HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Brioukhanov, A. L. Articles by Eggen, R. I. L. Search for Related Content PubMed PubMed Citation Articles by Brioukhanov, A. L. Articles by Eggen, R. I. L. Agricola Articles by Brioukhanov, A. L. Articles by Eggen, R. I. L.

The catalase and superoxide dismutase genes are transcriptionally up-regulated upon oxidative stress in the strictly anaerobic archaeon Methanosarcina barkeri

¹ Department of Microbiology, Biological Faculty, Lomonosov Moscow State University, Vorob'evy Hills 1/12, 119992 Moscow, Russia
² Department of Environmental Toxicology, Swiss Federal Institute of Aquatic Science and Technology (EAWAG), Überlandstrasse 133, 8600 Dübendorf, Switzerland

Correspondence
Andrei L. Brioukhanov
brjuchanov{at}mail.ru

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Methanosarcina barkeri is a strictly anaerobic methanogenic archaeon, which can survive oxidative stress. The oxidative stress agent paraquat (PQ) suppressed growth of M. barkeri at concentrations of 50–200 µM. Hydrogen peroxide (H₂O₂) inhibited growth at concentrations of 0.4–1.6 mM. Catalase activity in cell-free extracts of M. barkeri increased about threefold during H₂O₂ stress (1.3 mM H₂O₂, 2–4 h exposure) and nearly twofold during superoxide stress (160 µM PQ, 2 h exposure). PQ (160 µM, 2–4 h exposure) and H₂O₂ (1.3 mM, 2 h exposure) also influenced superoxide dismutase activity in cell-free extracts of M. barkeri. Dot-blot analysis was performed on total RNA isolated from H₂O₂- and PQ-exposed cultures, using labelled internal DNA fragments of the sod and kat genes. It was shown that H₂O₂ but not PQ strongly induced up-regulation of the kat gene. PQ and to a lesser degree H₂O₂ induced the expression of superoxide dismutase. The results indicate the regulation of the adaptive response of M. barkeri to different oxidative stresses.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Methanosarcina barkeri belongs to the methanogenic archaea, very ancient micro-organisms which play a major role in the global production of methane as their end product of energy metabolism and represent the most important component in anaerobic degradation of organic compounds (Whitman et al., 1992). Methanogens live under extreme conditions of strict anaerobiosis in rice field soils, sludge sediments, swamped soils, plant xylem and the digestive tract of animals (Chin & Conrad, 1995). However, some species of methanogens can be relatively tolerant to oxygen exposure, surviving several hours in an oxygenated atmosphere during conditions of transient aerobiosis. Methanosarcina species can survive in oxygenated paddy soils, indicating that they are aerotolerant to some degree. Cells began to produce methane and to grow again as soon as anoxic conditions were re-established (Fetzer et al., 1993). Methanobrevibacter arboriphilus and Methanobacterium thermoautotrophicum remain viable upon exposure to air for up to 30 h and cells of Methanosarcina barkeri survive even longer because of the formation of cell flocs (Kiener & Leisinger, 1983). Some Methanogenium, Methanobacterium and Methanosarcina species were detected even in aerobic ecosystems such as surfaces of vegetables (Elstner, 1990) and desert soils (Peters & Conrad, 1995). Methanogens thus must have adaptive capacities to deal with the transient presence of oxygen and the concomitant oxidative stress. However, the mechanisms and regulation of oxidative stress responses in methanogenic archaea represent an almost completely unexplored area of research.

When strictly anaerobic micro-organisms come into contact with O₂, generally hydrogen peroxide (H₂O₂), superoxide radical () and hydroxyl radical (OH^.) are generated by the autoxidation of reduced iron–sulfur proteins and/or flavoproteins, catecholamines and quinones (Touati, 1997; Storz & Imlay, 1999). These reactive oxygen species are strong oxidants, which can destroy peptide bonds, and cause the depolymerization of nucleic acids and oxidation of polysaccharides and polyunsaturated fatty acids (Elstner, 1990; Fridovich, 1995) if not immediately removed by the main enzymes of antioxidative defence: superoxide dismutase and catalase. Anaerobic micro-organisms need defence systems to enable them to survive transient exposure to aerobic conditions or various pollutants in the environment.

