Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371     

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

A superoxide dismutase (SOD) gene was cloned from the thermophilic bacterium Rhodothermus sp. XMH10 for the first time and highly expressed in Escherichia coli. The Rhodothermus sp. XMH10 SOD (RhSOD) gene encodes 209 amino acids with a putative molecular weight of 23.6 kDa and a pI value of 5.53. The recombinant RhSOD was detected to be an iron type SOD and existed as a dimer on its natural status. Experiments revealed that this RhSOD showed high activity at 50–70 °C and pH 5.0. Compared to SODs from other thermophiles, it was highly thermostable, maintaining more than 90% of its activity after incubation at 70 °C for 12 h, only totally inactivated after more than 4-h incubation at 80 °C. It also showed much higher resistance to KCN, NaN₃ and H₂O₂ as compared to other SODs. Xin Wang and Haijie Yang contribute to this work equally.     

Cloning and Expression in Escherichia Coli of a Gene Encoding Superoxide Dismutase from Listeria Ivanovii

A chromosomal DNA fragment from the gram-positive bacterium Listeria ivanovii (ATCC 19119) encoding a superoxide dismutase (SOD) gene has been cloned in Escherichia coli QC779 (sodAsodB) using the plasmid vector pTZ19R. The DNA fragment inserted into the plasmid showed-high structural instability in E. coli QC779 (recA⁺). but turned out to be a stable 1.95 kbp DNA fragment when transformed into E. coli DHSa (recA-). The gene is expressed in both of these E. coli strains at high levels. Preliminary studies showed that the activity of the recombinant SOD within E. coli DHSα was up to 13-times the combined activity of both E. coli SODs. The recombinant SOD forms active hybrid SODS with both E. coli SODs in vivo.     

Effect of short- and long-term salinity on the activities of antioxidative enzymes and lipid peroxidation in tomato roots

Changes in the antioxidative enzyme activities (SOD, CuZnSOD, GSH-Px, GST), as well as TBARS content in 5-week-old tomato (Lycopersicon esculentum Mill. cv “Perkoz”) roots were examined 1, 3 h (short-term stress) and 1–14 days (long-term stress) after a single application of 50 mM (mild stress) and 150 mM NaCl (severe stress). The severe stress caused an increase in GST, GSH-Px and SODs activities from the beginning of the experiment while mild stress induced enhancement of GST activity from the second day of experiment. The maximum increase in SODs after both NaCl solutions were applied and in GST activity after the higher NaCl dose on the second day of the experiment was observed. Moreover, after 1 h of NaCl treatment with both tested NaCl solutions, the highest induction of GSH-Px activity appeared. TBARS content was elevated from the first hour of salt stress and decreased only 14 days after 50 mM NaCl application which was accompanied by high induction of GSH-Px activity. In conclusion, enhanced activities of tested enzymes indicate their involvement in early and late defence systems under salinity stress. Moreover, the dynamics of the changes in the antioxidant enzymes suggests that the second day following NaCl application is a crucial moment of the experiment with regard to salt-mediated oxidative stress.     

Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress

In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)‐mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast‐targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non‐transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.     

Cloning and characterization of a novel splicing isoform of the iron-superoxide dismutase gene in rice (Oryza sativa L.)

Superoxide dismutases (SODs) are ubiquitous metalloenzymes in aerobic organisms that play a crucial role in protecting organisms against ROS. Here, we report the molecular cloning and functional characterization of a novel alternatively spliced variant of the iron-superoxide dismutase gene, OsFe-SODb, from a rice panicle cDNA library. The alternative splicing event occurred in the fourth exon of the OsFe-SOD gene, and led to the translation of two isoforms of different sizes. The 5′ flanking region of the OsFe-SOD was cloned and many cis-acting regulatory elements were found that are involved in light responsiveness, including a G-box and an I-box. RT-PCR analysis showed that the two alternative forms of OsFe-SOD were expressed in both the vegetative and reproductive tissues of Cpslo17. Moreover, accumulation of both isoforms was upregulated by light induction. In addition, the alternative splicing of OsFe-SOD mRNA was sensitive to low temperature. High yield production of the two recombinant OsFe-SOD isoforms was achieved in Escherichia coli. SOD assays showed that C-terminal truncation in OsFe-SODb did not result in a loss of SOD enzyme activity.     

