Structural and functional characterization of an organic hydroperoxide resistance protein from Mycoplasma gallisepticum

As obligate parasites, Mycoplasma species are continuously exposed to oxidative damage due to host-generated peroxides and reactive oxygen species (ROS). In addition, the production of endogenous oxidants is believed to be a primary virulence mechanism of several Mollicute species, indicating that oxidative stress resistance is crucial to survival of these bacteria in the host milieu. Despite the abundance of oxidants at the site of infection, enzymes responsible for the detoxification of ROS have never been characterized in mycoplasmas. Here we characterize a homolog of the ohr (organic hydroperoxide resistance) family from Mycoplasma gallisepticum (encoding MGA1142). Unlike previously characterized ohr genes, the mga1142 gene is not upregulated in response to oxidative stress but displays a novel pattern of expression. Both organic and inorganic peroxides can act as substrates for MGA1142, but they are degraded with various efficiencies. Furthermore, cumene hydroperoxide, an aromatic peroxide metabolized with high efficiency by other Ohr proteins, was shown to rapidly inactivate MGA1142, accounting for the sensitivity of M. gallisepticum cells to this compound. Comparative modeling of the MGA1142 quaternary structure revealed that the active site of this molecule has a relatively wide conformation. These data indicate that the natural substrate for MGA1142 differs from that for previously characterized Ohr proteins. Triton X-114 partitioning demonstrated that MGA1142 is located in both cytosol and membrane fractions, suggesting that in vivo this molecule plays a role in the detoxification of both endogenous and exogenous peroxides. A model describing how MGA1142 is likely to be oriented in the cell membrane is presented.     

Activities of Alkyl Hydroperoxide Reductase Subunits C1 and C2 of Vibrio parahaemolyticus against Different Peroxides

Alkyl hydroperoxide reductase subunit C gene (ahpC) functions were characterized in Vibrio parahaemolyticus, a commonly occurring marine food-borne enteropathogenic bacterium. Two ahpC genes, ahpC1 (VPA1683) and ahpC2 (VP0580), encoded putative two-cysteine peroxiredoxins, which are highly similar to the homologous proteins of Vibrio vulnificus. The responses of deletion mutants of ahpC genes to various peroxides were compared with and without gene complementation and at different incubation temperatures. The growth of the ahpC1 mutant and ahpC1 ahpC2 double mutant in liquid medium was significantly inhibited by organic peroxides, cumene hydroperoxide and tert-butyl hydroperoxide. However, inhibition was higher at 12°C and 22°C than at 37°C. Inhibiting effects were prevented by the complementary ahpC1 gene. Inconsistent detoxification of H₂O₂ by ahpC genes was demonstrated in an agar medium but not in a liquid medium. Complementation with an ahpC2 gene partially restored the peroxidase effect in the double ahpC1 ahpC2 mutant at 22°C. This investigation reveals that ahpC1 is the chief peroxidase gene that acts against organic peroxides in V. parahaemolyticus and that the function of the ahpC genes is influenced by incubation temperature.     

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts

Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.     

Influence of oxyR on Growth,Biofilm Formation,and Mobility of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common marine food-borne enteropathogen. In this study, we examined the antioxidative activity, growth, biofilm formation, and cell mobility of an oxyR deletion mutant and its genetically complementary strain of V. parahaemolyticus. oxyR is the regulator of catalase and ahpC genes. Protection against extrinsic H₂O₂ and against the organic peroxides cumene hydroperoxide and tert-butyl hydroperoxide was weaker in the deletion mutant than in its parent strain. Expression of the major functional antioxidative genes, ahpC1 and VPA1418, was markedly decreased in the oxyR mutant. Growth of this mutant on agar medium was significantly inhibited by autoclaved 0.25% glucose and by 0.25% dipotassium hydrogen phosphate, 0.5% monosaccharides (glucose, galactose, xylose, and arabinose), or 114.8 mM phosphates. The inhibition of the growth of this oxyR mutant by extrinsic peroxides, autoclaved sugars, and phosphates was eliminated by the complementary oxyR gene or by the addition of catalase to the autoclaved medium, while no inhibition of growth was observed when filter-sterilized sugars were used. The formation of biofilm and swimming mobility were significantly inhibited in the oxyR mutant relative to that in the wild-type strain. This investigation demonstrates the antioxidative function of oxyR in V. parahaemolyticus and its possible roles in biofilm formation, cell mobility, and the protection of growth in heated rich medium.     

Polyphosphates and exopolyphosphatase activities in the yeast Saccharomyces cerevisiae under overexpression of homologous and heterologous PPN1 genes

The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.     

Role of glutathione peroxidase against oxidative stress in yeast: phenotypic character of lipid hydroperoxide-sensitive mutants derived from Hansenula mrakii

Wild type cells of Hansenula mrakii IFO 0895 were highly resistant to the oxidative stress caused by lipid hydroperoxide. The resistance was due to a glutathione peroxidase (GSHPx) which was induced when the yeast was cultured in a medium containing lipid hydroperoxide (Inoue, Y. et al., Agric. Biol. Chem., 54, 3289–3293, 1990). In order to investigate the role of GSHPx, two mutants sensitive to lipid hydroperoxide were isolated. The phenotypes of the mutants were temperature-dependent; i.e., the mutants could grow at 28°C, but not at 35°C in the presence of lipid hydroperoxide. The mutants failed to induce the GSHPx at 35°C. However, the enzyme induced at 28°C and prepared from both mutants was stable after incubation at 37°C for 1 h.     

