Superoxide-generating Agent Research Articles

Polymer/Nanoceria Hybrid Polyplexes for Gene and Antioxidant Delivery.

Various diseases, including cancers and inflammatory diseases, are characterized by a disruption of redox homeostasis, suggesting the need for synergistic treatments involving co-delivery of gene therapies and free radical scavengers. In this report, polyethylenimine (PEI), nanoceria (NC), and DNA were complexed to form nanoparticles providing simultaneous delivery of a gene and an antioxidant. NC was coated in citric acid to provide stable, 4 nm particles that electrostatically bound PEI/DNA polyplexes. The resulting ternary particles transfected HeLa cells with similar efficiency to that of ternary polyplexes comprising 15 kDa poly-l-α-glutamic acid/PEI/DNA while providing smaller particle sizes by more than 100 nm. NC/PEI/DNA polyplexes exhibited enhanced radical-scavenging activity compared to free NC, and oxidative stress from the superoxide-generating agent, menadione, could be completely reversed by the delivery of NC/PEI/DNA polyplexes. Transfection by NC/PEI/DNA polyplexes was demonstrated to occur efficiently through caveolin-mediated endocytosis and macropinocytosis. Co-delivery of genes encoding reactive oxygen species-scavenging proteins, transcription factors, growth factors, tumor suppressors, or anti-inflammatory genes with NC, therefore, may be a promising strategy in synergistic therapeutics.

Reactive Oxygen Species Contribute to Symptomless, Extreme Resistance to Potato virus X in Tobacco.

Here we show that in tobacco (Nicotiana tabacum cultivar Samsun NN Rx1) the development of Rx1 gene-mediated, symptomless, extreme resistance to Potato virus X (PVX) is preceded by an early, intensive accumulation of the reactive oxygen species (ROS) superoxide (O2·-), evident between 1 and 6 h after inoculation and associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities. This suggests a direct contribution of this ROS to virus restriction during symptomless, extreme resistance. Superoxide inhibition in PVX-inoculated leaves by infiltration of antioxidants (superoxide dismutase [SOD] and catalase [CAT]) partially suppresses extreme resistance in parallel with the appearance of localized leaf necrosis resembling a hypersensitive resistance (HR) response. F1 progeny from crosses of Rx1 and ferritin overproducer (deficient in production of the ROS OH·) tobaccos also display a suppressed extreme resistance to PVX, because significantly increased virus levels are coupled to HR, suggesting a role of the hydroxyl radical (OH·) in this symptomless antiviral defense. In addition, treatment of PVX-susceptible tobacco with a superoxide-generating agent (riboflavin/methionine) results in HR-like symptoms and reduced PVX titers. Finally, by comparing defense responses during PVX-elicited symptomless, extreme resistance and HR-type resistance elicited by Tobacco mosaic virus, we conclude that defense reactions typical of an HR (e.g., induction of cell death/ROS-regulator genes and antioxidants) are early and transient in the course of extreme resistance. Our results demonstrate the contribution of early accumulation of ROS (superoxide, OH·) in limiting PVX replication during symptomless extreme resistance and support earlier findings that virus-elicited HR represents a delayed, slower resistance response than symptomless, extreme resistance.

Differential correlations between changes to glutathione redox state, protein ubiquitination, and stress-inducible HSPA chaperone expression after different types of oxidative stress.

