An oxidative stress-specific bacterial cell array chip for toxicity analysis

An oxidative stress-specific bacterial cell array chip was fabricated and implemented in the analysis of various different chemicals. The chip consisted of twelve toxicity responsive strains that respond specifically to different oxidative toxicities such as the generation of the superoxide radical, except for strain EBMalK, which was included as a negative control. Each bioluminescent strain carried a fusion of a stress gene promoter (sodA, pqi-5, soxR, fumC, soxS, inaA, hmp, malK, katG, zwf, fpr or pgi) to the bacterial lux reporter genes. A total of nine chemicals were selected to exhibit the capabilities of this array when analyzing different oxidative toxicities. Each of the chemicals were categorized according to their structure and their ability to form radicals in vivo: (I) paraquat, an active radical producer, (II) structural analogs of paraquat that produce radicals, (III) chemicals that are distinct from paraquat but still produce radicals and (IV) chemicals having similar structures as paraquat but do not produce radicals. The results found that each strain was responsive to one or more of the compounds tested but, as a definitive factor, the responses from the chip were dependent upon the production of radicals, i.e., the strains were unresponsive to compounds that were similar in structure to paraquat but lacked the ability to generate radicals. The specificity of the strains used in the chip was also demonstrated by their ability to discriminate between the superoxide radical and hydrogen peroxide. Therefore, this cell array chip could be implemented in characterizing and understanding the toxic impacts of newly synthesized chemicals and drugs in terms of toxicity classification and the nature of oxidative damage experienced by cells.     

Characterization ofgltA: luxCDABE fusion inEscherichia coli as a toxicity biosensor

The use ofgltA gene, as a new biomarker for environmental stress biomonitoring, was investigated because of its key position as the first enzyme of the tricarboxylic acid (TCA) cycle. A recombinant bioluminescentEscherichia coli strain, EBJM2, was constructed using a plasmid carrying the citrate synthase (gltA) promoter transcribing thePhotorhabdus luminescens luxCDABE genes (gltA::luxCDABE). The responses from this strain were studied with five different classes of toxicants: DNA damage chemicals, phenolics, oxidative-stress chemicals, PAHs, and organic solvents. EBJM2 responded strongly to DNA damage chemicals, such as mitomycin C (MMC) and methyl-nitro-nitrosoguanidine (MNNG), and nalidixic acid with the strongest responses. In contrast, tests with several compounds from the other four classes of toxicants gave no significant response. Therefore, EBJM2 was found to be sensitive to DNA damage chemicals.     

Characterization of superoxide-stress sensing recombinant Escherichia coli constructed using promoters for genes zwf and fpr fused to lux operon

To measure the toxicity experienced by superoxide-generating compounds, two plasmids were constructed in which the superoxide-inducible fpr and zwf promoters from Escherichia coli were fused to promoterless Vibrio fischeri luxCDABE operon present in plasmid pUCD615. The bioluminescent response of E. coli harboring these constructs was studied as a function of the toxicity and was shown to be specific for superoxide generating chemicals. The two promoters employed, fpr and zwf, responded differentially to the redox-chemicals tested. Furthermore, a ΔmarA strain bearing the fpr::luxCDABE fusion had a weaker response to paraquat (methyl viologen) than its isogenic parent strain, whereas zwf induction was not inhibited in ΔmarA or Δrob strains. The fpr and zwf promoters were also induced by alkylating agents but were unresponsive in ΔmarA or Δrob strains. Using optimized assay conditions, the abilities of these strains to differentially respond to superoxide stress and alkylating agents that may be present in contaminants proves them to be good biosensor candidates for monitoring toxicity.     

Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli cells

Aims: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. Methods and Results: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild‐type strain OW6, compared with the CTAB‐resistant strain OW66. The activity of manganese–superoxide dismutase (Mn–SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real‐time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. Conclusions: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn–SOD activity. Significance and Impact of the Study: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.     

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.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Effects of the free radical generator paraquat on differentiation, superoxide dismutase, glutathione and inorganic peroxides in microplasmodia of Physarum polycephalum

The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. The responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. The addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. The white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. The results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Effects of the Free Radical Generator Paraquat On Differentiation, Superoxide Dismutase, Glutathione and Inorganic Peroxides In Microplasmodia of Physarum Polycephalum

Abstract. The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. the responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. the addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. the white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. the results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli

5-Aminolevulinic acid (ALA) is a value-added compound with potential applications in the fields of agriculture and medicine. Although massive efforts have recently been devoted to building microbial producers of ALA through metabolic engineering, few studies focused on the cellular response and tolerance to ALA. In this study, we demonstrated that ALA caused severe cell damage and morphology change of Escherichia coli via generating reactive oxygen species (ROS), which were further determined to be mainly hydrogen peroxide and superoxide anion radical. ALA treatment activated the native antioxidant defense system by upregulating catalase (CAT) and superoxide dismutase (SOD) expression to combat ROS. Further overexpressing CAT (encoded by katG and katE) and SOD (encoded by sodA, sodB, and sodC) not only improved ALA tolerance but also its production level. Notably, coexpression of katE and sodB in an ALA synthase expressing strain enhanced the biomass and final ALA titer by 81% and 117% (11.5 g/L) in a 5 L bioreactor, respectively. This study demonstrates the importance of tolerance engineering in strain development. Reinforcing the antioxidant defense system holds promise to improve the bioproduction of chemicals that cause oxidative stress.     

