View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Directory of Open Access Journals Hindawi Publishing Corporation Archaea Volume 2012, Article ID 315153, 10 pages doi:10.1155/2012/315153 Research Article Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase Stefanie Berger, Cornelia Welte, and Uwe Deppenmeier Institute for Microbiology and Biotechnology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany Correspondence should be addressed to Uwe Deppenmeier, [email protected] Received 16 May 2012; Accepted 30 June 2012 Academic Editor: Francesca Paradisi Copyright © 2012 Stefanie Berger et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl- CoA, AMP, and pyrophosphate (PPi) and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell. 1. Introduction 7–70 μM is needed [7]. Therefore, Methanosaeta species pre- vail over members of the genus Methanosarcina in low acetate Methanogenic archaea are of high ecological importance as environments frequently encountered in natural habitats. they are responsible for closure of the global carbon cycle Important biotechnological habitats are biogas facilities [8– and production of the greenhouse gases CO2 and methane 12], where Methanosaeta species are of special importance [1–3]. They are also an integral part of biogas reactors and for reactor performance and stability [12, 13]. contribute to the production of the combustible gas methane In acetate-degrading (aceticlastic) methanogenesis, ace- that is a source of renewable energy [4, 5]. Methanogenic tate first has to be activated at the expense of ATP. This reac- archaea use end products of anaerobic bacterial degradation tion can be catalysed by the high activity but low affinity ace- processes like H2/CO2 and acetate as substrates for growth. tate kinase/phosphotransacetylase (AK/PTA) system that is It is estimated that about two thirds of the methane used by Methanosarcina sp. [14, 15] or by the low-activity but produced by methanogenic archaea on earth derives from high-affinity AMP-dependent acetyl-CoA-synthetases (ACS) acetate degradation [6]. But despite its high abundance [16–18]. While the AK/PTA system generates ADP, Pi and only two genera are able to use acetate as substrate for acetyl-CoA from ATP, CoA, and acetate [15, 19, 20], the methanogenesis, namely, Methanosarcina and Methanosaeta. ACSconvertsATP,CoA,andacetatetoacetyl-CoA,AMPand While Methanosarcina species are metabolically versatile, pyrophosphate (PPi)[16, 18]. In the first step of aceticlastic members of the genus Methanosaeta are specialized on methanogenesis, acetyl-CoA is cleaved into its methyl and acetate utilization. This is reflected in a very high affinity for carbonyl moiety by the action of a CO dehydrogenase/acetyl- the substrate. For growth, a minimal concentration of only CoA synthase. In the course of this reaction, the carbonyl 2 Archaea group is oxidized to CO2 and electrons are transferred to to the manufacturer’s instructions (Ambion, Darmstadt, ferredoxin [21–23]. The methyl group is donated to the Germany). Preparations were treated with DNAse I to reduce methanogenic cofactor tetrahydrosarcinapterin and subse- DNA contaminations. Cleaning and concentration of RNA quently transferred to coenzyme M (CoM) by a membrane were achieved using the SurePrep RNA Cleanup and Con- bound Na+ translocating methyltransferase. Reduction of centration kit (Fisher Scientific, Schwerte, Germany). RNA the methyl group to methane with coenzyme B as electron purity was quantified spectrophotometrically by examining donor leads to the formation of the so-called heterodisul- the 260 nm/280 nm ratio as well as by denaturing agarose gel fide (CoM-S-S-CoB). Only recently we demonstrated that electrophoresis. Methanosaeta (Mt.) thermophila uses the heterodisulfide as Primers for qRT-PCR were designed using the Primer3 terminal electron acceptor in an anaerobic respiratory chain software (http://frodo.wi.mit.edu/primer3/input.htm). For with reduced ferredoxin as the sole electron donor [24]. the highly homologous ACS genes, the