In primary bovine fibroblasts with an hspa1b/luciferase transgene, we examined the intensity of heat-shock response (HSR) following four types of oxidative stress or heat stress (HS), and its putative relationship with changes to different cell parameters, including reactive oxygen species (ROS), the redox status of the key molecules glutathione (GSH), NADP(H) NAD(H), and the post-translational protein modifications carbonylation, S-glutathionylation, and ubiquitination. We determined the sub-lethal condition generating the maximal luciferase activity and inducible HSPA protein level for treatments with hydrogen peroxide (H2O2), UVA-induced oxygen photo-activation, the superoxide-generating agent menadione (MN), and diamide (DA), an electrophilic and sulfhydryl reagent. The level of HSR induced by oxidative stress was the highest after DA and MN, followed by UVA and H2O2 treatments, and was not correlated to the level of ROS production nor to the extent of protein S-glutathionylation or carbonylation observed immediately after stress. We found a correlation following oxidative treatments between HSR and the level of GSH/GSSG immediately after stress, and the increase in protein ubiquitination during the recovery period. Conversely, HS treatment, which led to the highest HSR level, did not generate ROS nor modified or depended on GSH redox state. Furthermore, the level of protein ubiquitination was maximum immediately after HS and lower than after MN and DA treatments thereafter. In these cells, heat-induced HSR was therefore clearly different from oxidative stress-induced HSR, in which conversely early redox changes of the major cellular thiol predicted the level of HSR and polyubiquinated proteins.

Regulation by SoxR of mfsA, Which Encodes a Major Facilitator Protein Involved in Paraquat Resistance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia MfsA (Smlt1083) is an efflux pump in the major facilitator superfamily (MFS). Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin) treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084) is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression.

Different influences of extracellular and intracellular superoxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries.

Superoxide production is increased in diseased blood vessels, which is considered to lead to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway. To investigate the respective influence of extracellular and intracellular superoxide on vascular function through the NO/sGC/cGMP pathway, mechanical responses of rat external iliac arteries without endothelium were studied under exposure to a superoxide-generating agent, pyrogallol, or menadione. Exposure to pyrogallol impaired the relaxation induced by acidified NaNO2 (exogenous NO) but not that by nitroglycerin (organic nitrate), BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), or 8-Br-cGMP (cGMP analog). Superoxide dismutase (SOD) and tempol restored the impaired relaxation by acidified NaNO2. Superoxide production in the bathing solution, but not in artery segments, was significantly increased by exposure to pyrogallol, which was abolished in the presence of SOD or tempol. However, exposure to menadione impaired the relaxant response to acidified NaNO2, nitroglycerin, or BAY 41-2272, whereas it augmented that to BAY 60-2770. Also, this exposure had no effect on the 8-Br-cGMP-induced vasorelxation. Superoxide production in artery segments was dramatically enhanced by exposure to menadione, whereas that in the bathing solution was not affected. This increase in vascular superoxide production was normalized by tempol but not by SOD. These findings suggest that extracellular superoxide reacts with NO only outside the cell, whereas intracellular superoxide not only scavenges NO inside the cell but also shifts the sGC redox equilibrium.

Effective stimulation of the biotechnological potential of the medicinal white rot fungus: Phellinus pini by menadione-mediated oxidative stress.

The effect of menadione (MQ; 2-methyl-1,4-naphtoquinone), a superoxide-generating agent, on the natural biodegradation system in the medicinal white rot fungus Phellinus pini was determined. While measuring the activities of extracellular manganese-dependent peroxidase (MnP) and intracellular chitinase, it was found that the application of MQ (0.75 mM) distinctly stimulated the activities of these enzymes in comparison to the control values (without MQ). Using the capillary electrophoresis (CE) method, an increase in the extracellular oxalic acid (OXA) concentration was detected during the first days after the addition of MQ. It was observed that the rate of intracellular proteolysis at pH 3.5 evidently decreased under oxidative stress conditions. Contrary to these results, the activities of serine proteases at pH 9.5 measured against fluorogenic peptide substrates distinctly increased in stressed cultures. The MQ treatment also caused an evident increase in the catalase (CAT) activity, as well as the levels of superoxide anion radicals (SORs), formaldehyde (FA), and phenolic compounds (PHC) in the experimental cultures. The results obtained confirm that prooxidants may find application as an effective way to stimulate biotechnological production of MnP and chitinase by white rot fungi.

AraC/XylS family stress response regulators Rob, SoxS, PliA, and OpiA in the fire blight pathogen Erwinia amylovora.

Transcriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob-overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS-deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora. PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA, including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses.

