INTRODUCTION

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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 METHODS

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Organism, 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-MRF' and XLOLR, and Pfu DNA polymerase were from Stratagene. Ampicillin, kanamycin, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) were from Sigma. Synthetic oligonucleotides were obtained from MWG-Biotech. The digoxigenin labeling kit and the luminescence detection kit for nucleic acids were from Roche. All DNA-modifying enzymes were from United States Biochemicals. The Thermo Sequenase fluorescence-labeled primer cycle sequencing kit with 7-deaza-dGTP, the ALF Express DNA sequencer, fast protein liquid chromatography equipment and columns, and standard proteins for polyacrylamide gel electrophoresis (PAGE) and gel filtration columns were from Amersham Pharmacia Biotech.

Purification of catalases. M. arboriphilus strains AZ and DH1 were grown autotrophically with H₂ and CO₂ with shaking (130 rpm) at 37°C in a 2-liter bottle sealed with a butyl rubber stopper and a plastic screw cap containing 1 liter of medium as described previously (1). The gas phase was H₂-CO₂ (4:1) pressurized to 150 kPa. The cells were harvested in the late exponential phase at a cell concentration of 1 g (wet mass)/liter.

Molecular properties of catalase. Analytical gel filtration was performed by chromatography on a Superdex 200 (1- by 30-cm) column which was equilibrated with 100 mM potassium phosphate (pH 7.0). The column was calibrated by standard protein solution containing ferritin (440 kDa), catalase (bovine liver) (232 kDa), aldolase (158 kDa), ovalbumin (43 kDa), and RNase A (13.7 kDa). Native PAGE was performed using a gradient gel (4 to 15%) from Bio-Rad. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (bovine liver) (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa) were used for calibration of the gel. The molecular mass of the purified protein was determined by matrix-assisted laser desorption ionization-time of flight (mass spectrometry) (MALDI-TOF [MS]) using Voyager-DERD from Applied Biosystems. The N-terminal sequence of the protein was determined on a 477 A protein-peptide sequencer from Applied Biosystems.

Determination of enzyme specific activity. Catalase activity was routinely determined spectrophotometrically at 25°C by monitoring the decrease in absorbance at 240 nm (₂₄₀ = 39.4 M¹ cm¹) of 13 mM H₂O₂ in 50 mM Tris-HCl (pH 8.0) (2, 24). One unit is defined as the amount of enzyme that catalyzes the oxidation of 1 µmol of H₂O₂ min¹ under the assay conditions used here. Peroxidase activity was tested spectrophotometrically at 25°C in 50 mM Tris-HCl (pH 8.0) with 1.0 mM H₂O₂ and ABTS as substrates.

Generation of a hybridization probe for the kat gene. The oligonucleotides 5'-AATAAGAAGAAGTTTACTACTAATAAAGG (sense) and 5'-TCTCCTAGCATTGTGTAGTTTGTTCCTTT (antisense) were derived from the N-terminal amino acid sequence of purified catalase. The 25-µl PCR mixture contained 2.5 ng of genomic DNA of M. arboriphilus strain AZ, 1.3 U of Pfu DNA polymerase, 100 µM deoxynucleoside triphosphate, 2.0 mM MgCl₂, and 0.5 µM concentrations of each of the two primers. The temperature program was 5 min at 95°C, 30 cycles of 30 s at 94°C and 1 min at 40°C, and a final step of 5 min at 72°C. The PCR product was cloned into the pCR-Blunt vector using an Invitrogen Zero Blunt PCR cloning kit. The identity of the cloned fragment was determined by DNA sequencing. The following 26-base oligonucleotide probe was designed from the PCR product: 5'-GTAATGGATGCCTGATCATTAGGTAC. The oligonucleotide was subjected to 3' labeling with digoxigenin-dUTP following a protocol from Roche and then used for Southern hybridizations (29) of M. arboriphilus genomic DNA digested to completion with restriction endonucleases. The hybridization and washing conditions were 15 mM sodium citrate, pH 7.0, containing 150 mM NaCl and 50°C.

Cloning and sequencing of the kat gene. The DNA from M. arboriphilus strain AZ was partially digested with the restriction enzyme Sau3AI. The DNA fragments (6 to 10 kb) were purified, and a ZAP Express library was prepared as described previously (31). The 26-base probe for kat was used to screen the library. E. coli cells were infected with packaged ZAP DNA and plated onto Luria-Bertani agar (29). Positive clones were identified by plaque hybridization. Excision and recircularization of pBK-CMV and its insert generated plasmid clones. A DNA fragment cloned into pBK-CMV vector was sequenced using the dideoxynucleotide method with fluorescence-labeled primers by MWG AG Biotech.

Nucleotide sequence accession number. The nucleotide sequence of the kat gene from M. arboriphilus strain AZ is available under accession number AJ300838 in the EMBL database.

	RESULTS

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Cell extracts of M. arboriphilus strains AZ and DH1 grown in standard medium, which was supplemented with 0.1% yeast extract, exhibited weak catalase activity (Table 1). When hemin (30 µM) was added to the medium, the catalase activity was dramatically increased. In the case of a medium containing neither yeast extract nor hemin, no catalase activity was detected. The presence and absence of hemin in the growth medium did not have a significant effect on the growth rate and the cell concentration reached. The lag period after inoculation was, however, much more variable and generally longer when hemin was omitted. The catalase activity was associated with the soluble cell fraction. It was not sensitive to air. Therefore, the enzyme could be purified under oxic conditions.

