A compensatory increase in trehalose synthesis in response to desiccation stress in Saccharomyces cerevisiae cells lacking the heat shock protein Hsp12p

The effect of HSP12 deletion on the response of yeast to desiccation was investigated. The Deltahsp12 strain was found to be more desiccation tolerant than the wild-type strain. Furthermore, the increased intracellular trehalose levels in the Deltahsp12 strain suggested that this strain compensated for the lack of Hsp12p synthesis by increasing trehalose synthesis, which facilitated increased desiccation tolerance. Results obtained from flow cytometry using the membrane exclusion dye propidium iodide suggested that Hsp12p helped maintain plasma membrane integrity during desiccation. Analysis of the oxidative loads experienced by the wild-type and Deltahsp12 strains showed that during mid-exponential phase, the increased trehalose levels present in the Deltahsp12 cells resulted in increased protection of these cells against reactive oxygen species compared with wild-type cells. During stationary phase, lower levels of reactive oxygen species reduction by reduced glutathione was enhanced in the wild-type strain, which displayed lower intracellular trehalose concentrations. Comparison of the tolerance of the wild-type and Deltahsp12 strains with applied oxidative stress showed that the Deltahsp12 strain was more tolerant to exogenously applied H2O2, which we attributed to the higher intracellular trehalose concentration. Flow cytometry demonstrated that Hsp12p played a role in maintaining plasma membrane integrity during applied oxidative stress.     

A novel bioluminescent bacterial biosensor using the highly specific oxidative stress-inducible pgi gene

A new oxidative stress-responsive bacterial biosensor was constructed using the promoter of the pgi gene fused to the luxCDABE reporter. This strain (PGRFM) responded in a dose-dependent manner to methyl viologen (MV), a model redox chemical that results in oxidative stress. The responses of strain PGRFM to redox chemicals was strongly dependent on the available carbon source. For example, when the strain was grown under nutrient-limited conditions in the presence of glucose or gluconate it was capable of responding to low MV concentrations (0.6-19.3ppm), whereas the same cells grown in LB (a nutrient rich media) only responded to higher concentrations (4.9-625ppm). This allowed us to select PGRFM's growth conditions and extend the range of concentrations at which a stress-inducing chemical could be detected. Further, strain PGRFM responded to structural analogs of MV (i.e., ethyl and benzyl viologen), demonstrating that this strain is responsive to the presence of superoxide radicals, regardless of the chemical by which they are generated. Strain PGRFM's response patterns to these analogs were distinct from each other, which determined their strength to induce oxidative stress. As well, a significant induction was seen when this strain was exposed to hydrogen peroxide, illustrating that strain PGRFM is responsive in the presence of both the superoxide (O(2)(-)) and hydroxyl (OH) radicals.     

Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli

To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.     

The role of lager beer yeast in oxidative stability of model beer

Aims: In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. Methods and Results: Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma‐mass spectrometry, and the slowest formation of radicals in the model beers. Conclusions: A more oxidative stable beer is not obtained by a more‐oxidative‐stress‐tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less‐oxidative‐stress‐tolerant strain, exhibiting a higher iron uptake. Significance and Impact of the Study: To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin.     

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast

To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.     

Regulation of the oxidative stress response by the hpr gene in Bacillus subtilis

Bacillus subtilis mutants with null mutations in the spo0 A gene are resistant to oxidative stress during the exponential phase of growth. This resistance phenotype can be suppressed by mutations in the abrB gene, or in the hpr gene. Both of these gene products are negative regulatory proteins which are over-produced in a spo0 A strain, and the over-production of the hpr gene product results from over-production of the abrB gene product. The results suggested that the resistance to oxidative stress in a spo0 A strain is due to the lack of a protein directly controlled by the hpr negative regulator. Other mutations in the spo0 A gene conferring resistance to ethanol stress (eth) or suppressors of sporulation phenotypes (sof) had no effect on the sensitivity to oxidative stress of strains bearing them.     

Resistance to oxidative stress caused by ceftazidime and piperacillin in a biofilm of Pseudomonas.