Differential response to paraquat induced oxidative stress in two rice cultivars on antioxidants and chlorophyll a fluorescence

The responses of antioxidative system and photosystem II photochemistry of rice (Oryza sativa L.) to paraquat induced oxidative stress were investigated in a chilling-tolerant cultivar Xiangnuo no. 1, and a chilling-susceptible cultivar, IR-50. Electrolyte leakage and malondialdehyde (MDA) content of Xiangnuo no. 1 were little affected by paraquat, but they increased in IR-50. After paraquat treatment, superoxide dismutase (SOD) activity remained high in Xiangnuo no. 1, while it declined in IR-50. Activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) declined with oxidative stress in both cultivars, but Xiangnuo no. 1 had higher GR activity than IR-50. Under paraquat induced oxidative stress, ascorbic acid (AsA) and reduced glutathione (GSH) concentrations remained high in Xiangnuo no. 1, but decreased in IR-50. The results indicated that higher activities of SOD and GR and higher contents of AsA and GSH in Xiangnuo no. 1 under paraquat induced oxidative stress were associated with its tolerance to paraquat, while paraquat induced damage to IR-50 was related to decreased activities of SOD, APX and GR and contents of AsA and GSH. F _v/F _m, Φ _PSII, and qP remained high in Xiangnuo no. 1, while they decreased greatly in IR-50 under paraquat induced oxidative stress.     

Electrophoretic mobilities of superoxide dismutases from species ofPhotobacterium,beneckea, vibrio,and selected terrestrial enterobacteria

Cell-free extracts of 93 strains from 23 species and biotypes ofPhotobacterium, Beneckea, andVibrio were electrophoresed on polyacrylamide gels and then stained for superoxide dismutase (SOD) activity. All strains exhibited a single activity band with the exception ofPhotobacterium leiognathi, which had one major and two minor bands. Strains representative of all three genera were found to have SOD activities that were sensitive to treatment with H₂O₂, suggesting that they were iron-containing enzymes. Examination of the effect of gel concentration on relative mobility (Rm) suggested that all the iron-containing SODs had very similar molecular weight. Most species could be distinguished on the basis of differences in the Rm values of their SODs.Vibrio was readily separable fromBeneckea andPhotobacterium. A limited electrophoretic analysis of the SODs fromAeromonas, Serratia, Proteus, Erwinia, and other species of terrestrial enterobacteria indicated groupings that were in agreement with previous studies.     

Proline antioxidant role in the common ice plant subjected to salinity and paraquat treatment inducing oxidative stress

Leaves of 4-week-old (juvenile) and 9-week-old (adult) plants of the halophyte Mesembryanthemum crystallinum L. (the common ice plant), cultured under controlled conditions in the phytotron, were treated with paraquat (0.1 μM), which produces superoxide radical, and (or) paraquat combined with introduction of NaCl (100 mM) or proline (5 mM) into nutrient medium. After a 20-h dark period (23°C), plants were transferred into light (4 h at 54.1 W/m² of photosynthetically active radiation) for stimulation of O°₂⁻ formation in plastids. Activities of antioxidant enzymes, the contents of MDA, H₂O₂, chlorophyll, and free proline were measured in leaves. Plant responses in two age groups, which differed in the type of photosynthesis (juvenile plants had C₃ type of photosynthesis, whereas adult plants were at the transition stage to Crassulacean Acid Metabolism (CAM) photosynthesis), differed in the levels of constitutive proline and proline, induced by NaCl and paraquat, as well as in activities of superoxide dismutase (SOD) and catalase. Changes in SOD activity and proline accumulation in response to paraquat treatment combined with NaCl revealed opposite dependence to accumulation of proline: the more proline accumulated in leaves, the lower activity of the enzyme. In response to paraquat treatment, the content of chlorophylls a and b most drastically declined in juvenile plants. Negative effect of salinity on the content of chlorophylls was lower than that of paraquat and was almost the same in plants of both age groups. Protective effect of exogenous proline was most profound in the case of paraquat treatment. Exogenous proline decreased the rate of lipid peroxidation, the content of superoxide radical and, consequently, SOD activity (almost fivefold), and increased the content of chlorophylls (a and b) in leaves of adult plants. The obtained data suggest that stress-induced accumulation of proline in the common ice plant has both osmoprotectory and antioxidant functions.     

