Thioredoxin 2 from Escherichia coli is not involved in vivo in the recycling process of methionine sulfoxide reductase activities

Thioredoxins (Trx) 1 and 2, and three methionine sulfoxide reductases (Msr) whose activities are Trx-dependent, are expressed in Escherichia coli. A metB(1)trxA mutant was shown to be unable to grow on methionine sulfoxide (Met-O) suggesting that Trx2 is not essential in the Msr-recycling process. In the present study, we have determined the kinetic parameters of the recycling process of the three Msrs by Trx2 and the in vivo expression of Trx2 in a metB(1)trxA mutant. The data demonstrate that the lack of growth of the metB(1)trxA mutant on Met-O is due to low in vivo expression of Trx2 and not to the lower catalytic efficiency of Msrs for Trx2.     

Oxidative stress resistance of Escherichia coli strains deficient in glutathione biosynthesis

Sensitivity to various oxidants was determined for Escherichia coli strains JTG10 and 821 deficient in biosynthesis of glutathione (gsh-) and their common parental strain AB1157 (gsh+). The three strains showed identical sensitivity to H2O2. E. coli 821 was more resistant than AB1157 and JTG10 to menadione, cumene hydroperoxide, and N-ethylmaleimide. This resistance was not related to the gsh mutation because the other gsh- mutant and the parental strain showed similar sensitivity to these oxidants. The measured activities of NADPH:menadione diaphorase and glucose-6-phosphate dehydrogenase and the extracellular level of menadione suggested that the enhanced resistance of E. coli 821 to menadione might be due to decreased diaphorase activity, but not to a lowered rate of menadione uptake.     

A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyR(Xp)). The oxyRR2(Xp) mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H(2)O(2) and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2(Xp) mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2(Xp) mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyR(Xp) mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris.     

The role of a bifunctional catalase-peroxidase KatA in protection of Agrobacterium tumefaciens from menadione toxicity

Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants. The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A. tumefaciens from reactive oxygen species other than H(2)O(2) was evaluated in the katA mutant (PB102). Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione. The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level. Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102. A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes. Heterologous expression of X. campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102. The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA. This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

The molecular mechanism of menadione resistance in the <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 cyanobacterium

The effect of mutations in the genes encoding dehydrogenases and oxidases on the resistance of the Synechocystis sp. PCC 6803 cyanobacterium to menadione, an oxidative stress inducer, was studied. An enhanced sensitivity to menadione was observed in the mutants carrying inserts in the drgA gene encoding the NAD(P)H:quinone oxidoreductase (NQR) and in the ndhB gene encoding the subunit of NDH-1 complex. The menadione resistance in the mutants lacking oxidases (Ox), succinate dehydrogenase (SDH), and NDH-2 dehydrogenase do not differ from those in wild-type cells. An additional mutation in the drgA gene increased the sensitivity to menadione in the NDH-2 and Ox mutants. The double mutant that lacks both SDH and NQR was not viable. The expression of the drgA gene decreased during cell incubation in the dark but increased in the presence of glucose both in the dark and in light. Under photoautotrophic growth conditions, the dehydrogenase activity of the cells mainly depends on the NQR and NDH-1 functions. The re-reduction rate of the photosystem I reaction center (P700⁺) increased in wild-type and NDH-1 mutants after its oxidation with white light in the presence of DCMU after addition of menadione, and it decreased in the NQR mutant. The reduction of P700⁺ was accelerated in the presence of menadiol in all the strains studied. These results suggest that NQR provides defense of cyanobacterium cells from the toxic effect of menadione via its two-electron reduction to menadiol. An increased sensitivity of the NDH-1 mutant to menadione may result from the inhibition of respiration and the cyclic electron transport in photosystem I.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Comparative studies on the biochemical properties of the malic enzymes from Trypanosoma cruzi and Trypanosoma brucei

The construction of engineered bacterial cells with a reduced genome allows the investigation of molecular mechanisms that may be cryptic in wild-type strains and derivatives. Previously, a large-scale combined deletion mutant of Escherichia coli that lacked 29.7% of the parental chromosome was constructed by combining large chromosome deletions. In this work, we improved the system for making markerless-chromosomal deletions and obtained mutants with a genome that lacked up to 38.9% of the parental chromosome. Although the large-scale deletion mutants possessed genes needed for resistance to oxidative stress, including superoxide dismutase, catalase, and RpoS, they were sensitive to menadione, which induces reactive oxygen species during stationary phase. Small genome size did not necessarily correlate with greater sensitivity to menadione as several mutants with large deletions were more resistant to menadione. The sensitivity to menadione depended on whether the mutants were grown aerobically or anaerobically, suggesting that the mechanism governing menadione resistance depended on the oxygen tension of the growth medium. Further analysis of the large-scale deletion mutants should help identify the regulatory networks that are important for cellular defense against oxidative stress.     