Superoxide dismutase (SOD, EC 1.15.1.1) catalyses the disproportionation of 2 to O₂ and H₂O₂ and thus protects cells from free superoxide radicals, the products of univalent reduction of O₂. Generally, microbial species with high SOD activity have high or moderate aerotolerance in comparison to species with low or no SOD activity (Hewitt & Morris, 1975; Privalle & Gregory, 1979; Gregory & Dapper, 1980). Fe-containing SODs have been purified and characterized in the following methanogenic archaea: Methanobacterium bryantii (Kirby et al., 1981), Methanobacterium thermoautotrophicum (Takao et al., 1991), Methanosarcina barkeri (Brioukhanov et al., 2000) and Methanobrevibacter arboriphilus (Brioukhanov et al., 2006).

Catalase (EC 1.11.1.6) catalyses the conversion of 2 H₂O₂ to O₂ and H₂O, using H₂O₂ as the electron donor. Anaerobes that can endure only a short-term contact with O₂ do not need an obligatory catalase activity, unlike aerobic organisms, because H₂O₂ can be decomposed spontaneously or by non-enzymic mechanisms of defence (Hewitt & Morris, 1975; Fridovich, 1995). Nevertheless, catalase activity was found even in the cells of strictly anaerobic methanogenic archaea of the genus Methanobrevibacter (Leadbetter & Breznak, 1996). Haem-containing monofunctional catalases of Methanosarcina barkeri (Shima et al., 1999) and Methanobrevibacter arboriphilus (Shima et al., 2001) have been characterized, and the corresponding genes were cloned and sequenced.

Methanogenic archaea also contain additional enzymes of antioxidative defence as part of alternative oxidative stress response systems, such as the F₄₂₀ : H₂ oxidase of Methanobrevibacter arboriphilus, involved in O₂ detoxification (Seedorf et al., 2004), desulfoferrodoxin and rubrerythrin of Methanobacterium thermoautotrophicum (Smith et al., 1997), and desulfoferrodoxin of Methanococcus jannaschii (Bult et al., 1996); genes encoding these were found in the sequenced genomes.

In aerobic micro-organisms the physiological, biochemical and genetic responses to different oxidative stress conditions are complex and subtly regulated by two major transcriptional factors, OxyR and SoxRS, and intimately coupled to other regulatory networks in the cell; these responses have been quite well investigated to date (Lynch & Lin, 1996; Rosner & Storz, 1997; Storz & Imlay, 1999).

Significantly less is known about the molecular mechanisms and regulation of the oxidative stress responses that are required for the relative aerotolerance of strict anaerobic micro-organisms. Such responses have to be quite fast and fully induced, otherwise damage of the cell macromolecules by toxic oxygen derivatives will prevent the further expression of the antioxidative defence system. The oxidative stress responses in several species of strictly anaerobic bacteria have been studied in some detail. O₂ exposure leads to a strong increase of the specific activity of SOD in Bacteroides thetaiotaomicron (Pennington & Gregory, 1986). Aeration also induced the synthesis of SOD in the cells of Porphyromonas gingivalis, intensifying their virulence (Amano et al., 1992; Lynch & Kuramitsu, 1999). On treatment of Bacteroides fragilis with O₂, paraquat (PQ) or H₂O₂ an immediate de novo synthesis of more than 28 proteins starts, including catalase and SOD. The oxidative stresses induced by H₂O₂ or PQ are similar but not identical to the response induced by O₂ (Rocha & Smith, 1997; Rocha et al., 2003). These regulated and adaptive responses suggest the involvement of transcriptional regulators, one of which has been identified as OxyR (Rocha et al., 2003). An increase in the expression of the catalase of B. fragilis in response to aeration and to entry into the stationary growth phase is similar to adaptive response with involvement of hydroperoxidase II in Escherichia coli (Rocha & Smith, 1995, 1997; Rocha et al., 1996). Recently the genes involved in the adaptive response to oxidative stress in Clostridium perfringens were identified. Among them were the genes encoding SOD, catalase, alkyl hydroperoxide reductase and ATP-dependent RNA helicase (Briolat & Reysset, 2002; Jean et al., 2004).