Induction of superoxide dismutases in Escherichia coli B by metal chelators

The effects of metal salts, chelating agents, and paraquat on the superoxide dismutases (SODs) of Escherichia coli B were explored. Mn(II) increased manganese-containing SOD (MnSOD), whereas Fe(II) increased iron-containing SOD (FeSOD). Chelating agents induced MnSOD but decreased FeSOD and markedly increased the degree of induction seen with Mn(II). Paraquat also exerted a synergistic effect with Mn(II). High levels of MnSOD were achieved in the combined presence of Mn(II), chelating agent, and paraquat. All of these effects were dependent on the presence of oxygen. MnSOD, not ordinarily present in anaerobically grown E. coli cells, was present when the cells were grown anaerobically in the presence of chelating agents. These results are accommodated by a scheme which incorporates autogenous repression by the apoSODs and competition between Fe(II) and Mn(II) for the metal-binding sites of the apoSODs. It is further supposed that oxygenation and intracellular O2- production favor MnSOD production because O2- oxidizes Mn(II) to Mn(III), which competes favorably with Fe(II) for the apoSODs.     

Proline involvement in the common sage antioxidant system in the presence of NaCl and paraquat

To elucidate proline antioxidant properties in common sage (Salvia officinalis L.) plants, they were treated with paraquat (a producer of superoxide radical) and/or NaCl and also with paraquat and proline at the stage of 4–5 true leaves. The paraquat solution (1 ml containing 0.1 μmol of the agent) was applied to the leaf surface; NaCl (200 mM) and proline (the final concentration of 5 mM) were added to nutrient medium. Experimental plants were firstly kept in darkness for 12 h, then illuminated, and in 3, 6, and 12 h, leaves and roots were fixed for biochemical analyses. The results obtained are in agreement with the supposition of proline antioxidant properties. In particular, it was established that paraquat induced a slight increase in the proline level in the leaves during dark period of plant growth and also during subsequent 3 h after light switching on. This transient proline accumulation in the leaves was accompanied by its level decrease in the roots. Proline addition to the nutrient medium of paraquat-treated plants neutralized paraquat damaging action on the leaves. In the presence of paraquat, proline treatment reduced the accumulation in the roots of hydrogen peroxide and malondialdehyde, the product of membrane lipid peroxidation. It also affected indirectly the activities of superoxide dismutase (SOD) and free, covalently bound, and ionically bound peroxidases. Keeping in mind that, in the presence of paraquat, superoxide-induced changes in SOD activity in the roots were negatively correlated with the level of proline, which content was the highest during the last hours of experiments, we can conclude that proline antioxidant effects are manifested only after 12 h of stressor action, whereas antioxidant enzymes are involved in ROS scavenging during the earlier stage of damaging factor action.     

Molecular cloning and expression of a Cu/Zn-Containing superoxide dismutase from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Superoxide dismutases (SODs) constitute the first line of cellular defense against oxidative stress in plants. SODs generally occur in three different forms with Cu/Zn, Fe, or Mn as prosthetic metals. We cloned the full-length cDNA of the Thellungiella halophila Cu/Zn-SOD gene ThCSD using degenerate RT-PCR and rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the ThCSD gene (GenBank accession number EF405867) had an open reading frame of 456 bp. The deduced 152-amino acid polypeptide had a predicted molecular weight of 15.1 kDa, an estimated pI of 5.4, and a putative Cu/Zn-binding site. Recombinant ThCSD protein was expressed in Escherichia coli and assayed for SOD enzymatic activity in a native polyacrylamide gel. The SOD activity of ThCSD was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, confirming that ThCSD is a Cu/Zn-SOD. Northern blotting demonstrated that ThCSD is expressed in roots, stems, and leaves. ThCSD mRNA levels increased by about 30-fold when plants were treated with sodium chloride (NaCl), abscisic acid (ABA), and indole-acetic acid (IAA) and by about 50-fold when treated with UVB light. These results indicate that ThCSD is involved in physiological pathways activated by a variety of environmental conditions. These authors contributed equally to this work.     