Construction of flocculent industrial yeast by the yeast flocculation gene <Emphasis Type="Italic">FLO1</Emphasis>

The structure gene FLO1 from Saccharomyces cerevisiae W303-1A encoding a flocculation protein and the G418 resistance gene kanMX from plasmid pUG6 were amplified by PCR method. The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Angel yeast. The transformant Angel yeast F6 was obtained and showed strong and stable flocculation ability during 20 batches inoculation. And the flocculation ability of the transformant Angel yeast F6 showed no difference in the medium with the initial pH ranging from 3.5 to 6.0. Noteworthily, the flocculation onset of the transformant strain was in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And in the experiment the ethanol yield and other properties of the transformant Angel yeast F6 were similar to those of the wild-type strain, although its fermentation time was a little slower comparing with the wild-type strain. Those would be potential application for yeast cells to separate and recycle in the fuel ethanol industry.     

NrdH Redoxin Enhances Resistance to Multiple Oxidative Stresses by Acting as a Peroxidase Cofactor in Corynebacterium glutamicum

NrdH redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. Although NrdH redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. In this study, we confirmed that the Corynebacterium glutamicum NrdH redoxin catalytically reduces the disulfides in the class Ib ribonucleotide reductases (RNR), insulin and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), by exclusively receiving electrons from thioredoxin reductase. Overexpression of NrdH increased the resistance of C. glutamicum to multiple oxidative stresses by reducing ROS accumulation. Accordingly, elevated expression of the nrdH gene was observed when the C. glutamicum wild-type strain was exposed to oxidative stress conditions. It was discovered that the NrdH-mediated resistance to oxidative stresses was largely dependent on the presence of the thiol peroxidase Prx, as the increased resistance to oxidative stresses mediated by overexpression of NrdH was largely abrogated in the prx mutant. Furthermore, we showed that NrdH facilitated the hydroperoxide reduction activity of Prx by directly targeting and serving as its electron donor. Thus, we present evidence that the NrdH redoxin can protect against the damaging effects of reactive oxygen species (ROS) induced by various exogenous oxidative stresses by acting as a peroxidase cofactor.     

Constitutive expression of rice <Emphasis Type="Italic">ORGAN SIZE RELATED</Emphasis> genes in <Emphasis Type="Italic">Arabidopsis</Emphasis> results in organ size enlargement

In plants, organ size control is a fundamental process during development. The Arabidopsis ORGAN SIZE RELATED (OSR) gene family plays a key role in organ size regulation. To explore the roles of OSR orthologs in rice, a BLAST search in the rice genome was performed and five putative OSR orthologs were isolated and designated as OsOSR. Constitutive expression of OsOSR1, OsOSR2 and OsOSR4 in Arabidopsis resulted in enlarged organ sizes, as a consequence of enhanced cell number and cell size, while the increase of organ size in the OsOSR3 and OsOSR5-expressing plants was only due to cell enlargement. Our results suggest that the rice OsOSR genes possess the conserved organ growth-promoting function and may be involved in the coordination of cell proliferation and expansion during plant development.     

Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e₆ amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.     

Both Decrease in ACL1 Gene Expression and Increase in ICL1 Gene Expression in Marine-Derived Yeast Yarrowia lipolytica Expressing INU1 Gene Enhance Citric Acid Production from Inulin

In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.     

Toxicity of Linoleic Acid Hydroperoxide to Saccharomyces cerevisiae: Involvement of a Respiration-Related Process for Maximal Sensitivity and Adaptive Response

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho⁰] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho⁰] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 μg ml⁻¹). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37°C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.     

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.     

Molecular breeding of a sake yeast with a mutated ARO4 gene which causes both resistance to o-fluoro-dl-phenylalanine and increased production of β-phenethyl alcohol

A sake yeast, Saccharomyces cerevisiae, was transformed with a YCp vector containing a mutated ARO4 gene (ARO4-OFP) derived from S. cerevisiae which encodes the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase released from feedback inhibition by tyrosine. The transformant showed feedback-insensitive DAHP synthase activity and simultaneously acquired both o-fluoro-dl-phenylalanine (OFP)-resistance and increased production of β-phenethyl alcohol and tyrosine. The greater production of the alcohol and its acetate, rose-like flavor components, improved the aroma and flavor of sake prepared experimentally with the transformant. The ARO4-OFP gene was also demonstrated to be available as a dominant selection marker in yeast vectors for the transformation of industrial strains of S. cerevisiae.     

Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions

Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.     

Growth Response of Clostridium perfringens to Oxidative Stress

The sensitivity of Clostridium perfringens strain 13 to oxygen and its toxic derivatives was investigated in a new, defined medium (MMP). Exponentially growing cells in MMP medium were very sensitive to exposure to air by vigorous shaking. When exposed to air, the cells survived only 1hour and then rapidly died. Addition of cysteine, ascorbic acid, or yeast extract to the medium significantly increased vegetative cell survival without inducing sporulation. The level of toxicity of peroxyl and hydroperoxyl radicals, generated by H₂O₂, t-butyl hydroperoxide or ethanol, was very similar with and without air exposure. By contrast, plumbagin or menadione, which generate superoxide radicals in the presence of oxygen, caused high levels of cell death only in aerobiosic culture. Growth-arrested cells were more resistant to H₂O₂and to redox-cycling agents than were exponentially growing cells, but the resistance required de novo synthesis of proteins. An adaptive response to oxidative stress was also suggested by the higher level of cell resistance to H₂O₂and to ethanol when cells were pretreated with sublethal doses of these oxidants.     

The yeast glutaredoxins are active as glutathione peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.