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Applied and Environmental Microbiology, March 2002, p. 1173-1179, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1173-1179.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Differential Expression of Methanogenesis Genes of Methanothermobacter thermoautotrophicus (Formerly Methanobacterium thermoautotrophicum) in Pure Culture and in Cocultures with Fatty Acid-Oxidizing Syntrophs

Hong-Wei Luo, Hui Zhang, Toshihiko Suzuki, Satoshi Hattori, and Yoichi Kamagata^*

Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan

Received 18 July 2001/ Accepted 27 November 2001

Top Abstract Introduction Materials and Methods Results Discussion References

 
The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain TM, was investigated both in a pure culture sufficiently supplied with H₂ plus CO₂ and in a coculture with an acetate-oxidizing hydrogen-producing bacterium, Thermacetogenium phaeum strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the mcr gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas mcr and mrt, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (mtd and mth) for N⁵,N¹⁰-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (frh and mvh) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for Methanothermobacter thermoautotrophicus strain H cocultured with a thermophilic butyrate-oxidizing syntroph, Syntrophothermus lipocalidus strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of M. thermoautotrophicus strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Methanogens are the key microorganisms responsible for the final step of degradation of organic materials in the natural environment when alternate electron acceptors, such as sulfate or Fe(III), are limited. The substrates for methanogens are limited to a small number of small molecules, such as H₂-CO₂, formate, methanol, methylamines, and acetate. Most of the energy source is supplied by fermentative microbes that are capable of fermenting substrates such as carbohydrates to produce H₂ and acetate, both of which are subsequently converted to methane by methanogens.

It is well known that the oxidation of organic materials is strictly regulated by H₂ partial pressures. In particular, fatty acids such as acetate, propionate, and butyrate are very important intermediates in anaerobic processes and are known to be difficult substrates to degrade unless H₂ partial pressures are kept very low (25). In this respect, hydrogenotrophic methanogens play a crucial role in constantly eliminating H₂ and producing methane to make such substrate oxidation by proton-reducing, H₂-producing organisms energetically feasible. Hence, the syntrophic association between substrate oxidizers and H₂-scavenging methanogens is indispensable for sustaining the overall process of anaerobic degradation.

On the other hand, all research done so far on the genetics and biochemistry of methanogens has been based on pure cultures grown under high H₂ partial pressures, such as 10⁵ Pa, to yield sufficient amounts of cells for further study. Considering the fact that very limited amounts of H₂ are constantly available in the natural environment, there should be significant physiological differences between methanogens that are exposed to large amounts of H₂ and methanogens that are supplied far less H₂ in association with substrate-oxidizing H₂-producing syntrophic microorganisms.

Of the hydrogenotrophic methanogens, Methanothermobacter thermoautotrophicus (formerly Methanobacterium thermoautotrophicum) is the most extensively studied methanogen for the biochemistry and genetics of methanogenesis (4, 6, 17, 23, 29, 31, 32). Recent complete genome sequencing of this member of the Archaea is giving further insight into the gene arrangement of all machinery of methane formation and carbon assimilation and is permitting the study of comparative genomics between this organism and the other members of the Archaea. Before the complete genome analysis, several researchers reported that M. thermoautotrophicus harbors two isofunctional formylmethanofuran dehydrogenases (FWD and FMD, encoded by fwdHFGDACB and fmdECB, respectively) (10, 11), F₄₂₀-dependent and H₂-dependent N⁵,N¹⁰-methylene-tetrahydromethanopterin (H₄MPT) dehydrogenases (MTD and MTH, encoded by mtd and mth, respectively) (16, 33), and methyl coenzyme M reductases (MRI and MRII, encoded by mcrBDCGA and mrtBDGA, respectively), which are the enzymes responsible for the first, fourth, and final steps of methanogenesis from H₂-CO₂, respectively. In addition, two types of hydrogenases were found to be present in M. thermoautotrophicus strains (2, 7, 22, 30). These findings raised intriguing questions as to why such isofunctional gene sets are present on its compact genome and how methanogens regulate the expression of those genes. The gene expression of MRI and MRII was found to be dependent on growth phase, gassing rate (H₂ in the input gas or mixing speed of the fermentor impeller), medium constituents, pH, and temperature (15, 18, 19, 21). However, there have been no investigations on gene expression under syntrophic conditions in which H₂ partial pressures are kept extremely low.