Changes in organelle superoxide dismutase isoenzymes during air adaptation of submerged rice seedlings: Differential behaviour of isoenzymes in plastids and mitochondria

Changes in activities and levels of superoxide dismutase (SOD, EC 1.15.1.1) isoenzymes were studied during air adaptation of submerged rice (Oryza sativa L.) seedlings. Seeds were germinated for 6 d in the dark under water (submerged), and then for another day in air (air-adapted). For a control, seeds were germinated for 6 d throughout in air (aerobic). Staining for activity of SOD of shoot extracts showed a total of five major distinct forms of SOD: one mitochondrial Mn enzyme (mtSOD) and four CuZn enzymes, one of which was plastidic (plSOD) and the other three cytosolic (cytSOD). Activity of plSOD was much lower in submerged seedlings than in aerobic controls and increased after exposure to air. In contrast, mtSOD activity in submerged seedlings was as high as that found in aerobic controls, and did not increase upon exposure to air. One of the cytSODs showed responses similar to those of plSOD. The activities of another two cytSODs were slightly lower in submerged seedlings than those in aerobic controls, but decreased after 24 h of air adaptation. Western blot analysis revealed that these changes in activities of SODs are due to changes in the levels of their enzyme proteins. We also followed changes in the levels of cytochrome c and ferredoxin-NADP⁺ reductase (EC 1.6.99.4) as indices of the development of mitochondria and plastids, respectively. Organelle SODs were always present at higher levels than would be expected in view of the development of the electron-transport systems of the corresponding organelles during submergence and the subsequent air-adaptation period.Abbreviations cytSOD cytosolic CuZn-superoxide dismutase - FNR ferredoxin-NADP⁺ reductase - mtSOD mitochondrial Mn-superoxide dismutase - plSOD plastidic CuZn-superoxide dismutase - SOD superoxide dismutase We gratefully acknowledge Dr. A. Wada, Kyoto University, for the use of a densitometer for this work, and also would like to thank Y. Maki, Kyoto University, for help with the densitometry of the photographs.     

Recombinant superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum

 Superoxide dismutase (SOD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (a facultative aerobe) has been cloned and expressed in a mesophilic host (Escherichia coli) as a soluble tetrameric apoprotein. The purified apoprotein can be reconstituted with either Mn or Fe by heating the protein with the appropriate metal salt at an elevated temperature (95  °C). Both Mn- and Fe-reconstituted P. aerophilum SOD exhibit superoxide dismutase activity, with the Mn-containing enzyme having the higher activity. P. aerophilum SOD is extremely thermostable and the reconstitution with Mn(II) can be performed in an autoclave (122  °C, 18 psi). The Mn(III) optical absorption spectrum of Mn-reconstituted P. aerophilum SOD is distinct from that of most other MnSODs and is unchanged upon addition of NaN₃. The optical absorption spectrum of Fe-reconstituted P. aerophilum SOD is typical of Fe-substituted MnSODs and authentic FeSOD and exhibits a pH-dependent transition with an effective pK _a value higher than that found for Fe-substituted MnSOD from either E. coli or Thermus spp. Amino acid sequence analysis shows that the P. aerophilum SOD is closely related to SODs from other hyperthermophilic archaea (Aeropyrum pernix and Sulfolobus spp.), forming a family of enzymes distinct from the hyperthermophilic bacterial SOD from Aquifex pyrophilus and from mesophilic SODs. Received: 2 February 2000 / Accepted: 29 March 2000     

The Rate of Cu,Zn Superoxide Dismutase Evolution

The rate of amino acid replacement in Cu, Zn SOD greatly departs from the expectations of the molecular clock. We examine 27 Cu, Zn SOD sequences available and conclude that: (I) the SOD enzymes from different mammal families differ from each other by roughly the same number of replacements, which is consistent with a simultaneous mammalian radiation; (2) over the most recent 60 million years (MY) the rate of SOD evolution is fairly high (15aa/100aa/100MYR) and may be considered constant; (3) the rate of accumulation of amino acid replacements since the divergence of fungi. plants and animals to the present is inconstant along different branches of the evolutionary tree; moreover it steadily decreases with time, to the same extent in all lineages; (4) some comparisons exhibit divergences that are in any case greater than expected from a Poisson process on the assumption of a molecular clock; (5) plant chloroplast enzymes display fewer differences from each other than cytoplasmic ones; (6) bacteriocuprein (from Photobacterium leiognathi), fluke and human extracellular SOD are all three extremely remotely related to one another and to the SOD of other eukaryotes.

The process of consistent decline of the rate of evolution of Cu. Zn SOD can be described by a number of mathematical functions. We explore simple models that assume constant rates and might be applicable to other proteins or genes that apparently evolve at disparate rates.     

Generation and characterization of cells that can be conditionally depleted of mitochondrial SOD2

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.