The regulation of antioxidative defence system in anaerobes and the number of genes involved in oxidative stress response remain unclear, especially in the strictly anaerobic archaea. To the best of our knowledge there are no data in the literature on the regulation of catalase and SOD activities at the cellular and molecular levels in methanogenic archaea in response to transient oxic/anoxic conditions. In a first step towards the characterization of the oxidative stress response, and the regulation of expression of the main enzymes of antioxidative defence, we purified and characterized the haem-containing monofunctional catalase (Shima et al., 1999) and the iron SOD (Brioukhanov et al., 2000) from Methanosarcina barkeri. Subsequently, their encoding genes (kat and sod) were cloned and sequenced (Shima et al., 1999; Brioukhanov et al., 2000). In the present article we report on the regulation of catalase and SOD expression in M. barkeri under different oxidative stresses, at both the mRNA and protein (enzyme activity) level.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Strain and growth conditions.
Methanosarcina barkeri strain Fusaro (DSMZ 804) was from the Deutsche Sammlung von Mikroorganismen und Zellkulturen. It was grown anaerobically (Hungate, 1967) on 1.2 % (v/v) methanol at 37 °C in 250 ml bottles sealed with a butyl rubber stopper and an aluminium cap containing 100 ml medium as described previously (Karrasch et al., 1989). For induction of peroxide stress and PQ stress responses the cultures were treated with freshly prepared and filter-sterilized anaerobic solutions of H₂O₂ and PQ (methyl viologen dichloride hydrate, Fluka Chemie). Growth was followed by measuring OD₅₅₀ at regular intervals (1 cm path-length cuvette, Jasco V-550 spectrophotometer).

Preparation of cell-free extracts.
To measure specific activities of SOD and catalase, cell-free extracts of M. barkeri were made. Cells were harvested (9000 g, 4 °C, 20 min) in the late-exponential phase of growth and suspended in ice-cold 50 mM potassium phosphate buffer, pH 7.8. Cells were disrupted by ultrasonication, using a Branson Sonifier 450 (8x30 s with 2 min interim cooling in ice). Cell debris was removed by centrifugation for 20 min at 20 000 g and 4 °C, and the supernatant was stored at –20 °C.

SOD specific activity determination.
SOD activity was determined spectrophotometrically at 25 °C (1 cm cuvette, Jasco V-550 spectrophotometer) by the xanthine oxidase–cytochrome c method (McCord & Fridovich, 1969). The 0.7 ml assay mixture contained: 50 mM potassium phosphate, pH 7.8; 0.1 mM EDTA; 50 µM xanthine (sodium salt, Serva); 1.7 mU xanthine oxidase (Serva); 10 µM cytochrome c (Merck). The reduction of cytochrome c by , which was generated from O₂ by reduction with xanthine, was followed by measuring A₅₅₀. One unit (U) of SOD activity was defined as the amount of enzyme required to inhibit the reduction rate of cytochrome c by 50 % (McCord & Fridovich, 1969).

Catalase specific activity determination.
Catalase activity was determined spectrophotometrically at 25 °C (1 cm cuvette, Jasco V-550 spectrophotometer) by monitoring the decrease in A₂₄₀ (₂₄₀=39.4 M^–1 cm^–1) of 13 mM H₂O₂ in 50 mM Tris/HCl buffer, pH 8.0 (Beers & Sizer, 1952; Nelson & Kiesow, 1972). One unit (U) of activity was defined as the amount of enzyme that catalyses the oxidation of 1 µmol H₂O₂ min^–1 under the assay conditions.

Protein concentrations were determined by the method of Bradford (1976) using standard reagents (Bio-Rad) and bovine serum albumin as standard.

Gene amplification.
Probes for the sod and kat genes were obtained by PCR using as template genomic DNA from M. barkeri, isolated as described previously (Jarrell et al., 1992). The nucleotide sequences of the sod and kat genes from M. barkeri strain Fusaro are available under accession numbers AJ272498 and AJ005939, respectively, in the GenBank/EMBL/DDBJ database. The oligonucleotide primers 5'-AAACCCGGGATGGCCAAAGAATTGTACAA-3' (forward for sod), 5'-AAACTCGAGTTATTTCCTCATTTTCCTGAA-3' (reverse for sod), 5'-AAACCCGGGATGGGTGAAAAGAATTCTTC-3' (forward for kat) and 5'-AAACTCGAGTTATCTCTCAGTTGCTCGG-3' (reverse for kat) were derived from the nucleotide sequences of the corresponding genes (Microsynth). The corresponding primers were located on the both ends of the coding sequence of the whole gene. The 25 µl PCR mixture contained 2.5 ng genomic DNA of M. barkeri strain Fusaro, 1.3 U Taq DNA polymerase (Stratagene), 250 µM deoxynucleoside triphosphates, 1.5 mM MgCl₂ and 0.5 µM concentrations of each of the two primers. The temperature programme was 5 min at 95 °C, 30 cycles of 30 s at 94 °C, 1 min at 55 °C and 1 min at 72 °C, and a final step of 5 min at 72 °C. The PCR products were isolated from the agarose gel using the JetSorb Gel Extraction kit (Genomed) and suspended in 20 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). Probes for the sod and kat genes were cloned into pGEM-T Easy vector (Promega Catalys), using E. coli DH5 competent cells, and re-isolated by digestion with SmaI and XhoI restriction enzymes (Stratagene).

