Evolutionary relationships of superoxide dismutases and glutamine synthetases from marine species of Alteromonas,Oceanospirillum, Pseudomonas and Deleya

Evolutionary relationships among marine species assigned to the genera Alteromonas, Oceanospirillum, Pseudomonas, and Alcaligenes were determined by an immunological study of their Fe-containing superoxide dismutases (FeSOD) and glutamine synthetases (GS), two enzymes with differentially conserved amino acid sequences which are useful for determining intermediate and distant relationships, respectively. Five reference antisera were prepared against the FeSODs from Alteromonas macleodii, A. haloplanktis, Oceanospirillum commune, Pseudomonas stanieri, and Deleya pacifica. For GS, a previously prepared antiserum to the enzyme from Escherichia coli was employed. Amino acid sequence similarities for both enzymes were determined by the quantitative microcomplement fixation technique and the Ouchterlony double diffusion procedure. Six evolutionary groups were detected by FeSOD sequence similarities: three subgroups within the genus Alteromonas, the genera Oceanospirillum and Pseudomonas, and a new genus, Deleya (to accommodate marine Alcaligenes). Only four groupings were delineated by the GS data: the latter three genera and one group composed of all the species of Alteromonas. Evidence that all of these subgroups are derived from the evolutionary lineage defined by the purple sulfur photosynthetic bacteria is presented.Abbreviations Alt Alteromonas - anti-Amac, anti-Ahal, anti-Ocom, anti-Psta, anti-Dpac antisera to the Fe-containing superoxide dismutases from Alteromonas macleodii 107, Alteromonas haloplanktis 121, Oceanospirillum commune 8, Pseudomonas stanieri 146, Deleya pacifica 62 - FeSOD Fe-containing superoxide dismutase - G+C guanine plus cytosine - GS glutamine synthetase - ImD immunological distance - MnSOD Mn-containing superoxide dismutase - Oce Oceanospirillum - Pse Pseudomonas - Rm relative mobility - rRNA ribosomal RNA - SOD superoxide dismutase Dedicated to the memory of Professor Roger Y. Stanier     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Expression of the Cu,Zn superoxide dismutase of Aspergillus fumigatus as determined by immunochemistry and immunoelectron microscopy

Abstract A polyclonal antibody against purified Cu,Zn superoxide dismutase (SOD) from the pathogen Aspergillus fumigatus was raised in a sheep. This antibody recognised purified A. fumigatus SOD, together with a single band of 19 kDa in A. fumigatus cytoplasmic antigen, by immunodevelopment of Western blots. The polyclonal serum did not recognise either the manganese or iron containing forms of the enzyme; however, it was reactive against putative Cu,Zn SODs in other members of the genus Aspergillus . Immunofluorescent staining of A. fumigatus cultures demonstrated expression of the Cu,Zn SOD in conidia and hyphae, with the cell wall staining particularly intensely. Conidiophores were stained in an uniformly intense pattern. Immunoelectron microscopy confirmed that the SOD was present within the hyphal cell wall, although there was also labelling in the cytoplasm. SOD may protect Aspergillus against oxidants produced by immune effector cells and these observations demonstrate that the enzyme is available to perform its antioxidant function within the cell wall.     

Possible involvement of nodule superoxide dismutase and catalase in leghemoglobin protection

Superoxide anion is able to oxidize oxyleghemoglobin prepared from soybean nodules. Furthermore, ferrileghemoglobin is oxidized to leghemoglobin (IV) by hydrogen peroxide and this irreversible reaction leads to a complete inactivation of the hemoprotein. In scavenging O ₂ ^- and H₂O₂, superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) are able to limit these oxidations. The occurrence of these enzymes within soybean nodules and their main characteristics are reported here. A general scheme taking into account their roles in leghemoglobin protection in vivo is proposed.Abbreviations Lb leghemoglobin - SOD superoxide dismutase     

Construction of an Escherichia coli K-12 strain deleted for manganese and iron superoxide dismutase genes and its use in cloning the iron superoxide dismutase gene of Legionella pneumophila.

Summary An Escherichia coli K-12 strain deleted for sodA and sodB (manganese and iron superoxide dismulases) was constructed and characterized by Southern blotting, enzyme assays, and physiological analyses. The sod deletion strain was used to clone the iron superoxide dismutase gene of Legionella pneumophila by complementation to paraquat resistance.     