The capacity to form a biofilm was evaluated in Pseudomonas aeruginosa isolated from patients with lung and urinary infections. Adherence, development of microcolonies and slime formation varied in the studied strains. P. aeruginosa P63 isolated from cystic fibrosis (CF) exhibited important microcolony formation with the densest biofilm, and was selected to study the oxidative stress produced with ceftazidime and piperacillin by means of chemiluminescence (CL) in cell suspensions and biofilm. P. aeruginosa strain P63 was compared with P69; both were sensitive to ceftazidime and showed increase of reactive species of oxygen (ROS) in the presence of this antibiotic. P. aeruginosas P69 exhibited resistance to piperacillin and low ROS production, while piperacillin-sensitive strain P63 showed high oxidative stress with this antibiotic. Piperacillin stimulated oxidative stress, increasing ROS production only in the sensitive strain. Higher antibiotic concentrations were necessary to augment ROS in bacteria biofilm than in suspension. Incubation of P63 strain with ceftazidime or piperacillin in the presence of its own extracellular matrix (EM) or sodium alginate stimulated lesser oxidative stress and slower decrease of ROS than in the absence of these polysaccharides. A variant, V(10), obtained from strain P63 showed more sensitivity to the antibiotics than the wild-type, and concomitantly exhibited higher production of ROS in the presence of both the antibiotics studied.     

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

A yeast mutant lacking the two major cytosolic sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1p) and NADP+-specific isocitrate dehydrogenase (Idp2p), has been demonstrated to lose viability when shifted to medium with acetate or oleate as the carbon source. This loss in viability was found to correlate with an accumulation of endogenous oxidative by-products of respiration and peroxisomal beta-oxidation. To assess effects on cellular protein of endogenous versus exogenous oxidative stress, a proteomics approach was used to compare disulfide bond-containing proteins in the idp2Deltazwf1Delta strain following shifts to acetate and oleate media with those in the parental strain following similar shifts to media containing hydrogen peroxide. Among prominent disulfide bond-containing proteins were several with known antioxidant functions. These and several other proteins were detected as multiple electrophoretic isoforms, with some isoforms containing disulfide bonds under all conditions and other isoforms exhibiting a redox-sensitive content of disulfide bonds, i.e., in the idp2Deltazwf1Delta strain and in the hydrogen peroxide-challenged parental strain. The disulfide bond content of some isoforms of these proteins was also elevated in the parental strain grown on glucose, possibly suggesting a redirection of NADPH reducing equivalents to support rapid growth. Further examination of protein carbonylation in the idp2Deltazwf1Delta strain shifted to oleate medium also led to identification of common and unique protein targets of endogenous oxidative stress.     

Sclerotial biomass and carotenoid yield of Penicillium sp. PT95 under oxidative growth conditions and in the presence of antioxidant ascorbic acid

AIMS: To determine the effect of oxidative stress and exogenous ascorbic acid on sclerotial biomass and carotenoid yield of Penicillium sp. PT95. METHODS: In this experiment, high oxidative stress was applied by the inclusion of FeSO(4) in the growth medium and exposure to light. Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Supplementation of exogenous ascorbic acid (as antioxidant) to the basal medium caused a concentration-dependent delay of sclerotial differentiation (up to 48 h), decrease of sclerotial biomass (up to 40%) and reduction of carotenoid yield (up to 91%). On the contrary, the exogenous ascorbic acid also caused a concentration-dependent decrease of lipid peroxidation in colonies of this fungus. CONCLUSIONS: Under high oxidative stress growth condition, the sclerotial biomass and carotenoid yield of PT95 strain in each plate culture reached 305 mg and 32.94 microg, which were 1.23 and 3.71 times higher, respectively, than those at low oxidative stress growth condition. These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that strain PT95 may be used for solid-state fermentation of carotenoid production under high oxidative stress growth conditions.     

Relative contribution of detoxifying enzymes to pyrethroid resistance in a resistant strain of Helicoverpa armigera

Abstract:  The relative contribution of oxidases and esterases to pyrethroid resistance was studied in a YS-FP strain of Helicoverpa armigera from China. The YS-FP strain was derived from a field-collected strain (YS) by 16 generations of selection with a mixture of fenvalerate and phoxim. Compared with the YS strain, the YS-FP strain showed 1850- to >7140-fold resistance to four ester-bonded phenoxybenzyl alcohol pyrethroids (fenvalerate, deltamethrin, cypermethrin and cyhalothrin), >205-fold resistance to a non-ester phenoxybenzyl alcohol pyrethroid (etofenprox) and only 19-fold resistance to an ester-bonded methylated biphenyl alcohol pyrethroid (bifenthrin). The oxidase inhibitor piperonyl butoxide eliminated most the of resistance to fenvalerate, deltamethrin, cypermethrin, cyhalothrin and etofenprox, whereas the esterase inhibitor S,S,S -tributylphosphorothioate had a small synergistic effect for fenvalerate and cyhalothrin only. This suggests that the resistance to these pyrethroids in the YS-FP strain was mainly because of enhanced oxidative detoxification. The monooxygenase activities of the midguts of sixth-instar larvae of the YS-FP strain to substrates p -nitroanisole, ethoxycoumarin and methoxycoumarin were 3.7-, 4.7- and 10-fold, respectively, compared with that of the YS strain. Glutathione S -transferase activity and esterase activity were not significantly altered in the YS-FP strain. This confirms that enhanced oxidative detoxification was a major mechanism contributing to pyrethroid resistance in the YS-FP strain.     

