Spathiphyllum 12 weeks after transfer from in vitro, while in Calathea plants an additional Mn-SOD form was present from the second until the fourth week after transplantation. The observed changes reflect the plants' capacity to develop antioxidant mechanisms during acclimatization. These findings indicate that the adaptation of micropropagated plants to ex vitro conditions is more extended in time than generally accepted.     

Characterization of the antioxidant system during the vegetative development of pea plants

The antioxidative system was studied during the development of pea plants. The reduced glutathione (GSH) content was higher in shoots than in roots, but a greater redox state of glutathione existed in roots compared with shoots, at least after 7 d of growth. The 3-d-old seedlings showed the highest content of oxidised ascorbate (DHA), which correlated with the ascorbate oxidase (AAO) activity. Also, the roots exhibited higher DHA content than shoots, correlated with their higher AAO activity. The activities of antioxidant enzymes were much higher in shoots than in roots. Ascorbate peroxidase (APX) activity decreased during the progression of growth in both shoots and roots, whereas peroxidase (POX) activity strongly increased in roots, reflecting a correlation between POX activity and the enhancement of growth. Catalase activity from shoots reached values nearly 3 or 4-fold higher than in roots. The monodehydroascorbate reductase (MDHAR) activity was higher in young seedlings than in more mature tissues, and in roots a decrease in MDHAR was noticed at the 11^th day. No dehydroascorbate reductase (DHAR) was detected in roots from the pea plants and DHAR values detected in seedlings and in shoots were much lower than those of MDHAR. In shoots, GR decreased with the progression of growth, whereas in roots an increase was seen on the 9^th and 11^th days. Finally, superoxide dismutase (SOD) activity increased in shoots during the progression of growth, but specific SOD activity was higher in roots than in shoots.     

Enzyme activities associated with oxidative stress in Metarhizium anisopliae during germination, mycelial growth, and conidiation and in response to near-UV irradiation

Metarhizium anisopliae isolates have a wide insect host range, but an impediment to their commercial use as a biocontrol agent of above-ground insects is the high susceptibility of spores to the near-UV present in solar irradiation. To understand stress responses in M. anisopliae, we initiated studies of enzymes that protect against oxidative stress in two strains selected because their spores differed in sensitivity to UV-B. Spores of the more near-UV resistant strain in M. anisopliae 324 displayed different isozyme profiles for catalase-peroxidase, glutathione reductase, and superoxide dismutase when compared with the less resistant strain 2575. A transient loss in activity of catalase-peroxidase and glutathione reductase was observed during germination of the spores, whereas the intensity of isozymes displaying superoxide dismutase did not change as the mycelium developed. Isozyme composition for catalase-peroxidases and glutathione reductase in germlings changed with growth phase. UV-B exposure from lamps reduced the activity of isozymes displaying catalase-peroxidase and glutathione reductase activities in 2575 more than in 324. The major effect of solar UV-A plus UV-B also was a reduction in catalase-peroxidases isozyme level, a finding confirmed by measurement of catalase specific activity. Impaired growth of M. anisopliae after near-UV exposure may be related to reduced abilities to handle oxidative stress.