Associations of genes encoding allozymes peroxidase and superoxide dismutase in poplar and spruce species

Summary Isozymes of peroxidase (PER) and superoxide dismutase (SOD) were analyzed in vegetative buds or very young leaves of seven species and two interspecific hybrids of Populus, in progenies of seven controlled crosses of three Populus species, and in needles of five Picea species and one putative hybrid. One to three PER, and one or two SOD zones of activity were observed. Electrophoretic mobility (EM) and banding phenotypes of isozymes of one PER locus were identical to those of one SOD locus in vegetative buds of five Populus species and hybrid. In leaves of the four Populus species and hybrid and progenies of controlled crosses, EM and phenotypes of isozymes of two PER loci were identical to those of two SOD loci. In Picea species, EM of isozymes of the only SOD locus was somewhat similar but not identical to that of one PER locus, and isozyme phenotypes of all individuals at the SOD locus were not identical to those at a PER locus. Chi-square tests verified the single-gene Mendelian control of the segregating allozyme variants at each of Per-L1 and Sod-1 in the three Populus species. The results of joint two-locus segregation tests indicated a very tight linkage and no recombination between Per-L1 and Sod-1 in three Populus species. Genes coding for isozymes of one or two PER loci are either presumably the same as, or very tightly linked to, the genes coding for isozymes of one or two SOD loci in the Populus species.     

Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light

The effect of a chilling stress, at a moderate photon flux density for a few hours, on the peroxidation of membrane lipids and on superoxide dismutase (SOD) activity was compared in leaf slices of chilling-sensitive and chilling-insensitive plants. The aim was to determine if susceptibility to chill-temperature photoinhibition could be related to either damage to membrane lipids by superoxide and-or a decrease in activity of chloroplast SOD. Plants used were Nerium oleander L., grown at 45° C, and Cucumis sativus L., both susceptible to chill-temperature photoinhibition, and N. oleander, grown at 20° C and Spinacia oleracea L., both insensitive to chill-temperature photoinhibition. Lipid peroxidation was assessed by measuring the concentration of malondialdehyde (MDA). Leaf slices from all plants showed a basal level of MDA which decreased by about 15% when the leaf slices were chilled in the light. The level of MDA was not increased by the addition of either KHCO₃ or methyl viologen during chilling but it was increased, up to threefold, by the addition of Rose Bengal, which produces singlet oxygen. Chloroplast SOD activity was assessed in leaf extracts as the cyanide-sensitive production of H₂O₂ in a system which produced superoxide. Activity of SOD was similar in all the plants and was altered little by chilling. The results show that for the plants tested, chilling at a moderate photon flux density for 5 h does not increase the susceptibility of cell membranes to peroxidative damage nor does it decrease the activity of SOD. It was concluded that the susceptibility of chilling-sensitive plants to chill-temperature photoinhibition cannot be explained on the basis of differences in the vulnerability of membrane lipids to damage by superoxide or differences in SOD activity.Abbreviations Chl chlorophyll - MDA malondialdehyde - MV methyl viologen - O ₂ ^- superoxide - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density - SOD superoxide dismutase Deceased     

Biosynthesis of superoxide dismutase and catalase inSaccharomyces cerevisiae: effects of oxygen and cytochromec deficiency

Summary Two strains ofSaccharomyces cerevisiae were used to study the synthesis of superoxide dismutase. One strain (cytochromec-deficient) contained 5–10% of the normal amounts of total cytochromec, while the other strain was a wild type. The cytochromec-deficient mutant had lower specific growth rate, growth yield, and oxygen uptake than the wild type. The superoxide dismutase and catalase activities, in both strains, were significantly lower under anaerobic than under aerobic conditions. Furthermore, under aerobic conditions the mutant contained higher levels of superoxide dismutase than the wild type which may be attributed to the higher intracellular flux of superoxide radicals caused by the cytochromec deficiency. The mutant also showed a lower level of catalase which was due to glucose repression.Paper Number 10007 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, U.S.A. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.     

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.     

1H NMR investigation of reduced copper-cobalt superoxide dismutase

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through ¹H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.Abbreviations WEFT water eliminated Fourier transform - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - SOD superoxide dismutase - E₂Co(II)SOD SOD with empty copper site (E=empty) and with cobalt(II) in the Zinc(II) site Offprint requests to: I. Bertini     

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     

Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice

Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing LAB were evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohn's disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing LAB showed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered LAB that produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies.     

