This review considers the distribution of the main enzymes of antioxidative defense, superoxide dismutase (SOD) and catalase, in various groups of strictly anaerobic microorganisms: bacteria of the genus Clostridium, Bacteroides, sulfate-reducing and acetogenic bacteria, methanogenic archaea, etc. Molecular and biochemical properties of purified Fe-containing SODs, cambialistic SODs, and heme catalases are presented. The physiological role and origin of the enzymes of antioxidative defense in strict anaerobes are discussed. Physiological responses (induction of SOD and catalase) to factors provoking oxidative stress in the cells of strict anaerobes able to maintain viability under aerobic conditions are also considered.

The hyperthermophile Thermotoga maritima, although strictly anaerobic, is able to grow in the presence of low amounts of O(2). Here, we show that this bacterium consumes O(2) via a three-partner chain involving an NADH oxidoreductase (NRO), a rubredoxin (Rd) and a flavo-diiron protein (FprA) (locus tags: TM_0754, TM_0659 and TM_0755, respectively). In vitro experiments showed that the NADH-dependent O(2) consumption rate was 881.9 (± 106.7) mol O(2) consumed min(-1) per mol of FprA at 37°C and that water was the main end-product of the reaction. We propose that this O(2) reduction chain plays a central role in the O(2) tolerance of T. maritima. Phylogenetic analyses suggest that the genes coding for these three components were acquired by an ancestor of Thermotogales from an ancestor of Thermococcales via a single gene transfer. This event likely also involved two ROS scavenging enzymes (neelaredoxin and rubrerythrin) that are encoded by genes clustered with those coding for FprA, NRO and Rd in the ancestor of Thermococcales. Such genomic organization would have provided the ancestor of Thermotogales with a complete set of enzymes dedicated to O(2)-toxicity defence. Beside Thermotogales and Thermococcales, horizontal gene transfers have played a major role in disseminating these enzymes within the hyperthermophilic anaerobic prokaryotic communities, allowing them to cope with fluctuating oxidative conditions that exist in situ.

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 mM. Hydrogen peroxide (H 2 O 2 ) inhibited growth at concentrations of 0?4-1?6 mM. Catalase activity in cell-free extracts of M. barkeri increased about threefold during H 2 O 2 stress (1?3 mM H 2 O 2 , 2-4 h exposure) and nearly twofold during superoxide stress (160 mM PQ, 2 h exposure). PQ (160 mM, 2-4 h exposure) and H 2 O 2 (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 H 2 O 2 -and PQ-exposed cultures, using labelled internal DNA fragments of the sod and kat genes. It was shown that H 2 O 2 but not PQ strongly induced up-regulation of the kat gene. PQ and to a lesser degree H 2 O 2 induced the expression of superoxide dismutase. The results indicate the regulation of the adaptive response of M. barkeri to different oxidative stresses.

Methanosarcina barkeri is a methanogenic archaeon that can only grow under strictly anoxic conditions but which can survive oxidative stress. We have recently reported that the organism contains a monofunctional catalase. We describe here that it also possesses an active iron superoxide dismutase. The enzyme was purified in three steps over 130-fold in a 14% yield to a specific activity of 1500 U/mg. SDS-PAGE revealed the presence of only one band, at an apparent molecular mass of 25 kDa. The primary structure determined from the cloned and sequenced gene revealed similarity to iron- and manganese superoxide dismutases. The highest similarity was to the iron superoxide dismutase from Methanobacterium thermoautotrophicum. The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase. The enzyme was not inhibited by cyanide (10 mM), which is a property shared by all iron- and manganese superoxide dismutases. The presence of superoxide dismutase in M. barkeri is noteworthy since a gene encoding superoxide dismutase (sod) has not been found in Archaeoglobus fulgidus, a sulfate-reducing archaeon most closely related to the Methanosarcinaceae.

Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity. This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri.Methanogens are anaerobic microorganisms that form methane as the end product of their energy metabolism. They all belong to the Archaea (34, 36). Five taxonomic orders of methanogens have been identified (4): Methanococcales, Methanobacteriales, Methanomicrobiales, Methanosarcinales, and Methanopyrales. Of these, the Methanosarcinales appear to have the most metabolic capabilities, as evidenced by a larger genome with twice as many open reading frames as other methanogens (see below), by the ability to utilize a broader spectrum of substrates (4), and by the possession of cytochromes (10,13,19) and of other heme-containing proteins such as catalase (30). Heme proteins appear to be lacking in members of the other orders (9,13,19,32) Given that only methanogens of the Methanosarcinales are capable of synthesizing heme, it was quite a surprise when Leadbetter and Breznak (20) reported that they had found catalase-like activity in Methanobrevibacter species that colonize the peripheral microoxic region of the hindgut of termites. The methanogens, which were shown to be relatively aerotolerant, reside on or near the hindgut epithelium. Catalase-like activity was also found in an authentic strain of Methanobrevibacter arboriphilus (formerly M. arboriphilicus) (1), indicating that the catalase-like activity was not restricted to the Methanobrevibacter species adapted to the hindgut of termites.One explanation for the findings in reference 20 could be that Methanobrevibacter species contain a nonheme catalase, such as the manganese catalase found in Thermus thermophilus (14) and lactic acid bacteria (12). This explanation was ruled out, however, by our finding, reported here, that catalase activity in M. arboriphilus strains was dependent on the presence of heme in the growth medium. We present evidence that M. arboriphilus contains a heme catalase which is formed from apoprotein synthesized by the methanogen and hemin taken up from the medium. MATERIALS AND METHODSOrganism, plasmid, phages, and chemicals. M. arboriphilus strains AZ (DSMZ 744) and DH1 (DSMZ 1125) were from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). The cloning vector ZAP Express, the helper phage ExAssist, Escherichia coli strains XL1 Blue-MRFЈ and XLOLR, and Pfu DNA polymerase were from Stratagene. Ampicillin, kanamycin, and 2,2Ј-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium ...

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