HSF1-mediated oxidative stress response to menadione in <i>Saccharomyces cerevisiae</i> KNU5377Y3 by using proteomic approach

The hat shock transcription factor HSF1 inthe yeast Saccharomyces cerevisiae regulates a wide range of genes and functions in diverse cellular reactions. To investigate the physiological response of HSF1 inthe presence of menadione (MD) in S. cerevisiae KNU 5377Y3, wild-type (k3wt) and isogenic hsf1 mutant (k3h1) cells were introduced. HSF1 was induced when k3wt cells were exposed to the superoxide-generating agent MD and k3h1 cells were hypersensitive to MD. Under MD stress, k3h1 cells down-regulated the expression of metabolic enzymes (Hxk, Fba1, Pgk1, Eno2, and Adh1), antioxidant enzymes (Trx2 and porin), and molecular chaperones and their cofactors (Hsp104, Ssb1, Hsp60, Hsp42, Hsp26, Hsp12, Cpr1, and Sti1). In addition, k3h1 cells increased cellular hydroperoxide levels and protein carbonylation under MD stress as compared to k3wt cells. However, there was a moderate difference in the wild-type (b3wt) and mutant (b3h1) cells derived from S. cerevisiae S288Cunder the same conditions. Thus, these results show that HSF1 is an important component of the stress response system, acting as an activator of cell rescue genes in S. cerevisiae KNU5377Y3, and its expression protects the cells from MD-induced oxidative damage by maintaining redox homeostasis and proteostasis in the presence of MD.

Contributions of Individual σ B -Dependent General Stress Genes to Oxidative Stress Resistance of Bacillus subtilis

The general stress regulon of Bacillus subtilis comprises approximately 200 genes and is under the control of the alternative sigma factor σ(B). The activation of σ(B) occurs in response to multiple physical stress stimuli as well as energy starvation conditions. The expression of the general stress proteins provides growing and stationary nonsporulating vegetative cells with nonspecific and broad stress resistance. A previous comprehensive phenotype screening analysis of 94 general stress gene mutants in response to severe growth-inhibiting stress stimuli, including ethanol, NaCl, heat, and cold, indicated that secondary oxidative stress may be a common component of severe physical stress. Here we tested the individual contributions of the same set of 94 mutants to the development of resistance against exposure to the superoxide-generating agent paraquat and hydrogen peroxide (H(2)O(2)). In fact, 62 mutants displayed significantly decreased survival rates in response to paraquat and/or H(2)O(2) stress compared to the wild type at a confidence level of an α value of ≤ 0.01. Thus, we were able to assign 47 general stress genes to survival against superoxide, 6 genes to protection from H(2)O(2) stress, and 9 genes to the survival against both. Furthermore, we show that a considerable overlap exists between the phenotype clusters previously assumed to be involved in oxidative stress management and the actual group of oxidative-stress-sensitive mutants. Our data provide information that many general stress proteins with still unknown functions are implicated in oxidative stress resistance and further support the notion that different severe physical stress stimuli elicit a common secondary oxidative stress.

Different Reactive Oxygen Species Lead to Distinct Changes of Cellular Metal Ions in the Eukaryotic Model Organism Saccharomyces cerevisiae

Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al3+) level rose up to 50-fold after the diamide treatment. Cellular potassium (K+) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al3+ accumulation was further validated by the enhanced Al3+ uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al3+ uptake, suggesting Al3+-specific transporters could be involved in Al3+ uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.

Structural-Functional Characterization and Physiological Significance of Ferredoxin-NADP+ Reductase from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP+ reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of •O2 -. Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762.

NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5′-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans

8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.

Chromium resistance strategies and toxicity: what makes Ochrobactrum tritici 5bvl1 a strain highly resistant

Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.