Superoxide protects Escherichia coli from bleomycin mediated lethality

Superoxide and its products, especially hydroxyl radical, were recently proposed to be instrumental in cell death following treatment with a wide range of antimicrobials. Surprisingly, bleomycin lethality to Escherichia coli was ameliorated by a genetic deficiency of superoxide dismutase or by furnishing the superoxide generator plumbagin. Rescue by plumbagin was similar in strains containing or lacking recA or with inactive, inducible, or constitutive soxRS regulons. Thus, superoxide interferes with bleomycin cytotoxicity in ways not readily explained by genetic pathways expected to protect from oxidative damage.     

An<Emphasis Type="Italic"> Escherichia coli</Emphasis> biosensor capable of detecting both genotoxic and oxidative damage

A two-plasmid dual reporter Escherichia coli biosensor was developed using the genes for bacterial bioluminescence and a mutant of the green fluorescent protein, GFPuv4. To achieve this, the two plasmids, which were derivatives of pBR322 and pACYC184, had compatible origins of replication and different antibiotic selection markers: ampicillin and tetracycline. The parent strains DK1 and ACRG43, each carrying a single plasmid with one of the fusion genes (strain DK1 harboring a fusion of the katG promoter to the lux operon while in ACRG43, the recA promoter was fused with the GFP gene), were responsive to oxidative and DNA damage, respectively, resulting in higher bioluminescence or fluorescence under the relevant toxic conditions. The responses of the dual sensor strain, DUAL22, to various toxicants, e.g., mitomycin C, N-methyl-N-nitro-N-nitrosoguanidine, hydrogen peroxide and cadmium chloride, were characterized and compared with the responses of the parent strains to the same chemicals. Finally, several chemical mixtures that cause various stress responses were tested to demonstrate the ability of this biosensor to detect specific stress responses within a multiple toxicity environment.     

Enhancement of the multi-channel continuous monitoring system through the use of Xenorhabdus luminescens lux fusions

The enhancement of the multi-channel continuous toxicity monitoring system developed previously was studied. To achieve better and more stable results from the system, the use of thermo-lux fusion strains that express the luxCDABE genes from Xenorhabdus luminescens was evaluated. A total of six recombinant Escherichia coli strains with the promoters from three oxidative-stress responsive genes, i.e. the katG, sodA and pqi-5 genes, fused to either the lux genes from Vibrio fischeri or X. luminescens were characterized and their responses to different chemicals compared. It was found that the basal level bioluminescence (BL) from the thermo-lux fusion strains was always higher while that of the V. fischeri lux strains were always near or below the lower limit of detection of the system. For example, the katG::V. fischeri lux strain, DPD2511, gave no discernible response due to its low level expression while a fusion of the katG promoter with the X. luminescens lux operon was clearly responsive and capable of detecting hydrogen peroxide down to about 1 ppm. The use of the thermo-lux strains found them to be as sensitive as the V. fischeri lux strains while providing a brighter, more stable basal level bioluminescence, making the analysis and monitoring of water-borne toxicity more reliable.     

Isolation of paraquat-resistant mutants of Escherichia coli: lack of correlation between resistance and the activity of superoxide dismutase

Abstract Paraquat-resistant Escherichia coli mutants were isolated. The mutants were 10- to 50-fold more resistant to paraquat than the wild type. The wild type was more responsive to the presence of paraquat by inducing higher levels of the manganese-containing superoxide dismutase (MnSOD). Thus, in minimal medium, 0.1 mM paraquat caused a 5-fold increase in MnSOD in the wild type while it had no effect on the level of MnSOD in the mutants. Yet, 50 mM paraquat exerted a dramatic induction of SOD in the mutant strains when grown in trypticase soy yeast extract (TSY) medium. In TSY medium, catalase was not significantly affected by paraquat in all the strains tested. Resistance to paraquat in these mutant strains is, therefore, unrelated to their capacity to detoxify superoxide or hydrogen peroxide.     

Phototoxic effect of UVR on wild type, ebony and yellow mutants of Drosophila melanogaster: Life Span, fertility, courtship and biochemical aspects

Melanin plays an important role in protecting organisms from ultraviolet radiation (UVR). Therefore, it is possible that differently colored strains can show different sensitivities to UVR. In the present work, life span, fertility and courtship behavior of wild type (w), ebony (e) and yellow (y) strains of Drosophila melanogaster were studied to evaluate their sensitivity to ultraviolet (UV). Because a range of phototoxic effects of UVR are mediated through generation of free radicals, levels of free radicals, lipid peroxide (malondialdehyde, MDA) and superoxide dismutase (SOD) activity of three strains were examined to indicate their antioxidant defending ability and oxidative status. It was shown that w always had the highest lifespan and fertility not only in the control but also in UV-exposed groups. Moreover, lifespan and fertility of e were significantly higher (P<0.0001) than those of y in the UV-exposed groups, but not for the control. On the other hand, UV exposure had an adverse effect on courtship of flies. Stronger electron paramagnetic resonance (EPR) signals could be detected in w, e and y exposed to 5 min UV. And there were more significant changes of EPR signals in y than in w and e. UVR had no significant (P=0.1782) effect on the SOD activities. After pooling data from the control and UV-exposed groups, we found that w had a significantly (P<0.05) higher level of SOD activity, but e and y were nearly at the same levels (P>0.05). MDA levels were increased in the UV dose-dependent manner (P=0.0495). In conclusion, our results suggested that UVR can decrease life span and fertility of flies and do harm to courtship, which may be due to oxidative damage to flies tissues (e.g. central nervous system) induced by free radicals. w had the highest tolerance to UVR, which may be ascribed to its advantage of survival under the natural condition and at high level of SOD activity. Then differences of pigment between e and y in absorbing UV, shielding against UV and scavenging free radicals produced by UVR should be responsible for their different sensitivity to UVR.     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.     

Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox ^−⁄− and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.