Multiple superoxide dismutases in Agrobacterium tumefaciens: functional analysis, gene regulation, and influence on tumorigenesis

Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme. sodBI is expressed at a high levels throughout all growth phases. sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner. sodBIII is expressed only during stationary phase. Analysis of the physiological function of sods reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sods contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.     

Characterization of stress processes of Phaffia rhodozyma stress-resistant mutant

A carotenoid-less Phaffia rhodozyma mutant (MCP 325) exhibited significantly higher resistance to oxidative stressors such as menadione, H2O2 and K2Cr2O7 than its astaxanthin-producing parental strain (MCP 324). The absence of carotenoids in the mutant did not explain this phenomenon. The cause of the decreased superoxide, hydroxyl radical and glutathione contents, the increased peroxide concentration and the elevated specific activity of catalase under uninduced conditions may be a second mutation. Peroxide treatment induced specific catalase activity in the mutant but not in the parental strain. Regulation of these processes led to the result that, in spite of the mutations, the two strains exhibited the same multiplication rate and generation time.     

Role of vitamin K2 in the organization and function of Staphylococcus aureua membranes.

A mutant of Staphylococcus aureus auxotrophic for menadione (a vitamin K2 precursor) was used to study the effects of menadione deprivation on the structure and function of the cell membrane. The phospholipid composition and metabolism was essentially unaltered by menadione deprivation. Removal of this percursor caused cellular levels of the cytochromes, protoheme, vitamin K2, and several membrane-bound flavoprotein dehydrogenase activities to decrease as a function of growth dilution. The cytochromes were enzymatically reducible and maintained in the same proportions as menadione-supplemented cells. Oxidative phosphorylation, however, was reduced more than 10-fold and membrane vesicles obtained from menadione-deprived cells were unable to couple glycine transport to L-lactate oxidation. Succinic dehydrogenase and adenosine 5' triphosphate hydrolysis appeared unaffected by menadione deprivation. These data suggest that menadione deprivation in the mutant stops the synthesis of vitamin K2 and other electron transport chain components and prosthetic groups. Although individual electron transport chain members remained fully functional during menadione deprivation, the overall efficiency of the chain, measured in terms of its function in electron transport, oxidative phosphorylation, and electron transport chain-linked transport, dropped greatly. This suggests that the synthesis of vitamin K2 is modulated to the synthesis of other components of the electron transport system, and that their organization into a functional system requires a specific concentration of vitamin K2 with respect to total membrane lipid.     

Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.     

Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia coli K-12.

The degradation of mRNA in Escherichia coli is thought to occur through a series of endonucleolytic and exonucleolytic steps. By constructing a series of multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and ams-1 (altered message stability) alleles, it was possible to study general mRNA turnover as well as the degradation of specific mRNAs. Of most interest was the ams-1 pnp-7 rnb-500 triple mutant in which the half-life of total pulse-labeled RNA increased three- to fourfold at the nonpermissive temperature. RNA-DNA hybridization analysis of several specific mRNAs such as trxA (thioredoxin), ssb (single-stranded-DNA-binding protein), uvrD (DNA helicase II), cat (chloramphenicol acetyltransferase), nusA (N utilization substance), and pnp (polynucleotide phosphorylase) demonstrated two- to fourfold increases in their chemical half-lives. A new method for high-resolution Northern (RNA) analysis showed that the trxA and cat mRNAs are degraded into discrete fragments which are significantly stabilized only in the triple mutant. A model for mRNA turnover is discussed.     

The CcmE protein from Escherichia coli is a haem-binding protein

We previously reported that a 17.5-kDa haem-binding polypeptide accumulates in Escherichia coli K-12 mutants defective in an essential gene for cytochrome c assembly, ccmF , and speculated that this polypeptide is either CcmE or CcmG. The haem-containing polypeptide, which is associated with the cytoplasmic membrane, has now been identified by N-terminal sequencing to be CcmE. The haem-dependent peroxidase activity of CcmE is clearly visible not only in a ccmF mutant, but also in ccmG and ccmH mutants, implying that CcmE functions either before or in the same step as CcmF, CcmG and CcmH in cytochrome c maturation. A trxA mutant, like the dipZ mutant, was unable to assemble c -type cytochromes or catalyse formate-dependent nitrite reduction: both activities were restored in the trxA and dipZ , but not ccmG , mutants by the reducing agent, 2-mercaptoethanesulphonic acid. Our data suggest that haem transferred across the cytoplasmic membrane by the CcmABCD complex becomes associated with CcmE, possibly by a labile covalent bond, before it is transferred to the cytochrome c apoproteins by the periplasmic haem lyase encoded by ccmF and ccmH . We further propose that CcmG is essential to reduce the disulphide bonds formed in cytochrome c apoproteins by DsbA, before haem is attached by the haem lyase. Electrons for disulphide bond reduction are supplied from thioredoxin in the cytoplasm via DipZ in the membrane, but can be replaced by the chemical reductant, 2-mercaptoethanesulphonic acid. According to this model, CcmG is the last protein in the reducing pathway which interacts stereospecifically with the apoprotein.