RNA isolation and dot-blot analysis.
Total RNA was isolated using the following procedure. Cultures were grown to late-exponential phase (OD₅₅₀ 1.5) and 2 ml aliquots were immediately centrifuged at 10 000 g for 10 min at 4 °C. After centrifugation, the cell pellet was suspended in 450 µl sterile AE buffer (30 mM sodium acetate pH 5.5, 10 mM Na₂EDTA and 1.5 %, w/v, SDS). Then immediate extraction with 450 µl TRIzol reagent (Life Technologies) was carried out at 65 °C for 5 min followed by centrifugation at 10 000 g for 10 min. The aqueous phase was precipitated with 250 µl cold 96 % ethanol. Then the RNeasy Mini kit (Qiagen) was used for the final isolation of RNA (steps 5–10 according to the RNeasy Mini protocol for isolation of total RNA from bacteria). The RNA pellet was dissolved in RNase-free water and stored at –70 °C. RNA samples with RNA loading dye (50 %, w/v, sucrose solution in 30 %, v/v, glycerol, 15 mg bromphenol blue ml^–1) were checked by electrophoresis (5–6 V cm^–1, 20 °C) in 1 % (w/v) agarose gel containing 1x TAE buffer (40 mM Tris/acetate pH 8.3, 1 mM EDTA). The concentration of total RNA was determined by measuring A₂₆₀.

RNA samples (10 µg) from the cultures exposed to different oxidative stress conditions were transferred to a nylon membrane (Qiagen) by capillary action in 10x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and cross-linked by UV irradiation (Sambrook et al., 1989). The probes (10 ng) used for dot-blot analysis were a 612 bp SmaI–XhoI DNA fragment of the sod gene and a 1518 bp SmaI–XhoI DNA fragment of the kat gene. Probes were denatured by boiling in a water bath for 10 min and labelled with DIG (Digoxigenin-11-dUTP) by random primer reaction with a DIG High Prime DNA labelling and detection starter kit (Roche Diagnostics). Hybridization, immunological detection of the hybridized probes with anti-digoxigenin-AP Fab fragments and then visualization with the chemiluminescence substrate CSPD were performed following the instruction manual from the kit. Autoradiographs were obtained by exposing the membranes to X-ray film (Eastman Kodak) for 30 min at 20 °C in the dark.

Chemicals.
Chemicals were from Fluka Chemie and of analytical grade.

Statistical analysis.
Initial growth inhibition experiments were replicated three times, while the stress treatments involving the enzyme activity measurements were replicated five times. The data were analysed by the SigmaPlot and SigmaStat programs (Systat Software). Dot-blot hybridization experiments were replicated twice. The densitometric quantification of the signals after dot-blot hybridization was done with the help of the ImageJ program (Wayne Rasband).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Determination of growth-inhibitory concentrations of PQ and H₂O₂
To investigate the influence of PQ as the source of superoxide radicals on the growth of Methanosarcina barkeri and determine the appropriate sublethal doses of PQ to be used for subsequent experiments, the following experiment was carried out. M. barkeri cultures were grown under standard conditions for 24 h, after which various amounts of PQ solution were added to the culture. Growth was not influenced by 25 µM PQ; between 50 and 100 µM PQ growth was retarded and at 200 µM PQ it was completely inhibited (Fig. 1a).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Effect of PQ (a) and H2O2 (b) on growth of M. barkeri.The data plotted are means±SD from three independent experiments.

Based on these results, subsequent exposure experiments were performed with 0.4 mM and 1.3 mM H₂O₂, and with 50 µM and 160 µM PQ, representing effective but non-lethal concentrations.

