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1.
Superoxide dismutase (SOD) is an essential enzyme protecting cells against oxidative stress. However, its specific role under different conditions is not clear. To study the possible role of SOD in the cell during respiration, Saccharomyces cerevisiae single and double mutants with inactivated SOD1 and/or SOD2 genes growing on ethanol as an energy and carbon source were used. Activities of antioxidant and associated enzymes as well as the level of protein carbonyls were measured. SOD activity was significantly higher in a Mn-SOD deficient strain than that in the wild-type parental strain, but significantly lower in a Cu, Zn-SOD mutant. A strong positive correlation between SOD and catalase activities (R(2) = 0.99) shows possible protection of catalase by SOD from inactivation in vivo and/or decrease in catalase activity because of lower H(2)O(2) formation in the mutant cells. SOD deficiency resulted in a malate dehydrogenase activity increase, whereas glucose-6-phosphate dehydrogenase (G6PDH) activity was lower in SOD-deficient strains. Linear and non-linear positive correlations between SOD and isocitrate dehydrogenase activities are discussed. No changes in the activity of glutathione reductase and protein carbonyl levels support the idea that SOD-deficient cells are not exposed to strong oxidative stress during exponential growth of yeast cultures on ethanol.  相似文献   

2.
In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isoform (peroxisomal catalase), the mutant showed the same levels of total catalase activity as the control strain. Furthermore, in cells deficient in Ctt1, the reduced glutathione:oxidized glutathione ratio (GSH:GSSG) of dry cells was higher than that of the control strain, indicating a compensatory mechanism of defense in response to dehydration. Even so, desiccation tolerance of the ctt1 strain was significantly lower than in the control strain. Using a fluorescent probe sensitive to oxidation, we observed that cells of the ctt1 strain showed levels of intracellular oxidation 70% higher than those of control strain, suggesting that Ctt1 plays a role in the maintenance of the intracellular redox balance during dehydration and, therefore, in tolerance against a water stress.  相似文献   

3.
Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.  相似文献   

4.
Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.  相似文献   

5.
Vattanaviboon P  Mongkolsuk S 《Gene》2000,241(2):259-265
Analysis of the Xanthomonas campestris pv. phaseoli (Xp) catalase profile using an activity gel revealed at least two distinct monofunctional catalase isozymes denoted Kat1 and Kat2. Kat1 was expressed throughout growth, whereas Kat2 was expressed only during the stationary phase of growth. The nucleotide sequence of a previously isolated monofunctional catalase gene, Xp katE, was determined. The deduced amino acid sequence of Xp KatE showed a high percentage identity to an atypical group of monofunctional catalases that includes the well-characterized E. coli katE. Expression of Xp katE was growth phase-dependent but was not inducible by oxidants. In addition, growth of Xp in a carbon-starvation medium induced expression of the gene. An Xp katE mutant was constructed, and analysis of its catalase enzyme pattern showed that Xp katE coded for the Kat2 isozyme. Xp katE mutant had resistance levels similar to the parental strain against peroxide and superoxide killing at both exponential and stationary phases of growth. Interestingly, the level of total catalase activity in the mutant was similar to that of the parental strain even in stationary phase. These results suggest the existence of a novel compensatory mechanism for the activity of Xp catalase isozymes.  相似文献   

6.
The katEkatG mutant of E. coli, UM1, had no assayable catalase activities in the extract and showed increased (about 20 fold) sensitivity to killing by H2O2 when compared with its parental strain CSH7. The mutant strain was able to reactivate H2O2-damaged lambda phage. On the other hand, recA and polA mutants were also highly sensitive to H2O2, but they had normal level of catalase activities. RecA derivatives of UM1 were much more sensitive to H2O2 than UM1 and recA strains. The induction of umu operon occurred in UM1 at lower (1/10-1/20) doses of H2O2 than in CSH7. From the results it is concluded that the lethal effect of H2O2 is due to DNA damage induced by it and that catalase and DNA repair systems have a distinct role in protection against H2O2 in E. coli.  相似文献   