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ?sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H₂O₂, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ?sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ?sigCDE than in the control strain. These results indicate that ?sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ?sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ?sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ?sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ?sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.     

Increased burn-induced immunosuppression in lipopolysaccharide-resistant mice

Severe burns induce a state of immunosuppression, and the inflammatory response after burn injury may play a role in this phenomenon. This study examined the effect of the inflammatory response to endotoxin on burn-induced immunosuppression and oxidative stress. An endotoxin-resistant mouse strain (C3H/HeJ) and a normally responding mouse strain (C3H/HeN) were compared. The mice were separated into three groups of five animals for each experimental day: (1) saline, (2) buprenorphine, and (3) buprenorphine and 20% total body surface area burn. All animals were fed ad libitum. The inflammatory response was studied at 1, 4, 7, 10, and 14 days postburn. Proliferation of activated splenocytes in burn mice was significantly lower on days 7, 10, and 14 for the C3H/HeJ strain and on days 4 and 10 for the C3H/HeN strain. Globally, C3H/HeJ presented stronger immune suppression than C3H/HeN. Oxidative stress parameters (liver malonaldehyde, spleen metabolic activity, and thiol concentrations) were higher in endotoxin-resistant mice than in the control strain. Impairment of the inflammatory response was more pronounced and oxidative stress was greater in endotoxin-resistant burn mice than in normal burn controls. Buprenorphine administration was not related to depression of these immune parameters. The inflammatory response following burn injury may be beneficial to the immune system.     

The role of Corynebacterium glutamicum spiA gene in whcA-mediated oxidative stress gene regulation

The Corynebacterium glutamicum WhcA protein, which inhibits the expression of oxidative stress response genes, is known to interact with the SpiA protein. In this study, we constructed and analyzed spiA mutant cells with the goal of better understanding the function of the spiA gene. A C. glutamicum strain overexpressing the spiA gene showed retarded cell growth, which was caused by an increased sensitivity to oxidants. Expression of the spiA and whcA genes was repressed by oxidant diamide, indicating coordinate regulation and dispensability of the genes in cells under oxidative stress. In the spiA-overexpressing cells, the trx gene, which encodes thioredoxin reductase, was severely repressed. Deletion of whcA in spiA-overexpressing cells (or vice versa) produced phenotypes similar to the wild-type strain. Collectively, these data demonstrate a negative regulatory role of the spiA gene in whcA-mediated oxidative stress response and provide additional clues on the mechanism by which the whcA gene is regulated.     

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.     

Ascorbic acid might play a role in the sclerotial differentiation of Sclerotium rolfsii

Certain phytopathogenic fungi differentiate by forming sclerotia by an unclear biochemical mechanism. We have proposed that sclerotial differentiation might be regulated by fungal antioxidant defense. Part of this defense might be ascorbic acid, which in its reduced form is a well-known antioxidant. This natural antioxidant was studied in Sclerotium rolfsii in relation to oxidative-growth conditions, developmental stages and strain-differentiating ability. The transition of a sclerotial strain from the undifferentiated to the differentiated stage was accompanied by a sharp shift in the ratio of reduced/oxidized ascorbate toward the oxidized form. Ascorbate profiles and lipid peroxidation levels were different between the sclerotial strain grown under high- and low-oxidative stress conditions, as well as between a nonsclerotial S. rolfsii strain grown under high-oxidative stress conditions. In addition, the ratio of reduced/oxidized ascorbate in the nonsclerotial strain remained unchanged throughout growth. Lipid peroxidation under high-oxidative stress conditions in sclerotial S. rolfsii colonies one day before differentiation was 3.6-fold higher than in same-day colonies of this strain grown under low-oxidative stress conditions and 2.5-fold higher than in similar-day colonies of the nonsclerotial strain grown under high-oxidative stress conditions. Exogenous ascorbate caused a concentration-dependent reduction of lipid peroxidation and a proportional inhibition of the degree of sclerotial differentiation in the sclerotial strain grown under high-oxidative stress conditions by lowering its lipid peroxidation before differentiation to levels similar to the strain grown under low-oxidative stress conditions and to the nonsclerotial strain. Ascorbic acid might be produced by the sclerotial strain to reduce oxidative stress, although less efficiently than the nondifferenting strain. The data of this study support our theory that oxidative stress might be the triggering factor of sclerotial differentiation in phytopathogenic fungi.