Very recently, two thermophilic syntrophic microorganisms, Thermacetogenium phaeum (8) and Syntrophothermus lipocalidus (27), were isolated. The former is an acetate-oxidizing bacterium, and the latter is a butyrate-oxidizing bacterium. Both require an H₂-scavenging methanogenic partner to grow on the respective substrate. These cultures allowed us to study the expression of methanogenesis genes under naturally occurring H₂ partial pressures. In this report, we compare the expression of genes involved in methanogenesis under syntrophic conditions to that in pure cultures in the presence of much higher concentrations of H₂.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Source of microorganisms.
M. thermoautotrophicus (formerly M. thermoautotrophicum) H (DSM1053) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). A pure culture of a thermophilic H₂- and formate-utilizing methanogen, M. thermoautotrophicus TM, and a thermophilic syntrophic acetate-oxidizing bacterium, T. phaeum strain PB^T, in coculture with M. thermoautotrophicus TM were from our culture collection (8). A thermophilic syntrophic butyrate-oxidizing bacterium, S. lipocalidus strain TGB-C1^T (27), in coculture with M. thermoautotrophicus H was a gift from Yuji Sekiguchi.

Media and culture conditions.
M. thermoautotrophicus strain H and strain TM were grown in a minimal salts medium which contained the following (liter^-1): 1.0 g of NH₄Cl, 0.3 g of KH₂PO₄, 0.6 g of NaCl, 0.1 g of MgCl₂ · 2H₂O, 0.08 g of CaCl₂ · 2H₂O, 3.5 g of KHCO₃, 0.001 g of sodium resazurin, 10 ml of vitamin solution, 5 ml of trace element solution, 0.3 g of Na₂S · 9H₂O, and 0.3 g of cysteine-HCl. The composition of the vitamin solution was as follows (liter^-1): 2.0 mg of biotin, 2.0 mg of folic acid, 10.0 mg of pyridoxine-HCl, 5.0 mg of thiamine-HCl, 5.0 mg of riboflavin, 5.0 mg of nicotinic acid, 5.0 mg of DL-calcium pantothenate, 0.1 mg of vitamin B₁₂, 5.0 mg of p-aminobenzoate, and 5.0 mg of lipoic acid. The trace element solution contained the following (liter^-1): 12.8 g of nitrilotriacetic acid, 1.35 g of FeCl₃ · 6H₂O, 0.1 g of MnCl₂ · 4H₂O, 0.024 g of CoCl₂ · 6H₂O, 0.1 g of CaCl₂ · 2H₂O, 0.1 g of ZnCl₂, 0.025 g of CuCl₂ · 2H₂O, 0.01 g of H₃BO₃, 0.024 g of Na₂MoO₄ · 4H₂O, 1.0 g of NaCl, 0.12 g of NiCl₂ · 6H₂O, 4.0 mg of Na₂SeO₃ · 5H₂O, and 4.0 mg of Na₂WO₄ · 2H₂O. The pH of the trace element solution was adjusted to 6.5. All cultivations were carried out at 55°C without agitation in a 1.3-liter serum bottle containing 500 ml of medium under an atmosphere of H₂-CO₂ (80:20 [vol/vol]).

T. phaeum PB, a thermophilic acetate-oxidizing syntroph, was precultivated in pure culture by using pyruvate as previously described (8). For the growth of the syntrophic acetate-oxidizing coculture, T. phaeum PB and M. thermoautotrophicus TM were grown with the same medium as that described above, except that 80 mM sodium acetate was added and the headspace was N₂-CO₂ (80:20 [vol/vol]).