Effect of exposure to PQ and H₂O₂ on the specific activities of SOD and catalase
The specific activities of the enzymes of antioxidative defence in cell-free extracts of M. barkeri after treatment of cultures with PQ and H₂O₂ were measured as an initial investigation of the oxidative stress response. PQ was added to the culture medium at concentrations of 50 µM (25 % growth inhibition concentration) and 160 µM (80 % growth inhibition concentration) for 30 min, 2 h, 4 h and 8 h before the cultures were harvested for measurements. H₂O₂ stress was induced by adding 0.4 mM H₂O₂ (25 % growth inhibition concentration) and 1.3 mM H₂O₂ (80 % growth inhibition concentration), also for 30 min, 2 h, 4 h and 8 h before activity measurements.

Upon addition of PQ (final concentration 160 µM), the specific activity of SOD increased nearly twofold after 2 h exposure, and was about 1.5-fold higher than the control after 4 h exposure (Fig. 2a). The lower concentration (50 µM) of PQ had an insignificant effect on specific activity of SOD (0.5–8 h exposure). The specific activity of SOD increased already after 0.5 h exposure of cells to H₂O₂ at a final concentration of 1.3 mM and reached its maximum (nearly twofold increase) after 2 h exposure (Fig. 2b).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Activity of SOD in cell-free extracts of M. barkeri after addition of PQ (a) or H2O2 (b) to the culture. The data plotted are means±SD from five independent experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Activity of catalase in cell-free extracts of M. barkeri after addition of PQ (a) or H2O2 (b) to the culture. The data plotted are means±SD from five independent experiments.

PQ at a final concentration of 160 µM significantly induced the synthesis of sod mRNA. The maximum (fivefold) up-regulation of the sod gene was observed already after 0.5 h and 2 h exposure to PQ (Fig. 4a), which corresponds to the increase of specific activity of SOD (Fig. 2a). H₂O₂ at a final concentration of 1.3 mM also influenced the synthesis of sod mRNA after 2 h exposure (fourfold up-regulation), which resembled the increase in SOD specific activity (Fig. 2b). The addition of lower concentrations of the two oxidizing agents did not strongly induce the expression of the sod gene.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Dot-blot hybridization analysis of RNA from M. barkeri (late-exponential phase of growth) after exposure to PQ or H2O2. Dot-blots were probed with sod (a) and kat (b) genes. Numbers below the spots represent the integrated density as compared to the reference probes (sod and kat from anaerobic cultures: bottom panels).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Methanosarcina barkeri Fusaro is a very strictly anaerobic methanogen which needs a redox potential of less than –0.3 V for growth (Hungate, 1967). Methanosarcina species can, however, survive transient aerobiosis and remains viable in the presence of O₂ for at least 48 h through the formation of cell flocs (Zhilina, 1972; Kiener & Leisinger, 1983). We have shown here that M. barkeri can grow after addition of 0.8 mM H₂O₂ or of 100 µM PQ to the culture medium. M. barkeri has a SOD activity which is two times lower than the specific SOD activity in Methanobrevibacter arboriphilus (Brioukhanov & Netrusov, 2004), and M. barkeri is nearly five times more sensitive to PQ exposure compared to M. arboriphilus (Brioukhanov et al., 2006). This confirms the relationship between SOD activity in the cells and aerotolerance of the micro-organism.

It should be noted that the mechanism of PQ toxicity in anaerobic conditions still remains unclear. One explanation is that culture medium and PQ solution even after standard preparation according to the Hungate technique could contain traces of oxygen, enough to give rise to superoxide. Alternative explanations are that under anaerobic conditions PQ can cause the oxidation of formate, deoxyribose, etc. (Winterbourn & Sutton, 1984), or can react as a strong redox-cycling agent with different electron acceptors, traces of H₂O₂ or transition metals (Weidauer et al., 2002), which leads to accumulation of reactive oxygen species under anaerobiosis.

The specific activity of catalase in cell-free extracts of M. barkeri increased 2.5-fold after 2 h exposure to H₂O₂ (final concentration of 1.3 mM); this does not represent a very fast response, in comparison with the 6–8 h doubling time of M. barkeri. Lower concentration of H₂O₂ (0.4 mM) had almost no effect on the activities of the enzymes of antioxidative defence, indicating that small amounts of H₂O₂ are probably decomposed by constitutive catalase, by non-enzymic mechanisms of antioxidative defence or spontaneously. Generally, it should be noted that the increase of specific activities even under the most severe tested stress conditions (160 µM PQ, 1.3 mM H₂O₂) was not high (two-fold for SOD and three-fold for catalase) and the maximal activities decreased after 2–4 h exposure to PQ or H₂O₂.

