Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyR-controlled) regulon

The synthesis of manganese-superoxide dismutase in response to hydrogen peroxide and to paraquat was examined in strains of Escherichia coli with different mutations in the oxyR gene. Hydrogen peroxide treatment did not induce manganese-superoxide dismutase, but did induce the oxyR regulon. Paraquat induced this enzyme in a strain compromised in its ability to induce the defense response against oxidative stress (oxyR deletion) as well as in a strain that is constitutive and overexpresses the oxyR regulon. Catalase (HPI), but not manganese-superoxide dismutase, was over-expressed under anaerobic conditions in a strain harboring a constitutive oxyR mutation. The data clearly demonstrate that the induction of manganese-superoxide dismutase is independent of the oxyR-controlled regulon.     

Increased superoxide radical production evokes inducible DNA repair in Escherichia coli

Paraquat induced the SOS response in Escherichia coli. This was measured in terms of acquired resistance towards UV lethality in a wild-type strain and in terms of appearance of beta-galactosidase activity in a din::Mu d(Ap lac) fusion strain. However measured, the induction of the SOS response by paraquat was entirely dioxygen-dependent; whereas induction of the SOS response by mitomycin C was independent of the presence of dioxygen. As expected, recA(Def) and lexA(Ind-) isogenic strains did not show the SOS response. It appears likely that O-2, whose intracellular production is increased by paraquat, leads to DNA damage which in turn induces the SOS response.     

The role of redox in the regulation of manganese-containing superoxide dismutase biosynthesis in Escherichia coli

The manganese-containing superoxide dismutase in Escherichia coli is an inducible enzyme that protects cells against oxygen toxicity. The manganese-enzyme is induced by oxygen, nitrate, redox active compounds that react with oxygen to generate superoxide radicals, as well as iron chelators. In order to test the hypothesis that the redox state of the cell is involved in regulating manganese-superoxide dismutase biosynthesis, we studied the effects of several oxidants on growth and superoxide dismutase biosynthesis. The data showed, that under anaerobic conditions, the active manganese-enzyme is induced in the presence of potassium ferricyanide, copper-cyanide complex, ammonium persulfate, and hydrogen peroxide. Western blot analysis revealed that the induction of manganese-superoxide dismutase by the oxidants is associated with de novo protein biosynthesis. Potassium ferricyanide and hydrogen peroxide induced the enzyme under aerobic conditions as well. It is concluded that the redox state of the cell possibly influences the biosynthesis and/or activity of an iron-containing repressor protein that serves to negatively regulate manganese-superoxide dismutase biosynthesis.     

Regulation of the manganese-containing superoxide dismutase gene from fission yeast

The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen. To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14. Restriction mapping and nucleotide sequencing confirmed its construction. The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide. It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP). However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid. The beta-galactosidase synthesis appeared to be independent of the Pap1 protein. These results suggest that some metals, oxidative stress, and nitric oxide regulate the S. pombe MnSOD gene.     

Induction of the manganese-containing superoxide dismutase in Escherichia coli by nalidixic acid and by iron chelators

Abstract Nalidixic acid caused a significant increase in the Mn-containing superoxide dismutase (MnSOD) of Escherichia coli . The maximum stimulatory effect of nalidixic acid on MnSOD biosynthesis was observed at 0.1 mM. The stimulatory effect of nalidixic acid was not due to increases in the intracellular flux of O⁻₂, but rather to its ability to chelate Fe²⁺. Furthermore, 2,2'-dipyridyl and 1,10-phenanthroline were shown to cause a 7- to 20-fold increase in the MnSOD of E. coli . It is proposed that the repressor for MnSOD is an iron-containing protein.     

Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect.

Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.     

Catalase and superoxide dismutase in Escherichia coli

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.     

Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function

This review is concerned with the effects of environmental perturbations on the expression of the two superoxide dismutase (SOD) genes in Escherichia coli (sodA, MnSOD; sodB, FeSOD). Early studies using SOD activity, showed that MnSOD levels respond to changes in oxygen tension, type of substrate, redox active compounds, iron concentration, the nature of the terminal oxidant, and the redox potential of the medium. FeSOD levels appeared nominally insensitive to these perturbations. More recent molecular genetic studies revealed that sodA expression is subject to regulation by three major regulatory systems: fur (ferric uptake regulation) and arcA arcB (aerobic respiratory control) mediate repression of sodA, while a relatively new system, soxR soxS (superoxide response), mediates activation of sodA expression. By contrast, sodB expression, which is much less studied at this time, appears to be positively activated in trans by fur. A rudimentary gene regulation model is presented which rationalizes past observations, is experimentally testable, and should serve as a guide to future research in this area.     

Secretion of human superoxide dismutase in Escherichia coli using the condensed single-protein-production system

A secretion vector, pColdV for the Single-Protein-Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co-expressed with MazF, an ACA-specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ～20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two S-S bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope-labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.     

High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12

Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [³H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells. The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical.     

Chemical modification of iron- and manganese-containing superoxide dismutases from Escherichia coli

The manganese-containing (MnSOD) and iron-containing (FeSOD) superoxide dismutases from Escherichia coli are extensively (greater than 95%) inactivated by treatment with phenylglyoxal. The relatively high concentrations of phenylglyoxal and high pH required for optimal inactivation suggest that inactivation may be due to modification of an arginine with a "normal" elevated pKa, i.e., one not in an active site cavity where the pKa is likely to be lowered because of lower solvent accessibility and decreased polarity of the local environment. Treatment of either enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2-hydroxy-5-nitrobenzyl bromide, m-chloroperoxybenzoate, or tetranitromethane causes no inactivation, while 2,4,6-trinitrobenzenesulfonate, N-acetylimidazole, or diethyl pyrocarbonate cause 55-75% inactivation of each enzyme. Failure of hydroxylamine to reverse inactivation by the latter two suggests that in each instance loss of activity is due to lysine modification. The previously reported inactivation of FeSOD by H2O2 was further investigated, and no evidence was found for an affinity mechanism, i.e., a reversible binding of peroxide that precedes inactivation.     

Induction of manganese-containing superoxide dismutase is required for acid tolerance in Vibrio vulnificus

The manganese-containing superoxide dismutase (MnSOD) of Vibrio vulnificus, normally detected after the onset of the stationary phase, is expressed during the lag that immediately follows the transfer of cells grown exponentially to a fresh medium acidified to pH 5.0, whereas Fe-containing SOD is constitutively expressed. The signal triggering the growth lag and MnSOD induction therein is not low pH but intracellular superoxide accumulated under these conditions, since addition of a superoxide scavenger not only shortened the lag but also abrogated the MnSOD induction. If the lysine decarboxylase reaction proceeds in the presence of sufficient lysine, the broth is rapidly neutralized to abolish the generation of oxidative stress. Accordingly, the acid tolerance response was examined without the addition of lysine. SoxR regulates MnSOD induction. Lack of MnSOD caused by mutations in soxR or sodA resulted in low tolerance to low pH. The fur mutant derepressing MnSOD showed better tolerance than the wild type. Thus, an increase in total cytosolic SOD activity through MnSOD induction is essential for the cell to withstand the acid challenge. The contribution of cuprozinc-containing SOD to acid tolerance is not significant compared with those of cytosolic SODs.     

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.     

The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic,manganese-containing enzyme

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.     

Regulation of Escherichia coli superoxide dismutase genes (sodA and sodB) by oxygen

Summary Two types of superoxide dismutase genes, sodA and sodB, were fused to -galactosidase gene (lacZ), in order to quantitatively study the effect of oxygen concentration on the gene expression of sodA and sodB. -Galactosidase activity derived from the sodA-lacZ fusion was induced by shifting from anaerobic condition to aerobic condition. Maximum activity (9.4×10³ U/OD₆₆₀) was observed when oxygen partial pressure was. 0.6 atm. On the contrary, gene expression level for the sodB-lacZ gene fusion was about two times higher during anaerobic condition than that during aerobic condition. From these results it was concluded that oxygen positively affected the gene expression of sodA and negatively affected the gene expression of sodB. An inducible expression vector using the sodA regulatory region was also constructed.