The culture medium used for the syntrophic butyrate-oxidizing coculture (S. lipocalidus TGB-C1 and M. thermoautotrophicus H) was prepared as described previously (27). It contained the following (liter^-1): 0.535 g of NH₄Cl, 0.136 g of KH₂PO₄, 0.174 g of K₂HPO₄, 0.102 g of MgCl₂ · 6H₂O, 0.0441 g of CaCl₂ · 2H₂O, 0.002 g of FeCl₂, 1 ml of trace element solution, 5 ml of vitamin solution, 0.001 g of sodium resazurin, 2.52 g of NaHCO₃, 0.3 g of Na₂S · 9H₂O, and 0.3 g of cysteine-HCl. The components of the vitamin solution and the trace element solution were the same as those used in the minimal salts medium. Sodium butyrate (20 mM) was added after autoclaving. The gas phase for the coculture was N₂-CO₂ (80:20 [vol/vol]).

Measurements of methane, hydrogen, and substrates.
The methane formed in the headspace was determined by using a gas chromatograph (Shimadzu model GC-8AIT) with a molecular sieve 60/80-mesh column and a thermal conductivity detector. Argon was used as the carrier gas. Hydrogen was determined by using a gas chromatograph (model GS-15; Sensortech Co., Ltd., Shiga, Japan) with a molecular sieve 13x 60/80-mesh column and a semiconductor detector. Air was used as the carrier gas. Acetate, butyrate, and fermentation products were measured by using a high-performance liquid chromatograph (Shimadzu model LC-6A) fitted with an ion exclusion column (Shimadzu SCR-101) and a UV detector (Shimadzu model SPD-6A). The column was operated at 40°C, and 17 mM HClO₄ was used as the carrier gas at a flow rate of 1 ml min^-1.

Probe design for Northern analysis.
We made probes for Northern analysis to target three sets of isofunctional genes involved in methanogenesis: those for methyl coenzyme M reductases (mcr and mrt), N⁵,N¹⁰-methylene-H₄MPT dehydrogenases (mth and mtd), and hydrogenases (frh and mvh). These probes were made by amplifying the partial sequences of the target genes by using the primer sets shown in Fig. 1. PCR products were labeled with a digoxigenin DNA labeling kit (Roche Molecular Biochemicals).

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FIG. 1. Methanogenesis from H2 and CO2 and primer sets for making probes to detect target genes by Northern analysis. Isoenzymes and functionally equivalent enzymes are shown in bold. Coenzymes: MF, methanofuran; H4MPT, 5,6,7,8-tetrahydromethanopterin; CoM-SH, coenzyme M; HTP-SH, N-7-mercaptoheptanoyl-L-threonine phosphate; CoM-S-S-HTP, heterodisulfide of CoM-SH and HTP-SH. Enzymes: MVH, methyl viologen-reducing hydrogenase; FRH, F420-reducing hydrogenase; MTD, F420-dependent methylene-H4MPT dehydrogenase; MTH, hydrogen-dependent methylene-H4MPT dehydrogenase; MCR and MRT, methyl coenzyme M reductases; FWD, W-containing formylmethanofuran dehydrogenase; FMD, Mo-containing formylmethanofuran dehydrogenase.

RNA isolation and Northern analysis.
We isolated total RNAs from cells of M. thermoautotrophicus TM grown under different conditions: H₂-CO₂-grown cells and cells grown syntrophically with an acetate oxidizer, T. phaeum, on acetate. We also isolated RNAs from M. thermoautotrophicus H cells grown syntrophically with a butyrate oxidizer, S. lipocalidus. For the cocultures, we directly extracted RNAs without separation of methanogen cells from syntroph cells.

Triplicate or quadruplicate bottles (1.3 liters) containing 500 ml of medium were prepared for each of three different cultivations (M. thermoautotrophicus TM, M. thermoautotrophicus TM plus T. phaeum, and M. thermoautotrophicus H plus S. lipocalidus), and one bottle of those cultures was sacrificed at an appropriate interval of incubation for RNA extraction and subsequent Northern analysis. The experiments were repeated at least twice to verify reproducibility.