Lifespan extension and paraquat resistance in a ubiquinone-deficient Escherichia coli mutant depend on transcription factors ArcA and TdcA

We recently reported a genome-wide screen for extended stationary phase survival in Escherichia coli. One of the mutants recovered is deleted for ubiG, which encodes a methyltransferase required for the biosynthesis of ubiquinone. The ubiG mutant exhibits longer lifespan, as well as enhanced resistance to thermal and oxidative stress compared to wt at extracellular pH9. The longevity of the mutant, as well as its resistance to the superoxide-generating agent paraquat, is partially dependent on the hypoxia-inducible transcription factor ArcA. A microarray analysis revealed several genes whose expression is either suppressed or enhanced by ArcA in the ubiG mutant. TdcA is a transcription factor involved in the transport and metabolism of amino acids during anaerobic growth. Its enhanced expression in the ubiG mutant is dependent on ArcA. Our data are consistent with the hypothesis that ArcA and TdcA function in the same genetic pathway to increase lifespan and enhance oxidative stress resistance in the ubiG mutant. Our results might be relevant for the elucidation of the mechanism of lifespan extension in mutant mice and worms bearing mutations in ubiquinone biosynthetic genes.

Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170U/mg protein. The enzyme was inactivated by KCN and H(2)O(2) but not by NaN(3), confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60min and the half-life was 22 and 7min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.

Carnitine supplementation has protective and detrimental effects in Saccharomyces cerevisiae that are genetically mediated

l-Carnitine plays a well-documented role in eukaryotic energy homeostasis by acting as a shuttling molecule for activated acyl residues across intracellular membranes. This activity, supported by carnitine acyl-transferases and transporters, is referred to as the carnitine shuttle. However, several pleiotropic and often beneficial effects of carnitine in humans have been reported that appear to be unrelated to shuttling activity, but little conclusive evidence regarding molecular mechanisms exists. We have recently demonstrated a role of carnitine, independent of the carnitine shuttle, in yeast stress protection. Here, we show that carnitine specifically protects against oxidative stress caused by H(2)O(2) and the superoxide-generating agent menadione. Surprisingly, carnitine has a detrimental effect on survival when combined with thiol-modifying agents. Central elements of the oxidative stress response, specifically the transcription factors Yap1p and Skn7p, are shown to be required for carnitine's protective effect, but several downstream effectors are dispensable. A DNA microarray-based analysis identifies Cyc3p, a cytochrome c heme lyase, as being important for carnitine's impact during oxidative stress. These findings establish a direct genetic link to a carnitine-related phenotype that is independent of the shuttle system and suggests that Saccharomyces cerevisiae should provide a useful model for further elucidation of carnitine's physiological roles.

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress inSaccharomyces cerevisiae

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.

The Chromate-Inducible chrBACF Operon from the Transposable Element Tn OtChr Confers Resistance to Chromium(VI) and Superoxide

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

The Fusarium oxysporum sti35 gene functions in thiamine biosynthesis and oxidative stress response

The sti35 gene of the vascular wilt fungus Fusarium oxysporum was originally identified based on induced expression under stress conditions. In this study, the transcriptional regulation and biological function of sti35 were examined in the tomato pathogen F. oxysporum f.sp . lycopersici. Expression of sti35 was repressed by thiamine and induced by high temperatures. Sti35 transcripts were detected both during early and late stages of infection of tomato plants by F. oxysporum. Heterologous expression of the sti35 cDNA restored thiamine prototrophy in a Saccharomyces cerevisiae thi4 mutant and increased UV tolerance in a uvr − mutant of Escherichia coli. Targeted Δ sti35 knockout mutants of F. oxysporum exhibited a thiamine auxotrophic phenotype and reduced tolerance to the superoxide-generating agent menadione, indicating that Sti35 has a dual role in thiamine biosynthesis and oxidative stress response. RT-PCR analysis revealed the presence of differential RNA splicing of the second 5′-UTR intron, suggesting that thiamine may regulate sti35 expression via a post-transcriptional mechanism. F. oxysporum transformants carrying a transcriptional fusion of the sti35 promoter to the lacZ reporter gene produced high levels of β-galactosidase activity when grown in the absence, but not in the presence of thiamine. Thus, the sti35 promoter represents a useful tool for the controlled expression of genes of interest in F. oxysporum.