Purification. The catalase was purified in five steps to 500-fold in a 30% yield (Table 2). From 160 mg of cell extract protein, 0.1 mg of purified enzyme with a specific activity of 120,000 U/mg was obtained. Sodium dodecyl sulfate (SDS)-PAGE of the purified enzyme revealed the presence of only one band at an apparent molecular mass of 60 kDa (Fig. 1).

View larger version (41K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of purified catalase from M. arboriphilus strain AZ. Protein was separated on a 12% polyacrylamide gel and subsequently stained with Coomassie brilliant blue. Lane 1, low-molecular-mass standards (Amersham Pharmacia Biotech); lane 2, 1 µg of purified catalase; lane 3, 2 µg of purified catalase.

Molecular properties. The purified catalase eluted from gel filtration columns (Superdex 200) with an apparent molecular mass of 260 kDa (data not shown). MALDI-TOF (MS) showed that the molecular mass of the monomer is 57.7 kDa. These data suggest that the catalase is a homotetrameric enzyme.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   UV/visible spectrum of catalase purified from M. arboriphilus strain AZ grown in the presence of hemin (30 µM). The solution was 30 µg of enzyme in 50 µl of 100 mM potassium phosphate, pH 7.0. The spectrum was recorded with a Zeiss Specord S10 diode array spectrophotometer; the light path was 0.3 cm.

Catalytic properties. The activity of the purified catalase was routinely assayed at a H₂O₂ concentration of 13 mM. When the concentration was increased, the specific activity increased from 120,000 U/mg to approximately 400,000 U/mg at 60 mM H₂O₂. The concentration dependence between 8 and 50 mM followed the Michaelis-Menten equation: a plot of 1/v versus 1/[S] was linear (v is the reaction rate; S is the subtrate) (data not shown). At H₂O₂ concentrations higher than 60 mM the rate no longer increased. Fifty percent of the maximal activity was observed at a H₂O₂ concentration of approximately 30 mM.

Catalase-encoding gene. From the N-terminal amino acid sequence of the purified catalase, two PCR primers were synthesized to obtain a homologous probe for the kat gene encoding the catalase. A Sau3AI gene bank from M. arboriphilus strain AZ was screened with the probe. Of 4,000 plaques, 14 were positive. A plasmid containing the gene with its flanking regions was obtained from one of the positive plaques and sequenced.

	DISCUSSION

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The results clearly indicate that M. arboriphilus strain AZ contains within its genome a kat gene which encodes a monofunctional heme catalase. Active catalase was, however, formed by the anaerobe only when hemin was present in the growth medium, indicating that M. arboriphilus cannot synthesize heme, which therefore has to be imported by the organism. The same results were obtained for M. arboriphilus strain DH1. Strain AZ has been isolated from digested sludge of an anaerobic sewage treatment plant near Zürich, Switzerland (38), and the type strain DH1 has been isolated from wet-wood cores of trees growing near Madison, Wis. (39). Hemin is predicted to be available at relatively high concentrations in both anaerobic habitats, since in anaerobic environments heme released upon degradation of heme-containing proteins is only very slowly metabolized: known pathways require molecular oxygen to initiate heme degradation (8). It is therefore very likely that both M. arboriphilus strains also synthesize active catalase in their natural environments despite the fact that they cannot synthesize heme.

The Methanobrevibacter strains RFM1, RFM2, and RFM3 isolated from the hindgut of termites are very close relatives of M. arboriphilus (20, 21). They were reported to exhibit catalase activity after growing on media predicted to contain heme from the additives yeast extract and rumen fluid. The catalase activity of strain RFM1 was determined to be 54 µmol of H₂O₂ decomposed per min per mg of cell extract protein (20). The specific activity was thus on the same order as that of cell extract of M. arboriphilus grown in the presence of low hemin concentrations (see Results).

This is not the first report on an organism capable of synthesizing heme catalase apoprotein but not heme. Examples include many lactic acid bacteria which, like M. arboriphilus, can grow in the absence of hemin but which form active catalase only when the growth medium is supplemented with hemin (11, 18). Haemophilus influenzae requires heme for growth and active catalase formation (3, 22), and so does Bacteroides fragilis (27). Lactic acid bacteria, H. influenzae, and B. fragilis have close relatives which can synthesize heme, indicating that this trait was lost in these organisms. The presence of a kat gene in their genome can thus be explained. Close relatives of Methanobrevibacter, however, cannot synthesize heme and do not contain heme proteins.

Sequence comparison reveals that the catalase sequence from M. arboriphilus is most similar to that of M. barkeri (Table 3). From codon usage differences, it has been deduced that the kat gene of M. barkeri is relatively new in this organism (30). A kat gene encoding a monofunctional heme catalase is not found in the genomes of Halobacterium species NRC1 (6, 25), Archaeoglobus fulgidus (16), Thermoplasma acidophilum (28), Aeropyrum pernix (15), and Sulfolobus solfataricus (http://www.cbr.nrc.ca/sulfhome/), which all can synthesize heme. Halobacterium species and A. fulgidus do contain bifunctional catalase-peroxidase enzymes (7, 16, 25), which are, however, phylogenetically not related to the monofunctional heme catalases (23). It thus appears that among the archaea the presence of heme catalase is restricted to Methanosarcina species and Methanobrevibacter species.

Where did the kat gene in Methanobrevibacter species come from? In rotten wet wood, these archaea live in close proximity to many other microorganisms, which may include cellular slime molds such as Dictyostelium. In the hindgut of termites, the close proximity of the methanogens to the epithelial cells of the insect and to the microorganisms of the intestinal flora would allow lateral gene transfer.