For RNA extraction, 500 ml of culture was transferred from the serum bottle to a precooled bucket-type tube, and the cells were harvested immediately by centrifugation at 11,000 x g and 4°C for 15 min (Beckman AvantiTM HP-25I). After the cells were washed with ice-cold 0.01 M sodium phosphate buffer (pH 7.2), they were resuspended in 3.5 ml of the same buffer and passed through a French pressure cell at 20,000 lb/in² (SLM-Aminco FA-003 minicell). The resultant lysate was collected directly in a 50-ml polypropylene tube containing 10.5 ml of Isogen-LS (Nippongene, Tokyo, Japan) and vortexed briefly. After the addition of 2.8 ml of chloroform, the mixture was shaken vigorously for 15 s and centrifuged at 22,000 x g and 4°C for 15 min. The aqueous phase containing RNAs was transferred to another tube, and the nucleic acids were precipitated with isopropanol. Concentrations of nucleic acids were determined by using a Beckman DU 640 spectrophotometer at an absorbance of 260 nm.

For Northern blotting, RNA samples were denatured by incubation for 15 min at 65°C in 20 mM sodium 3-(N-morpholino)-propanesulfonic acid (MOPS; Nacalai Tesque Inc., Kyoto, Japan) containing 6.0% (vol/vol) formaldehyde and 50% (vol/vol) formamide and then separated by electrophoresis on an agarose-formaldehyde (1%:6.0% [vol/vol] gel. RNAs were transferred to a Hybond-N⁺ positively charged nylon membrane (Amersham). RNAs on the membrane were fixed with UV Stratalinker 1800 (Stratagene). Before addition of the DNA probes, the membrane was incubated for at least 1 h in hybridization buffer at 42°C. Hybridization buffer contained 5x SSPE (0.75 M NaCl, 43.25 mM NaH₂PO₄, 6.25 mM EDTA), 50% formamide, 5x Denhardt's solution (Wako, Osaka, Japan), 0.5% sodium dodecyl sulfate (SDS), and 1 mg of denatured fragmented salmon sperm DNA ml^-1. The DNA probe was denatured at 100°C for 10 min and added to a hybridization tube immediately. Hybridization was allowed to proceed for 16 h. The membrane was washed twice in 2x SSC buffer (33.3 mM NaCl, 33.3 mM sodium citrate) containing 0.1% SDS at room temperature for 5 min, twice in 1x SSC buffer (16.65 mM NaCl, 16.65 mM sodium citrate) containing 0.1% SDS at 60°C for 20 min, and twice in 0.5x SSC buffer (8.325 mM NaCl, 8.325 mM sodium citrate) containing 0.1% SDS at 60°C for 20 min. The signals of the mRNAs were detected by using a digoxigenin nucleic acid detection kit (Roche) according to the manufacturer's directions.

Fractionation of methanogen cells and syntroph cells by Percoll gradient centrifugation for two-dimensional (2-D) gel electrophoresis of whole proteins.
Fractionation of methanogen cells and syntroph cells was performed according to previously described methods with some modifications (3, 24). Nine parts (volume/volume) of Percoll (Pharmacia) were added to 1 part of 2.5 M sucrose to give a final density of about 340 mosmol/kg of H₂O. The solution was placed in an anaerobic chamber (H₂-N₂-CO₂, 10:10:80 [vol/vol]; MACS Anaerobic Workstation, Don Whitley Scientific Ltd., West Yorkshire, United Kingdom) overnight to remove dissolved oxygen. The cells of the syntrophic acetate-oxidizing coculture (T. phaeum PB plus M. thermoautotrophicus TM) were harvested by centrifugation at 11,000 x g and 4°C for 10 min, washed with 10 mM Tris-HCl buffer (pH 7.6), and resuspended in 0.5 ml of the same buffer. The cell suspension was added to the 25.5-ml Percoll-sucrose solution inside the anaerobic chamber. The gradient was self-generated by centrifugation at 45,000 x g with colored marker beads (Pharmacia) to determine the buoyant density, and 3 h of centrifugation (Beckman L-70 ultracentrifuge; type 70 Ti rotor) gave two distinct bands with 0.8 cm of distance between them (buoyant densities, 1.096 and 1.107 g/ml). Epifluorescence microscopy revealed that the lower band was comprised mostly of M. thermoautotrophicus TM cells, with a few flocs being formed by T. phaeum PB and M. thermoautotrophicus TM. This fraction was carefully taken with a syringe, and Percoll particles were removed by washing with the same buffer as that used above.