It was shown earlier (Briukhanov et al., 2002) that the specific activity of SOD reached its maximum (twofold above the activity in the mid-exponential phase of growth) in the stationary phase during cultivation of M. barkeri on methanol under anaerobic conditions. Catalase activity practically did not change during the anaerobic growth on methanol (Briukhanov et al., 2002). Probably, the cells need to intensify the antioxidative defence not only under unfavourable oxic conditions, but also when cultures are approaching the stationary phase of growth (conditions of cell starvation with dormant metabolism), where the rate of cell death and the possibility of formation of superoxide radicals are high (Fridovich, 1995).

Similar effects of oxidative stresses on SOD and catalase have been described for other strict anaerobes. Aeration increased the specific activity of SOD and the level of corresponding mRNA two- and threefold in P. gingivalis (Amano et al., 1992; Lynch & Kuramitsu, 1999) and the SOD activity in B. thetaiotaomicron cells fourfold (Pennington & Gregory, 1986). Oxidative stress induced a tenfold increase in SOD activity (Abdollahi & Wimpenny, 1990) and catalase activity (Fareleira et al., 2003) in the sulfate-reducing bacterium Desulfovibrio desulfuricans. The increase of SOD activity, accompanied by the de novo synthesis of SOD, continued for 90 min after oxygen treatment of B. fragilis cells (Privalle & Gregory, 1979). Our data show that exposure of M. barkeri to H₂O₂ and PQ also causes de novo SOD synthesis (Fig. 4a). The level of katB mRNA increased more than 15-fold upon exposure to O₂, PQ or H₂O₂ in mid-exponential-phase cultures of B. fragilis (Rocha & Smith, 1997). In M. barkeri cells from the late-exponential phase of growth the level of kat mRNA increased almost 10-fold upon exposure to H₂O₂ (Fig. 4b).

The higher concentrations of PQ (160 µM) and H₂O₂ (1.3 mM) tested had statistically significant effects on the specific activities of SOD and catalase of M. barkeri. The same concentrations also induced the corresponding sod and kat genes. PQ (160 µM) and H₂O₂ (1.3 mM) showed similar effects on SOD specific activity. However, the specific activity of catalase was 1.5 times higher and the expression of the kat gene of M. barkeri was five times higher after exposure to 1.3 mM H₂O₂, which is the direct substrate for catalase, compared to 160 µM PQ in the culture medium. In the latter case the intracellular superoxide, produced by PQ, must be previously converted to H₂O₂. The PQ treatment had a more significant effect on catalase activity, as would be expected. The regulation of antioxidative defence system in the cells of M. barkeri may be more complex and PQ may strongly induce all components of the system, including catalase. It was also obvious that sublethal concentrations of PQ had a less strong effect on kat gene expression in comparison with sublethal concentrations of H₂O₂ (maximally 1.8-fold compared to 9.5-fold with H₂O₂), and PQ had a strong stimulating effect on transcriptional up-regulation of the sod gene (160 µM, 0.5 h exposure; Fig. 4a).

Here we have shown at the enzyme activity and transcriptional levels of SOD and catalase, the main enzymes of antioxidative defence, are up-regulated and induced in the cells of a strictly anaerobic methanogenic archaeon. These enzymes possibly play a significant role in the protection of M. barkeri against the toxic effects of reactive oxygen species ( and H₂O₂), providing the relatively high aerotolerance of this methanogen.

	ACKNOWLEDGEMENTS

 
The authors thank Ms Karin Rüfenacht and Mr Christoph Werlen for technical support and help in this research. This work was supported in part by INTAS grant YSF 03-55-1667 to A. L. B.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Abdollahi, H. & Wimpenny, J. W. T. (1990). Effects of oxygen on the growth of Desulfovibrio desulfuricans. J Gen Microbiol 136, 1025–1030.

Amano, A., Ishimoto, T., Tamagawa, H. & Shizukuishi, S. (1992). Role of superoxide dismutase in resistance of Porphyromonas gingivalis to killing by polymorphonuclear leukocytes. Infect Immun 60, 712–714.[Abstract/Free Full Text]

Beers, R. F. & Sizer, I. W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195, 133–140.[Free Full Text]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Briolat, V. & Reysset, G. (2002). Identification of the Clostridium perfringens genes, involved in the adaptive response to oxidative stress. J Bacteriol 184, 2333–2343.[Abstract/Free Full Text]

Brioukhanov, A. L. & Netrusov, A. I. (2004). Catalase and superoxide dismutase: distribution, properties and physiological role in cells of strict anaerobes. Biokhimiia 69, 949–962.