least homologous However, the way this organism conserves energy is not yet areas were used as templates to guarantee specificity of the fully understood. It can be estimated that the amount of ions primers. The genes encoding glyceraldehyde-3-phosphate translocated over the cytoplasmic membrane in the course dehydrogenase (GAP-DH, mthe 0701) and ribosomal pro- of aceticlastic methanogenesis could be sufficient for the tein S3P (mthe 1722) were chosen as reference genes. phosphorylation of two ADP molecules. Yet AMP-dependent Sequences of the primers used can be seen from Table 1. acetyl-CoA synthetase and soluble pyrophosphatase (PPiase) PCR reactions were performed according to the man- activities could be demonstrated for the closely related Mt. ufacturer’s instructions (http://www1.qiagen.com)withon concilii [16, 18, 25]. Taking non-energy coupled hydrolysis average 250 ng of RNA. The QuantiTect SYBR Green of pyrophosphate into account, two ATP equivalents are RT-PCR kit (Qiagen, Hilden, Germany) and the iCycler consumed in the course of the acetate activation reaction. (Bio-Rad, Munich, Germany) were used for labeling and According to this model, the obligate aceticlastic methanogen quantification, respectively. For data analysis, the Bio-Rad Mt. thermophila is not able to conserve energy during iCycler software was used. Each PCR product gave a single methanogenesis. To clarify this contradiction, the acetate narrow peak in the melting curve analysis. A relative value activation reaction in Mt. thermophila was reevaluated by (ΔCt) for the initial target concentration in each reaction gene expression analysis and characterization of ACS and was determined by subtracting Ct values of the reference PPiase. genes from those of the genes of interest. By subtracting ΔCt values, comparisons among the genes of interest could be accomplished. In addition, negative-control assays were 2. Materials and Methods included that were not incubated with reverse transcriptase. 2.1. Materials. All chemicals and reagents were purchased These assays contained only traces of DNA that were not from Sigma-Aldrich (Munich, Germany) or Carl Roth removed by DNase treatment. The Ct values of the negative GmbH (Karlsruhe, Germany). Restriction endonucleases, controls were analyzed and were at least five cycles higher T4 DNA ligase, Taq DNA polymerase, and PCR reagents than the assays with reverse transcriptase treatment. were purchased from Fermentas (St. Leon-Rot, Germany). Phusion DNA polymerase was purchased from New England 2.4. Cloning into Expression Vectors. Genes from Mt. Biolabs (Frankfurt am Main, Germany). Oligonucleotides thermophila were amplified from chromosomal DNA were synthesized by Eurofins (Ebersberg, Germany). extracted with CTAB [26]. Restriction endonuclease sites were inserted by PCR; Primers had the following sequences (recognition sites for restriction endonucleases 2.2. Bioinformatics. For Blast analyses, the respective tool are underlined): mthe 0236 for ATGGTAGGTCTCAAA- on NCBI (http://www.ncbi.nlm.nih.gov/) was used. For the TGGCAGATAATATCTATGTGGTCGGG, mthe 0236 rev batch Blast analysis, those proteins that had a threshold ATGGTAGGTCTCAGCGCTCTTCTTGAATGCGGA- E-value e−40 were referred to as homologous. The < CTCGAGC, mthe 1194 for ATGGTAACCTGCATTAGC- programs PsiPred (http://bioinf.cs.ucl.ac.uk/psipred/)and GCCGCTGAGACTGCAAAGACTGCTG, mthe 1194 rev InterPro (http://www.ebi.ac.uk/interpro/)wereutilizedfor ATGGTAACCTGCATTATATCAGACTATGAGCGG- bioinformatic analyses of CBS domains. GATGTTCTCG. For cloning of the pyrophosphatase gene (mthe 0236), Eco31I was used, for cloning of the AMP- 2.3. qRT-PCR. Total RNA from Mt. thermophila DSM 6194 dependent ACS gene (mthe 1194) BveI. Amplicons were cut was isolated by TRI Reagent extraction. 250 mL cultures were and ligated into pASK-IBA3 or pASK-IBA5 (IBA GmbH, grown anaerobically to the mid- to late- exponential growth Gottingen,¨ Germany) to produce pASK-mthe0236-3 and phase in DSMZ medium 387 at 55◦Cwith50mMsodium pASK-mthe1194-5, respectively. Both vectors