2-D gel electrophoresis of whole proteins.
2-D gel electrophoresis of whole proteins was performed by using M. thermoautotrophicus TM cells (pure culture) and M. thermoautotrophicus TM cells fractionated from the acetate-oxidizing coculture by Percoll gradient centrifugation as described above. The cells were suspended in 3.5 ml of 10 mM Tris-HCl buffer (pH 7.6) and ruptured by passage twice through a precooled French pressure cell at 20,000 lb/in². The resultant lysate was centrifuged at 35,000 x g for 20 min, and the supernatant was collected.

Whole proteins of M. thermoautotrophicus TM were separated with a 2-D gel electrophoresis system (Pharmacia) according to the manufacturer's directions. Immobiline DryStrip pH 3 to 10 (Pharmacia) was used for the first dimension of isoelectric focusing. Approximately 180 µg of protein sample was loaded on the Immobiline DryStrip. Second-dimension separation was performed with ExcelGel XL SDS12-14 polyacrylamide gels (Pharmacia). The SDS gels were visualized by Coomassie brilliant blue R-350 staining. We performed 2-D electrophoresis seven times by using different batches of pure cultures and cocultures, and the protein patterns were highly reproducible.

Determination of amino-terminal amino acid sequences.
Protein spots that appeared on the 2-D SDS gels were transferred to an Immobilon P^SQ membrane (Millipore Corporation, Bedford, Mass.) according to the manufacturer's directions. Electrotransfer was performed at 20 V for 40 min with a semidry Trans-Blot Cell (Bio-Rad Laboratories). The transfer buffer contained 48 mM Tris, 39 mM glycine, 20% methanol, and 0.0375% SDS. The spots were excised from the membrane, and the N-terminal amino acid sequence was determined by using a G1005A protein sequencing system (Hewlett-Packard). A homology search for the N-terminal amino acid sequence was carried out with the M. thermoautotrophicus H genome sequence project (http://www.biosci.ohio.-state.edu/genomes/) and National Center for Biotechnology Information database by using the BLAST program.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Changes in hydrogen concentrations during methanogenesis by M. thermoautotrophicus in pure culture and in coculture with an acetate oxidizer or a butyrate oxidizer.
M. thermoautotrophicus TM was grown in pure culture with H₂ plus CO₂ as sole carbon and energy sources. It grew rapidly, and methane production reached 7 mmol (in the headspace) after 6 days of incubation (Fig. 2). In a syntrophic coculture of M. thermoautotrophicus TM and T. phaeum PB grown on acetate, acetate was stoichiometrically converted to methane. The mean generation time was calculated to be approximately 3 days, and it took over 2 weeks (Fig. 3) to produce 7 mmol of methane, a quantity that M. thermoautotrophicus in pure culture was able to attain in 6 days. The reason was probably that the oxidation of acetate to H₂-CO₂ by T. phaeum PB was rate limiting. The apparent H₂ concentration reached a maximum of 3.45 µmol in the headspace (82 Pa) in the early stage of acetate oxidation and methanogenesis and then declined. We also found a difference in the morphology of M. thermoautotrophicus cells between pure culture and coculture. The cells in pure culture were long bent rods more than 10 µm long, whereas the cells in coculture were short straight rods less than 6 µm long.

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FIG. 2. H2 concentration (open squares) and methane production (solid squares) by M. thermoautotrophicus TM in a 1.3-liter serum bottle containing 500 ml of medium. Arrows indicate the sampling points for RNA preparation; 28.3, 37.6, and 46.8 mg of dry cells (arrows 1, 2, and 3, respectively) were harvested and used for total RNA extraction and subsequent Northern analysis. Error bars indicate standard deviations.

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FIG. 3. Time course of acetate degradation (solid circles), methane production (solid squares), and H2 concentration (open squares) in a syntrophic acetate-oxidizing coculture (M. thermoautotrophicus TM plus T. phaeum PB) grown with 80 mM acetate in a 1.3-liter serum bottle containing 500 ml of medium. Arrows indicate the sampling points for RNA preparation; 13.1 and 65.7 mg of dry cells (arrows 1 and 2, respectively) were harvested and used for total RNA extraction and subsequent Northern analysis. Error bars indicate standard deviations.

