Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism.

The consumption of molecular oxygen by Pseudomonas aeruginosa can lead to the production of reduced oxygen species, including superoxide, hydrogen peroxide, and the hydroxyl radical. As a first line of defense against potentially toxic levels of endogenous superoxide, P. aeruginosa possesses an iron- and manganese-cofactored superoxide dismutase (SOD) to limit the damage evoked by this radical. In this study, we have generated mutants which possess an interrupted sodA (encoding manganese SOD) or sodB (encoding iron SOD) gene and a sodA sodB double mutant. Mutagenesis of sodA did not significantly alter the aerobic growth rate in rich medium (Luria broth) or in glucose minimal medium in comparison with that of wild-type bacteria. In addition, total SOD activity in the sodA mutant was decreased only 15% relative to that of wild-type bacteria. In contrast, sodB mutants grew much more slowly than the sodA mutant or wild-type bacteria in both media, and sodB mutants possessed only 13% of the SOD activity of wild-type bacteria. There was also a progressive decrease in catalase activity in each of the mutants, with the sodA sodB double mutant possessing only 40% of the activity of wild-type bacteria. The sodA sodB double mutant grew very slowly in rich medium and required approximately 48 h to attain saturated growth in minimal medium. There was no difference in growth of either strain under anaerobic conditions. Accordingly, the sodB but not the sodA mutant demonstrated marked sensitivity to paraquat, a superoxide-generating agent. P. aeuroginosa synthesizes a blue, superoxide-generating antibiotic similar to paraquat in redox properties which is called pyocyanin, the synthesis of which is accompanied by increased iron SOD and catalase activities (D.J. Hassett, L. Charniga, K. A. Bean, D. E. Ohman, and M. S. Cohen, Infect. Immun. 60:328-336, 1992). Pyocyanin production was completely abolished in the sodB and sodA sodB mutants and was decreased approximately 57% in sodA mutants relative to that of the wild-type organism. Furthermore, the addition of sublethal concentrations of paraquat to wild-type bacteria caused a concentration-dependent decrease in pyocyanin production, suggesting that part of the pyocyanin biosynthetic cascade is inhibited by superoxide. These results suggest that iron SOD is more important than manganese SOD for aerobic growth, resistance to paraquat, and optimal pyocyanin biosynthesis in P. aeruginosa.     

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.     

Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria.

Pseudomonas aeruginosa is a strict aerobe which is likely exposed to oxygen reduction products including superoxide and hydrogen peroxide during the metabolism of molecular oxygen. To counterbalance the potentially hazardous effects of elevated endogenous levels of superoxide, most aerobic organisms possess one or more superoxide dismutases or compounds capable of scavenging superoxide. We have previously shown that P. aeruginosa possesses both an iron- and a manganese-cofactored superoxide dismutase (D. J. Hassett, L. Charniga, K. A. Bean, D. E. Ohman, and M. S. Cohen, Infect. Immun. 60:328-336, 1992). In this study, the genes encoding manganese (sodA)- and iron (sodB)- cofactored superoxide dismutase were cloned by using a cosmid library of P. aeruginosa FRD which complemented an Escherichia coli (JI132) strain devoid of superoxide dismutase activity. The sodA and sodB genes of P. aeruginosa, when cloned into a high-copy-number vector (pKS-), partially restored the aerobic growth rate defect, characteristic of the Sod- strain, to that of the wild type (AB1157) when grown in Luria broth. The nucleotide sequences of sodA and sodB have open reading frames of 612 and 579 bp that encode dimeric proteins of 22.9 and 21.2 kDa, respectively. These data were also supported by the results of in vitro expression studies. The deduced amino acid sequence of the P. aeruginosa manganese and iron superoxide dismutase revealed approximately 50 and 67% similarity with manganese and iron superoxide dismutases from E. coli, respectively. There was also remarkable similarity with iron and manganese superoxide dismutases from other phyla. The mRNA start site of sodB was mapped to 174 bp upstream of the ATG codon. A likely promoter with similarity to the -10 and -35 consensus sequence of E. coli was observed upstream of the ATG start codon of sodB. Regions sequenced 519 bp upstream of the sodA electrophoresis, sodA gene revealed no such promoter, suggesting an alternative mode of control for sodA. By transverse field electrophoresis, sodA and sodB were mapped to the 71- to 75-min region on the P. aeruginosa PAO1 chromosome. Strikingly, mucoid alginate-producing bacteria generated greater levels of manganese superoxide dismutase than nonmucoid revertants, suggesting that mucoid P. aeruginosa is responding to oxidative stress and/or changes in the redox status of the cell.     

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.     

Oxygen toxicity in Streptococcus mutans: manganese, iron, and superoxide dismutase.

When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated oxygen levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an Rf value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels.     

Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus

Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.     

The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.     

Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus

A gene encoding superoxide dismutase (SOD), sodM, from S. aureus was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the S. aureus SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The sodM gene expressed from a plasmid rescued an Escherichia coli double mutant (sodA sodB) under conditions that are otherwise lethal. SOD activity gels of S. aureus RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in a sodM mutant, while the two lower bands were absent in a sodA mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the sodA and sodM sodA mutants but not the sodM mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the sodM sodA mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an S. aureus sodA mutant are maintained.     

Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum

The superoxide dismutase (SOD) gene of Methanobacterium thermoautotrophicum (Takao, M., Oikawa, A., and Yasui, A. (1990) Arch. Biochem. Biophys. 283, 210-216), a strictly anaerobic archaebacterium, was expressed in Escherichia coli. The gene product accounted for more than 30% of the host's soluble protein. The purified protein was an active iron-containing tetrameric SOD with specific activity similar to known manganese-containing SODs (MnSODs) of aerobic archaebacteria. Although M. thermoautotrophicum SOD is an iron-containing SOD (FeSOD), it resembles MnSODs in amino acid sequence as judged by criteria distinguishing FeSODs from MnSODs. Moreover, M. thermoautotrophicum SOD is resistant to azide and hydrogen peroxide as MnSODs are, suggesting that its evolution is distinct from known eubacterial FeSODs.     

Overproduction of the rbo gene product from Desulfovibrio species suppresses all deleterious effects of lack of superoxide dismutase in Escherichia coli.

In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs. This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris. Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype. rbo overexpression suppressed all deleterious effects of SOD deficiency in E. coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction. Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired. The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants. Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH2-terminal domain of Rbo, complemented sod mutants. The physiological role of Rbo in sulfate-reducing bacteria is unknown. In E. coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters. It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage.     

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.     

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.     

Cloning, sequencing, expression and characterization of the manganese superoxide dismutase gene from Vibrio alginolyticus.

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector. The full-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.