Copper,Zinc-Superoxide Dismutase from Clinically Isolated <Emphasis Type="Italic">Escherichia coli</Emphasis>: Cloning,Analysis of <Emphasis Type="Italic">sodC</Emphasis> and Its Possible Role in Pathogenicity

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.     

Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 Cu,Zn Superoxide Dismutases

Background  

Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.     

A role for <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis> Cu,ZnSOD in resistance to heme toxicity

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.     

Molecular analysis of superoxide dismutase in Campylobacter lari

The superoxide dismutase (SOD) gene clusters, sodB and sodC, and their adjacent genetic loci from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain were analyzed molecularly, and compared with those of thermophilic campylobacters. The UPTC CF89-12 strain carried sodB [structural gene 654 base pairs (bp)] and sodC (540 bp) genes, as did the Campylobacter lari RM2100 reference strain. However, the other three thermophilic Campylobacter jejuni, C. coli and C. upsaliensis reference strains carried only a single sodB gene, and no sodC. Although sodB and sodC in the UPTC strain shared relatively high nucleotide sequence similarities (92.9 % and 91.7 %, respectively) with the corresponding genes in the C. lari RM2100 strain, the sodB gene in the UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni NCTC11168 (80.8 % and 81.7 %), C. coli RM2228 (82.0 % and 83.1 %) and C. upsaliensis RM3195 (75.9 % and 77.0 %), respectively. All PCR amplifications of sodB and sodC gene segments with 28 C. lari isolates, including 14 UPTC isolates, gave positive results. C. lari organisms were shown to carry both the sodB and sodC genes with extremely high frequency. More high-SOD activity was seen with the C. lari isolates (n?=?9), including UPTC, than was seen with the other three thermophilic Campylobacter and Helicobacter pylori organisms.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂⁻). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O₂⁻ or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ^−/−, inos^−/−, and phox^−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all F. tularensis subspecies is their ability to survive and replicate within macrophages (18) and other cell types (6, 11, 25, 28). While recent work has furthered our understanding of F. tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O₂⁻) anion to hydrogen peroxide (H₂O₂) and O₂ (26). The dismutation of O₂⁻ prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (27). The F. tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O₂⁻ and NO radicals (8, 9, 21, 32). Moreover, many virulent bacteria possess two copies of the sodC gene (4). The evolutionary maintenance of an extra sodC gene copy suggests that it serves some essential function in survival (4). As an intracellular pathogen, F. tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render F. tularensis sensitive to ROS and attenuate virulence in mice (2). However, the contribution of F. tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a F. tularensis sodC mutant (ΔsodC) and a F. tularensis sodBC double mutant (sodB ΔsodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of F. tularensis in mice.     

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene.     

Functional design of bacterial superoxide:quinone oxidoreductase

The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions. It can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and defense mechanisms to neutralize superoxide are important for cellular health. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b₅₆₁ (CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potentials of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in the context of described cytochrome b₅₆₁ proteins and potential physiological roles of CybB.     

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co²⁺, Zn²⁺, and Mn²⁺. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii

Methanobacterium bryantii contains a single electrophoretically discernible superoxide dismutase, which constitutes 0.4% of the extractable protein. This enzyme has been purified to electrophoretic and ultracentrifugal homogeneity. It appears to be a tetramer. The subunits were tenaciously, but noncovalently bonded and were of identical size. The molecular weight of the enzyme was found to be 91,000 ± 2000. The specific activity of this enzyme was identical to that previously noted for the corresponding enzyme from Escherichia coli. The enzyme contained 2.7 atoms of Fe, 1.7 atoms of Zn, and less than 0.2 atoms Mn per tetramer. Its amino acid composition placed this enzyme with the other Mn- and Fe-containing superoxide dismutases. The M. bryantii enzyme was also similar to previously described Fe-containing superoxide dismutases in its optical and electron paramagnetic resonance spectra and in its susceptibility to inactivation by H₂O₂. The M. bryantii enzyme was ininhibited by N₃^?, but was less sensitive towards this inhibitor than other iron-containing superoxide dismutases.     

Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA, from Aspergillus oryzae KBN616

β-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the β-xylosidase level in A. oryzae to about 20% of that of the host strain.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Expression,purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.     

Excreted Leishmania peruviana and Leishmania amazonensis iron–superoxide dismutase purification: Specific antibody detection in Colombian patients with cutaneous leishmaniasis

Leishmania sp. survival in the vertebrate host depends on the host macrophage immune response as well as on the parasite’s defense against free radicals. Iron–superoxide dismutase (Fe-SOD) is a key antioxidant enzyme that contributes to radical superoxide dismutation, preventing the disease from surging and propagating itself. Leishmania sp. has various Fe-SOD isoforms, one of which (Fe-SODe) is excreted into the medium and, being highly immunogenic, can be considered a very good molecular marker. In this work, we purified the Fe-SOD enzymes excreted by L. peruviana and L. amazonensis and studied them as antigens in serodiagnosis. We used ELISA and Western blot techniques to test 51 human cutaneous leishmaniasis sera from Colombia. All 51 patients presented with dermal injuries caused by unknown Leishmania species. The results observed with the purified proteins were compared with those obtained when total soluble lysate and unpurified Fe-SODe were used as the antigen fraction. Thus, we conclude that the purified enzymes are more sensitive and specific than their unpurified counterparts and that there is no cross-reactivity between them.     

Protein trans-Splicing To Produce Herbicide-Resistant Acetolactate Synthase

Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of E. coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E. coli cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.