Results similar to those obtained with the acetate-oxidizing coculture were obtained with a butyrate-oxidizing coculture (M. thermoautotrophicus H and S. lipocalidus TGB-C1), although the maximum H₂ concentration (5.45 µmol in the headspace [136 Pa]) was slightly higher and the growth was faster than those obtained with the acetate-oxidizing coculture (data not shown).

Transcription of three sets of isofunctional genes responsible for methanogenesis by M. thermoautotrophicus in pure culture and in cocultures.
The expression of three sets of isofunctional genes, methyl coenzyme M reductase genes (mcrBDCGA and mrtBDGA), N⁵,N¹⁰-methylene-H₄MPT dehydrogenase genes (mtd and mth), and hydrogenase genes (frhADGB and mvhDGAB), was analyzed by using Northern blot hybridization. The pure culture and a coculture of M. thermoautotrophicus TM with an acetate-oxidizing syntroph, T. phaeum, at different growth stages (Fig. 2 and 3) were used for the preparation of total RNA. As shown in Fig. 4A, the mcrBDCGA (5.0-kb) and mrtBDGA (4.3-kb) genes were transcribed as a single transcriptional unit into polycistronic mRNA. In the pure culture, transcripts of both mcrBDCGA and mrtBDGA were detected in the early to middle stages of cultivation (Fig. 2), but they were no longer detected in the late stage of growth (Fig. 2). In contrast, in the coculture with the acetate oxidizer, only mcrBDCGA was preferentially expressed, and expression remained at the same levels in the early to late stages of growth (Fig. 3 and 4B).

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FIG. 4. Comparison of expression levels for three isofunctional gene sets responsible for methanogenesis in M. thermoautotrophicus strain TM. (A) Strain TM in pure culture. (B) Strain TM in syntrophic coculture. Aliquots (20 µg) of RNA preparations were used for Northern blot analysis. The lane numbers indicate the sampling points, which correspond to the numbers in Fig. 2 and 3.

Transcripts of two N⁵,N¹⁰-methylene-H₄MPT dehydrogenase genes (mtd, an F₄₂₀-dependent N⁵,N¹⁰-methylene-H₄MPT dehydrogenase gene, and mth, an H₂-dependent N⁵,N¹⁰-methylene-H₄MPT dehydrogenase gene) were detected in M. thermoautotrophicus cells in pure culture at the early to middle stages of cultivation, but the transcript of mtd was no longer detected at the late stage of cultivation (Fig. 4A). In contrast, the two genes were transcribed at the same expression levels in both the early and the late stages of growth in coculture with T. phaeum (Fig. 4B). Two NiFe hydrogenase-encoding genes (frhADGB, an F₄₂₀-reducing hydrogenase gene, and mvhDGAB, a methyl viologen-reducing hydrogenase gene) were also found to be transcribed in a manner similar to that of mtd in pure culture and coculture (Fig. 4).

We also attempted to determine the levels of transcription of the three sets of isofunctional genes in M. thermoautotrophicus H, a well-studied methanogen, in coculture with T. phaeum for comparison with previous reports on the expression of methane genes with this strain. The growth of the coculture was, however, too slow and unstable to perform Northern analysis, probably because the syntrophic oxidation of acetate may involve formate as an intermediate but M. thermoautotrophicus H lacks the ability to metabolize formate while M. thermoautotrophicus TM is able to use it (9).

We therefore determined the levels of transcription of the gene sets by using M. thermoautotrophicus H in coculture with S. lipocalidus, a thermophilic butyrate-oxidizing H₂-producing syntroph. Experiments were carried out in exactly the same way as for the coculture with T. phaeum by harvesting cells at two growth stages. Coincident with the gene expression pattern for M. thermoautotrophicus TM in coculture with T. phaeum, mcr, mrt, mtd, mth, frh, and mvh in M. thermoautotrophicus H cells in coculture were found to be transcribed at the same expression levels in the early to late stages of growth (data not shown).