7.
We compared measurements of cell survival and DNA single-strand breaks (SSBs) caused by hydrogen peroxide (H2O2) and UVA radiation (365-nm) in both a parental and a H2O2-resistant variant of the Chinese hamster ovary HA1 line derived by culturing cells in progressively higher concentrations of H2O2. Both RNA slot blot analysis and enzyme analysis confirmed that the variant possesses high levels of both catalase activity and mRNA. The variant was completely resistant to the lethal effects of H2O2 over the concentration range tested (up to 480 microM), whereas the parental strain showed less than 1% survival at this concentration. Similarly, the H2O2-resistant strain exhibited far fewer SSBs after exposure to H2O2 than the parental strain. Addition of o-phenanthroline to the parental cells during H2O2 exposure almost completely inhibited SSB induction, evidence that these SSBs are produced via the Fenton pathway of Haber-Weiss reactions. Very little difference was found between the variant and the parent after exposure to 365-nm radiation: only a minor difference in survival kinetics and no difference is SSB induction were observed between the two cell lines. These results are consistent with a hypothesis that most lethal events caused in cells by UVA occur by pathways that do not involve the H2O2 that is produced by sensitized reactions within the cells.  相似文献   

8.
9.
Helicobacter pylori induces a severe inflammatory response in the gastric mucosa. It is able to withstand the inflammatory response by producing proteins such as KatA and KapA. The C-terminus of KatA possesses a unique tetra-lysine motif not found in other catalases or other known protein sequences. Mutants deficient in this motif were constructed by site-directed mutagenesis. Cytoplasmic and periplasmic catalase activities were measured for the parental strain, a truncated KatA mutant (deficient in the unique C-terminal tetra-lysine motif) and a previously constructed KapA-deficient mutant (confirming previous observations regarding the possible periplasmic localisation of KatA). No differences were observed in the cytoplasmic catalase activities, however, the KapA-deficient mutant had approximately 5.5 times less catalase activity in the periplasmic extract when compared to the periplasmic preparations of either parental strain or KatA truncated mutant. N-terminal sequencing of KatA revealed no cleaved N-terminal signal peptide, indicating Sec-independent transport. These findings support previous reports that there is some form of interaction between KatA and KapA of H. pylori, an interaction which still needs to be characterised.  相似文献   

10.
Zymomonas mobilis ZmCytC as a peroxidase bearing three heme c-binding motifs was investigated with ΔZmcytC constructed. The mutant exhibited filamentous shapes and reduction in growth under a shaking condition at a high temperature compared to the parental strain and became hypersensitive to exogenous H(2)O(2). Under the same condition, the mutation caused increased expression of genes for three other antioxidant enzymes. Peroxidase activity, which was detected in membrane fractions with ubiquinol-1 as a substrate but not with reduced horse heart cytochrome c, was almost abolished in ΔZmcytC. Peroxidase activity was also detected with NADH as a substrate, which was significantly inhibited by antimycin A. NADH oxidase activity of ΔZmcytC was found to be about 80% of that of the parental strain. The results suggest the involvement of ZmCytC in the aerobic respiratory chain via the cytochrome bc(1) complex in addition to the previously proposed direct interaction with ubiquinol and its contribution to protection against oxidative stress.  相似文献   

11.
12.
13.
In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from 20 degrees C to 60 degrees C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's K(m) value and V(max) of the catalase for H2O2 were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of A406 to A280 for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.  相似文献   