Growth of Chlorella sorokiniana in the presence of sulfite elevates cell content of superoxide dismutase and imparts resistance towards paraquat

1. Growth of Chlorella sorokiniana in the presence of 7.5 mM sulfite, which halved the growth rate while doubling the superoxide dismutase (SOD; EC 1.15.1.1) content per cell, rendered the cells resistant to the toxic effects of 30 M paraquat. 2. While increasing total SOD content, sulfite increased the relative amount of the H₂O₂-resistant manganese-containing SOD. 3. It appears that O₂ ^– may be involved in mediating the toxicity of SO₂ in this green alga.Abbreviations SOD superoxide, dismutase - FeSOD ironcontaining superoxide dismutase - MnSOD manganese-containing superoxide dismutase     

Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture

Production of bioluminescence theoretically represents a cost, energetic or otherwise, that could slow Vibrio fischeri growth; however, bioluminescence is also thought to enable full symbiotic colonization of the Euprymna scolopes light organ by V. fischeri. Previous tests of these models have proven inconclusive, partly because they compared nonisogenic strains, or undefined and/or pleiotropic mutants. To test the influence of the bioluminescence-producing lux operon on growth and symbiotic competence, we generated dark luxCDABEG mutants in strains MJ1 and ES114 without disrupting the luxR-luxI regulatory circuit. The MJ1 luxCDABEG mutant out-competed its visibly luminescent parent approximately 26% per generation in a carbon-limited chemostat. Similarly, induction of luminescence in the otherwise dim ES114 strain slowed growth relative to DeltaluxCDABEG mutants. Some culture conditions yielded no detectable effect of luminescence on growth, indicating that luminescence is not always growth limiting; however, luminescence was never found to confer an advantage in culture. In contrast to this conditional disadvantage of lux expression, ES114 achieved approximately fourfold higher populations than its luxCDABEG mutant in the light organ of E. scolopes. These results demonstrate that induction of luxCDABEG can slow V. fischeri growth under certain culture conditions and is a positive symbiotic colonization factor.     

Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria

It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.Non-Standard Abbreviation AB medium autoinducer bioassay medium     

Transgenic cucumber fruits that produce elevated level of an anti-aging superoxide dismutase

Superoxide dismutase (SOD) plays an important role in cellular defense against oxidative stress in aerobic organisms. To generate cucumber (Cucumis sativus L.) fruits producing high yields of SOD for an anti-aging cosmetic material as a plant bioreactor, the CuZnSOD cDNA (mSOD1) from cassava was introduced into cucumber fruits by Agrobacterium-mediated transformation using the ascorbate oxidase promoter with high expression in fruits. The bialaphos-resistant shoots were selected on medium containing MS basal salts, 2 mg l^–1 BA, 0.1 mg l^–1 IAA, 300 mg l^–1 claforan, and 2 mg l^–1 bialaphos. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 1 mg l^–1 IAA, 300 mg l^–1 claforan, 2 mg l^–1 bialaphos to induce roots. Southern blot analysis confirmed that the mSOD1 gene was properly integrated into the nuclear genomes of three cucumber plants tested. The mSOD1 gene was highly expressed in the transgenic cucumber fruits, whereas it was expressed at a low level in the transgenic leaves. The SOD specific activity (units/mg protein) in transgenic fruits was approximately 3 times higher than in those of non-transgenic plants.     

Antigenicity of filarial superoxide dismutase in human bancroftian filariasis

Superoxide dismutase activity was measured in different stages of growth of filarial parasites (human and cattle). The activity was almost undetected or very low in microfilarial stage but in adult worms, the enzyme activity was high. The enzyme was characterized to be a Cu/Zn superoxide dismutase. Most of the enzyme activity was associated with a detergent extractable fraction of adult (Setaria) parasite. The enzyme was also detected in thein vitro released products of adult worms. The superoxide dismutase activity was completely inhibited with IgG antibody from chronic filarial patients in contrast to IgG from normal people. Filarial patients particularly have high IgG and IgM antibody levels to purified enzyme. However, individuals from non-filarial regions of Orissa are sero-negative for superoxide dismutase antibodies. Antibody response to superoxide dismutase could thus be used for filarial diagnosis.     

Anti-oxidant activity of superoxide dismutase and glutathione peroxidase enzymes in skeletal muscles from slaughter cattle infected with Taenia saginata

It is known that highly reactive oxygene species produced during normal cellular metabolism represent a powerful effector mechanism against parasites. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) belong to the main defense anti-oxidants that prevent the formation of new free radical species. The aim of this study was to assess the activities of SOD and GPx in cattle tissues infected with Taenia saginata. We observed a statistically significant increase in the SOD and GPx activities (p = 0.00003, 0.00008, respectively, Student’s t-test) in skeletal muscles infected with T. saginata in spectrophotometric analysis. With the use of western blot technique, SOD synthesis stimulation has appeared in the host tissues containing cysticerci in contrast with the control samples. There was no statistically significant increase in the GPx band intensity observed in the studied samples in comparison to controls (Gene Tools Version 4.01 program). These results support the significance of anti-oxidant processes in host defense mechanism during parasitic infections.