2-D electrophoretic analysis of the protein expression patterns for M. thermoautotrophicus TM in pure culture and in syntrophic coculture.
Cell extracts of M. thermoautotrophicus TM prepared from pure culture and coculture with T. phaeum were subjected to 2-D SDS gel electrophoresis. The protein patterns were clearly different between pure culture cells and coculture cells (Fig. 5). Ten major spots were extracted from the membrane for determination of N-terminal amino acid sequences. All of the sequences were retrievable from the M. thermoautotrophicus genome sequence database and could be identified as the enzymes involved in methane production. The protein expression levels for two spots, identified as N⁵-methyl-H₄MPT:HS-CoM methyltransferase H (mtrH) and N⁵,N¹⁰-methylene-H₄MPT reductase (mer), were not significantly different between pure culture cells and syntrophically grown cells, whereas the expression levels for the methyl viologen-reducing hydrogenase gamma subunit (mvhG) and heterodisulfide reductase B(hdrB) in pure culture cells were slightly higher than those in syntrophically grown cells. The levels of synthesis of the MRI , ß, and subunits of M. thermoautotrophicus TM in syntrophically grown cells were found to be significantly higher than those in pure culture cells. These data suggested that the intracellular amounts of the MRI and MRII isoenzymes are strongly influenced by culture conditions.

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FIG. 5. Comparison of protein expression patterns determined for M. thermoautotrophicus TM in pure culture (A) and syntrophic coculture (B) by 2-DE analysis. Gels were stained with Coomassie brilliant blue R-350. The molecular masses of the markers are shown on the right. The pH gradient on the gels was from 10 (left) to 3 (right). The arrows indicate the proteins for which N-terminal amino acid sequences were determined. Each spot was identified as follows: 1, methyl-H4MPT:HS-CoM methyltransferase subunit H (MtrH); 2, F420-dependent N5,N10-methylene-H4MPT reductase (Mer); 3, MRI ; 4, MRI ß; 5 and 9, MRI ; 6, MRII ; 7, methyl viologen-reducing hydrogenase gamma subunit (MVH ); 8, heterodisulfide reductase chain B (HDR); 10, MRII .

Top Abstract Introduction Materials and Methods Results Discussion References

 
To investigate the differential expression of methanogenesis genes, we used M. thermoautotrophicus as an H₂-scavenging partner for syntrophic oxidation of acetate and butyrate. Acetate oxidation to H₂-CO₂ (CH₃COO^- + 4H₂O 2HCO₃^- + 4H₂ + H⁺) is extremely endoergonic (+94.9 kJ/mol) under standard conditions. The reaction can occur only when the H₂ partial pressure is kept low enough by coupling with an H₂-consuming reaction. The apparent H₂ partial pressures observed in our coculture (T. phaeum plus M. thermoautotrophicus TM) were less than 82 Pa during active acetate oxidation and subsequent methanogenesis (Fig. 3), similar to the results for a previously described acetate-oxidizing coculture (13). This coculture allowed us to investigate the differential expression of methanogenesis genes in M. thermoautotrophicus in comparison with the pure culture of the same methanogenic strain grown with high H₂ concentrations.

Northern blot analysis showed that of the two methyl coenzyme M reductase genes, only the MRI gene was transcribed in coculture, whereas both the MRI and the MRII genes were transcribed in pure culture. In contrast to the results obtained for these two genes, two isofunctional genes, for N⁵,N¹⁰-methylene-H₄MPT dehydrogenases (MTD and MTH), and two different genes, for hydrogenases (FRH and MVH), were found to be expressed in both pure culture and coculture in the early to late stages of cultivation.