Brioukhanov, A., Netrusov, A., Sordel, M., Thauer, R. K. & Shima, S. (2000). Protection of Methanosarcina barkeri against oxidative stress: identification and characterization of an iron superoxide dismutase. Arch Microbiol 174, 213–216.[CrossRef][Medline]

Briukhanov, A. L., Thauer, R. K. & Netrusov, A. I. (2002). Catalase and superoxide dismutase in the cells of strictly anaerobic microorganisms. Mikrobiologiia 71, 330–335.[Medline]

Brioukhanov, A. L., Nesatyy, V. J. & Netrusov, A. I. (2006). Purification and characterization of a Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ. Biokhimiia 71, 546–553.

Bult, C. J., White, O., Olsen, G. J. & 20 other authors (1996). Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273, 1058–1073.[Abstract]

Chin, K. J. & Conrad, R. (1995). Intermediary metabolism in methanogenic paddy soil and the influence of temperature. FEMS Microbiol Ecol 18, 85–102.[CrossRef]

Elstner, E. F. (1990). Biochemie der Sauerstoffaktivierung und aktivierte Sauerstoffspezies. In Der Sauerstoff: Biochemie, Biologie, Medizin, pp. 5–55. Edited by E. F. Elstner. Mannheim, Germany: BI-Wissenschaftsverlag.

Fareleira, P., Santos, B. S., Antonio, C., Morades-Ferreira, P., LeGall, J., Xavier, A. V. & Santos, H. (2003). Response of a strict anaerobe to oxygen: survival strategies in Desulfovibrio gigas. Microbiology 149, 1513–1522.[Abstract/Free Full Text]

Fetzer, S., Bak, F. & Conrad, R. (1993). Sensitivity of methanogenic bacteria from paddy soil to oxygen and desiccation. FEMS Microbiol Ecol 12, 107–115.

Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annu Rev Biochem 64, 97–112.[CrossRef][Medline]

Gregory, E. M. & Dapper, C. H. (1980). Chemical and physical differentiation of superoxide dismutases in anaerobes. J Bacteriol 144, 967–974.[Abstract/Free Full Text]

Hewitt, J. & Morris, J. G. (1975). Superoxide dismutase in some obligately anaerobic bacteria. FEBS Lett 50, 315–318.[Medline]

Hungate, R. E. (1967). A roll tube method for cultivation of strict anaerobes. Methods Microbiol 2B, 117–132.

Jarrell, K. F., Faguy, D., Herbert, A. M. & Kalmokoff, M. L. (1992). A general method of isolating high molecular weight DNA from methanogenic archaea (archaeobacteria). Can J Microbiol 38, 65–68.[Medline]

Jean, D., Briolat, V. & Reysset, G. (2004). Oxidative stress response in Clostridium perfringens. Microbiology 150, 1649–1659.[Abstract/Free Full Text]

Karrasch, M., Bott, M. & Thauer, R. K. (1989). Carbonic anhydrase activity in acetate-grown Methanosarcina barkeri. Arch Microbiol 151, 137–142.[CrossRef]

Kiener, A. & Leisinger, T. (1983). Oxygen sensitivity of methanogenic bacteria. Syst Appl Microbiol 4, 305–312.

Kirby, T. W., Lancaster, J. R., Jr & Fridovich, I. (1981). Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii. Arch Biochem Biophys 210, 140–148.[CrossRef][Medline]

Leadbetter, J. R. & Breznak, J. A. (1996). Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp. nov., isolated from the hindgut of the termite Reticulitermes flavipes. Appl Environ Microbiol 62, 3620–3631.[Abstract]

Lynch, M. C. & Kuramitsu, H. K. (1999). Role of superoxide dismutase activity in the physiology of Porphyromonas gingivalis. Infect Immunol 67, 3367–3375.[Abstract/Free Full Text]

Lynch, A. S. & Lin, E. C. (1996). Responses to molecular oxygen. In Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology, 2nd edn, pp. 1526–1539. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.

