Roles of the Two-MnSOD System of Stenotrophomonas maltophilia in the Alleviation of Superoxide Stress.
ABSTRACT: Manganese-dependent superoxide dismutase (MnSOD, SodA) and iron-dependent SOD (FeSOD, SodB) are critical cytosolic enzymes for alleviating superoxide stress. Distinct from the singular sodA gene in most bacteria, Stenotrophomonas maltophilia harbors two sodA genes, sodA1 and sodA2. The roles of SodA1, SodA2, and SodB of S. maltophilia in alleviating superoxide stress were investigated. The expression of sod genes was determined by promoter-xylE transcriptional fusion assay and qRT-PCR. SodA2 and sodB expressions were proportional to the bacterial logarithmic growth, but unaffected by menadione (MD), iron, or manganese challenges. SodA1 was intrinsically unexpressed and inducibly expressed by MD. Complementary expression of sodA1 was observed when sodA2 was inactivated. The individual or combined sod deletion mutants were constructed using the gene replacement strategy. The functions of SODs were assessed by evaluating cell viabilities of different sod mutants in MD, low iron-stressed, and/or low manganese-stressed conditions. Inactivation of SodA1 or SodA2 alone did not affect bacterial viability; however, simultaneously inactivating sodA1 and sodA2 significantly compromised bacterial viability in both aerobic growth and stressed conditions. SodA1 can either rescue or support SodA2 when SodA2 is defective or insufficiently potent. The presence of two MnSODs gives S. maltophilia an advantage against superoxide stress.
Project description:Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.
Project description:By using an oligonucleotide probe constructed from a conserved region of amino acids located in the carboxyl-terminal end of superoxide dismutase (SOD) proteins, four SOD genes were cloned from the cyanobacterium Plectonema boryanum UTEX 485. One of these genes, designated sodB, encoded an FeSOD enzyme, while the remaining three genes, designated sodA1, sodA2, and sodA3, encoded MnSOD enzymes. To investigate the expression of these four genes, total cellular RNA was isolated from P. boryanum UTEX 485 cells grown under various conditions and RNA gel blot analysis was carried out. Results indicated that sodB and sodA1 were constitutively expressed, although sodB expression was partially repressed in cells grown under conditions of iron stress. sodA2 transcripts, which were not detectable in control cells, accumulated to high levels in cells treated with methyl viologen or in cells grown under conditions of iron or nitrogen stress. However, under microaerobic conditions, iron and nitrogen stress failed to induce sodA2, indicating that multiple factors affect the regulation of sodA2. While discrete transcripts were not detected for sodA3, hybridization was observed under a number of conditions, including those which increased the accumulation of sodA2 transcripts. Additionally, there were high levels of the sodA3 transcript detected in a P. boryanum UTEX 485 mutant strain resistant to methyl viologen treatment.
Project description:Microarray analyses were conducted to evaluate the paraquat-induced global transcriptional response of Bacillus anthracis Sterne (34F(2)) to varying levels of endogenous superoxide stress. Data revealed that the transcription of genes putatively involved in metal/ion transport, bacillibactin siderophore biosynthesis, the glyoxalase pathway, and oxidoreductase activity was perturbed most significantly. A B. anthracis mutant lacking the superoxide dismutase gene sodA1 (Delta sodA1) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the Delta sodA1 mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and Delta sodA1 cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The Delta sodA1 mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in B. anthracis. A double mutant lacking both the sodA1 and sodA2 genes (Delta sodA1 Delta sodA2) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between sodA1 and sodA2, and a role for the sod genes in manganese homeostasis.
Project description:Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.
Project description:The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Delta sod15 Delta sodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Delta sod15 Delta sodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Delta sod15 Delta sodA1 Delta sodC Delta sodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.
Project description:BACKGROUND: Superoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism. METHODOLOGY/PRINCIPAL FINDINGS: This study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0-8.0) and temperature (4-70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA(-) SodB(-) Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB. CONCLUSIONS/SIGNIFICANCE: This is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles.
Project description: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.
Project description:In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.
Project description:Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. The sodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M(r) of 22,430 and pI of 5. 8. S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetic tac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H(2)O(2). The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl(2) or Fe(NH(4))(2)(SO(4))(2), demonstrating the cambialistic nature of the S. meliloti SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H(2)O(2). Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H(2)O(2) of this unusual Fe-type SOD.
Project description: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.