There are several reports on the effect of H₂ supply on the expression of MRI, MRII, MTD, MTH, and other related proteins. Bonacker et al. reported that a continuous supply of H₂-CO₂ at 90:10, 80:20, and 70:30 (vol/vol) gave essentially the same results for Methanothermobacter marburgensis; i.e., MRII was preferentially expressed in the exponential phase and MRI was expressed in the linear growth phase at 65°C (5). Morgan et al. found that an excess supply of H₂ for M. thermoautotrophicus H cells resulted in the preferential expression of the MRII and MTH genes, whereas under H₂-limited conditions directed by several means that allowed only very limited growth, the MRI and MTD genes were transcribed (15). As shown for methane formation in Fig. 2 and 3, acetate oxidation and subsequent methane formation were much slower than methane formation from H₂-CO₂ in pure culture, suggesting that the rate of hydrogen supply was very low. Hence, the situation occurring in the syntrophic substrate oxidation may be analogous to that occurring in the pure culture supplied with limited amounts of H₂, as in the study of Morgan et al. (15). However, we were able to detect transcripts of mtd and mth under both low-H₂-supply conditions (syntrophic coculture) and high-H₂-supply conditions (pure culture), whereas mth transcripts were observed only with a high H₂ supply in the experiments done by Morgan et al. (15). The reason why our results differed from those of Morgan et al. (15) is not the difference in the strains used, because we obtained the same results when we used a butyrate-oxidizing coculture in which the partner methanogen was strain H, the same strain used by Morgan et al. Recently, Afting et al. reported that in M. marburgensis cells grown under H₂-limited conditions, the specific activity of MTH was always very similar to that in cells grown under high-H₂-supply conditions. Furthermore, there was no significant difference in the transcription and translation of mth in cells grown under both types of conditions (1). Our results showing that MTH was expressed both in pure culture and in coculture in the early to late stages of cultivation are consistent with the results of Afting et al. (1).

2-D SDS gel electrophoresis of protein patterns ensured that under syntrophic conditions, MRI (spots 3, 4, and 5 in Fig. 5B) and F₄₂₀-dependent N⁵,N¹⁰-methylene-H₄MPT reductase (spot 2) were the most abundant proteins of all the detected proteins. It is generally known that methyl coenzyme M reductases represent more than 10% of cellular protein (6), but MRI seemed to be extraordinarily abundant in our coculture. MRI is characterized by a specific activity lower than that of MRII and by a substrate affinity higher than that of MRII. It is easy to envisage that when M. thermoautotrophicus is grown in coculture, the methanogen preferentially uses the higher-affinity enzyme under substrate-limited conditions and that, because of its lower activity, the organism has to keep producing the enzyme, resulting in a higher level of expression of the gene and thus a higher level of production of the enzyme. Nonetheless, the reason for such an overexpression of the gene remains unclear.

M. thermoautotrophicus may sense the availability of exogenous H₂ and transmit this signal to the methanogenesis gene promoter, but the molecular details of this regulation system are still unclear (15, 18, 20). Several genes which seem to encode two-component sensor kinases and response regulators have been found in the M. thermoautotrophicus genome (28). Recently, a novel multicomponent regulatory system that mediates H₂ sensing in Alcaligenes eutrophus was reported (14). The features of the H₂ receptor protein HoxBC resembled the typical features of NiFe hydrogenases.

Methanothermobacter is ubiquitous in anaerobic methanogenic environments and plays a particularly important role in methane fermentation reactors. Using an in situ hybridization technique combined with confocal laser scanning microscopy with a Methanothermobacter-specific probe, investigators have shown that Methanothermobacter cells are closely associated with fatty acid-oxidizing H₂-producing syntrophs (12, 26). This result indicates that low hydrogen levels are needed for the syntrophic partner to oxidize substrates. Considering such situations, it would not be surprising if the MRI gene is preferentially expressed in such methanogenic microbial ecosystems. To date, the differential expression of methanogenesis genes has been discussed primarily in conjunction with H₂ partial pressures and some other physicochemical factors. However, cell-cell interactions including syntrophic associations between substrate oxidizers and methanogens may provide further insight into the expression of methane genes.

 
We thank John N. Reeve at Ohio State University for helpful discussion.

 
* Corresponding author. Mailing address: Research Institute of Biological Resources (formerly National Institute of Bioscience and Human-Technology), National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba 305-8566, Japan. Phone: 81-298-61-6591. Fax: 81-298-61-6587. E-mail: y.kamagata{at}aist.go.jp.

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Applied and Environmental Microbiology, March 2002, p. 1173-1179, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1173-1179.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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