McCord, J. M. & Fridovich, I. (1969). Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244, 6049–6055.[Abstract/Free Full Text]

Nelson, D. P. & Kiesow, L. A. (1972). Enthalpy of decomposition of hydrogen peroxide by catalase at 25 °C (with molar extinction coefficients of H₂O₂ solutions in the UV). Anal Biochem 49, 474–478.[CrossRef][Medline]

Pennington, C. D. & Gregory, E. M. (1986). Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron. J Bacteriol 166, 528–532.[Abstract/Free Full Text]

Peters, V. & Conrad, R. (1995). Methanogenic and other strictly anaerobic bacteria in desert soil and other oxic soils. Appl Environ Microbiol 61, 1673–1676.[Abstract]

Privalle, C. T. & Gregory, E. M. (1979). Superoxide dismutase and O₂ lethality in Bacteroides fragilis. J Bacteriol 138, 139–145.[Abstract/Free Full Text]

Rocha, E. R. & Smith, C. J. (1995). Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis. J Bacteriol 177, 3111–3119.[Abstract/Free Full Text]

Rocha, E. R. & Smith, C. J. (1997). Regulation of Bacteroides fragilis katB mRNA by oxidative stress and carbon limitation. J Bacteriol 179, 7033–7039.[Abstract/Free Full Text]

Rocha, E. R., Selby, T., Coleman, J. P. & Smith, C. J. (1996). Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide. J Bacteriol 178, 6895–6903.[Abstract/Free Full Text]

Rocha, E. R., Herren, C. D., Smalley, D. J. & Smith, C. J. (2003). The complex oxidative stress response of Bacteroides fragilis: the role of OxyR in control of gene expression. Anaerobe 9, 165–173.

Rosner, J. L. & Storz, G. (1997). Regulation of bacterial responses to oxidative stress. Curr Top Cell Regul 35, 163–177.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Seedorf, H., Dreisbach, A., Hedderich, R., Shima, S. & Thauer, R. K. (2004). F₄₂₀ : H₂ oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F₄₂₀-dependent enzyme involved in O₂ detoxification. Arch Microbiol 182, 126–137.[Medline]

Shima, S., Netrusov, A., Sordel, M., Wicke, M., Hartmann, G. C. & Thauer, R. K. (1999). Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri. Arch Microbiol 171, 317–323.[CrossRef][Medline]

Shima, S., Sordel-Klippert, M., Brioukhanov, A., Netrusov, A., Linder, D. & Thauer, R. K. (2001). Characterization of a heme-dependent catalase from Methanobrevibacter arboriphilus. Appl Environ Microbiol 67, 3041–3045.[Abstract/Free Full Text]

Smith, D. R., Doucette-Stamm, L. A., Deloughery, C. & 22 other authors (1997). Complete genome sequence of Methanobacterium thermoautotrophicum H: functional analysis and comparative genomics. J Bacteriol 179, 7135–7155.[Abstract/Free Full Text]

Storz, G. & Imlay, J. A. (1999). Oxidative stress. Curr Opin Microbiol 2, 188–194.[CrossRef][Medline]

Takao, M., Yasui, A. & Oikawa, A. (1991). Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum. J Biol Chem 266, 14151–14154.[Abstract/Free Full Text]

Touati, D. (1997). Superoxide dismutases in bacteria and pathogen protists. In Oxidative Stress and the Molecular Biology of Antioxidant Defenses, pp. 447–493. Edited by J. G. Scandalios. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Weidauer, E., Morke, W., Foth, H. & Bromme, H. J. (2002). Does the anaerobic formation of hydroxyl radicals by paraquat monocation radicals and hydrogen peroxide require the presence of transition metals? Arch Toxicol 76, 89–95.[CrossRef][Medline]

Whitman, W. B., Bowen, T. L. & Boone, D. R. (1992). The methanogenic bacteria. In The Prokaryotes, pp. 719–767, 2nd edn. Edited by A. Balows and others. New York: Springer.

Winterbourn, C. C. & Sutton, H. C. (1984). Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: catalytic requirements and oxygen dependence. Arch Biochem Biophys 235, 116–126.[CrossRef][Medline]

Zhilina, T. N. (1972). Death of Methanosarcina in the air. Mikrobiologiia 41, 1105–1106.[Medline]

Received 23 September 2005; revised 3 February 2006; accepted 16 February 2006.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Brioukhanov, A. L. Articles by Eggen, R. I. L. Search for Related Content PubMed PubMed Citation Articles by Brioukhanov, A. L. Articles by Eggen, R. I. L. Agricola Articles by Brioukhanov, A. L. Articles by Eggen, R. I. L.