14.
15.
A comparison of Campylobacter jejuni VPI strain H840 (ATCC 29428), which can grow at O2 levels up to 15%, with variant strain MC711-01 (which can grow at O2 levels up to 21-26%) indicated that the specific activity of catalase in crude cell extracts was higher in the variant by a factor of 1.6 to 2.5, depending on cultural conditions. Smaller differences occurred with superoxide dismutase activity, while peroxidase activities were invariably lower in the variant strain. The variant strain was much more resistant than the wild type to the bactericidal effects of H2O2. The results suggest that catalase activity might be one of the factors associated with the greater tolerance of O2 by the variant strain. However, both strains became more susceptible to H2O2 when cultures were initially grown at 6% O2 and then shifted to 21% O2; thus the role of catalase in the oxygen tolerance of C. jejuni is probably minor.  相似文献   

16.
The role of catalase in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide under starvation was investigated. It was shown that under conditions used in this study 0.5 mM H2O2 did not change the number of viable cells in the wild strain YPH250, but this parameter was decreased by 15% in the acatalsaemic strain YWT1. Cells treatment with 0.5 mM H2O2 for 30 min did not modify the levels of carbonyl proteins in the parental strain, but caused its 1.4-fold increase in the defective strain. The observed 1.5-fold activation of catalase in the wild strain cells in response to H2O2-stress suggests that under starvation conditions catalase can be involved in the yeast cell protection, particularly they can prevent oxidative modification of some antioxidant and associated enzymes.  相似文献   

17.
Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.  相似文献   

18.
Respiratory mutants of the facultative photosynthetic bacterium Rhodopseudomonas capsulata were used to investigate the mechanism of (reversible) inhibition of bacteriochlorophyll (BChl) synthesis by molecular oxygen. Although mutant strain M5 lacks cytochrome oxidase activity, it closely resembles the parental wild-type strain in respect to the effect of O(2) on BChl formation. This observation does not support an earlier hypothesis that O(2) regulates BChl synthesis through an effect on the redox state of a component of the respiratory electron transport system. Mutant strain M2 shows normal cytochrome oxidase activity, but lacks both reduced nicotinamide adenine dinucleotide and succinate dehydrogenase activities; relative to the parental strain, BChl synthesis in M2 is more sensitive to O(2) inhibition. The foregoing and results of related experiments can be accounted for by a revised interpretation of the O(2) effect, which proposes that O(2) directly inactivates a "factor" necessary for BChl formation and that, at relatively low O(2) tension, the inactivation can be reversed by a flow of electrons (derived from reduced nicotinamide adenine dinucleotide and succinate) diverted from a portion of the electron transport system delimited by the mutational blocks in M2 and M5.  相似文献   

19.
The microaerophilic nature of Campylobacter species implies an inherent sensitivity towards oxygen and its reduction products, particularly the superoxide anion. The deleterious effects of exposure to superoxide radicals are counteracted by the activity of superoxide dismutase (SOD). We have shown previously that Campylobacter coli possesses an iron cofactored SOD. The sodB gene of C. coli UA585 was insertionally inactivated by the site-specific insertion of a tetO cassette. Organisms harboring the inactivated gene failed to produce a biologically functional form of the enzyme. While the ability of this mutant to grow in aerobic conditions was unchanged relative to the parental strain, its survival was severely compromised when nongrowing cells were exposed to air. Accordingly, the SOD-deficient mutant was unable to survive for prolonged periods in model foods. Furthermore, inactivation of the sodB gene decreased the colonization potential in an experimental infection of 1-day-old chicks. In contrast, strain CK100, which is deficient in catalase activity, showed the same survival and colonization characteristics as the parental strain. These results indicate that SOD, but not catalase, is an important determinant in the ability of C. coli to survive aerobically and for optimal colonization within the chicken gut.  相似文献   

20.
Human diploid cell strains develop progressively higher levels of specific catalase activity as they grow. Following subculture activity falls again. A diploid cell strain heterozygous for the gene for acatalasia I (acatalasemia) was found to develop specific catalase activity at proportionately the same rate as normal cell strains. Yet the mutant gene reduced the absolute level of specific catalase activity which the culture attained at any given point in time. In this respect the heterozygous acatalasia I strain resembles the homozygous acatalasia II strain previously reported.  相似文献   

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