Methanotrophy below pH 1 by a new Verrucomicrobia species PDF

Vol 450 | 6 December 2007 | doi:10.1038/nature06222 LETTERS Methanotrophy below pH 1 by a new Verrucomicrobia species Arjan Pol1, Klaas Heijmans1, Harry R. Harhangi1, Dario Tedesco2, Mike S. M. Jetten1 & Huub J. M. Op den Camp1 Mud volcanoes, mudpots and fumaroles are remarkable geological...

Vol 450 | 6 December 2007 | doi:10.1038/nature06222 LETTERS Methanotrophy below pH 1 by a new Verrucomicrobia species Arjan Pol1, Klaas Heijmans1, Harry R. Harhangi1, Dario Tedesco2, Mike S. M. Jetten1 & Huub J. M. Op den Camp1 Mud volcanoes, mudpots and fumaroles are remarkable geological at these sites are microbially oxidized into sulphuric acid, creating an features characterized by the emission of gas, water and/or semi- extremely acidic environment. The very acidic soil of the Solfatara liquid mud matrices1 with significant methane fluxes to the atmo- was shown to support significant methane consumption2, but so far it sphere (1021 to 103 t y21)2–4. Environmental conditions in these is unknown which microbes could be responsible for this consump- areas vary from ambient temperature and neutral pH to high tem- tion. Obligately aerobic methanotrophs are assumed to be a unique peratures and low pH. Although there are strong indications for group of bacteria, belonging to either the Alpha or Gamma subclass biological methane consumption in mud volcanoes4,5, no metha- of the Proteobacteria, which use methane as the sole source of energy notrophic bacteria are known that would thrive in the hostile and carbon10. So far, all aerobic methanotrophs have been shown to conditions of fumaroles (temperatures up to 70 6C and pH down contain a membrane-bound particulate methane mono-oxygenase to 1.8)2. The first step in aerobic methane oxidation is performed (pMMO), except for Methylocella sp. that was reported to have only by a soluble or membrane-bound methane mono-oxygenase. Here the soluble, cytoplasmic form of MMO (sMMO)11. The pmoA gene we report that pmoA (encoding the b-subunit of membrane-bound (encoding the 24 kDa b-subunit of this membrane bound MMO12) is methane mono-oxygenase) clone libraries, made by using DNA generally used as a phylogenetic marker for methanotrophic bacteria. extracted from the Solfatara volcano mudpot and surrounding Methanotrophs are widespread in nature and are mostly neutrophilic bare soil near the fumaroles, showed clusters of novel and distant and mesophilic. However, on the basis of molecular surveys, in the pmoA genes. After methanotrophic enrichment at 50 6C and last decade isolation and characterization of more extremophilic pH 2.0 the most distant cluster, sharing less than 50% identity with proteobacterial methanotrophs was initiated13. Thus far, the lowest any other described pmoA gene, was represented in the culture. pH values still supporting methanotrophic activity were reported for Finally we isolated an acidiphilic methanotrophic bacterium Acidi- bacteria isolated from peat bogs11,14,15. These bacteria belong to methylosilex fumarolicum SolV belonging to the Planctomycetes/ genera of the Alpha subclass of the Proteobacteria (Methylocella, Verrucomicrobia/Chlamydiae superphylum6, ‘outside’ the subphyla Methylocapsa and Methylocystis), and showed growth between of the Alpha- and Gammaproteobacteria containing the established pH 4.2 and 7.5 with a maximum methane-oxidizing activity around methanotrophs. This bacterium grows under oxygen limitation pH 5.0. on methane as the sole source of energy, down to pH 0.8—far The inner part of the Solfatara, characterized by a central mudpool below the pH optimum of any previously described methano- (fangaia) surrounded by bare, acid soil (pH 1–2), was sampled troph. A. fumarolicum SolV has three different pmoA genes, with and DNA was extracted to start a molecular survey of pmoA genes. two that are very similar to sequences retrieved from the mudpot. Here we report the presence of pmoA genes in an environmental Highly homologous environmental 16S rRNA gene sequences clone library constructed using this DNA as a PCR template for the from Yellowstone Park show that this new type of methanotrophic widely applied pmoA primer set A189/A682 (ref. 16), which also may bacteria may be a common inhabitant of extreme environments. amplify the gene of ammonium mono-oxygenase b subunit (amoA). This is the first time that a representative of the widely distributed We were only able to amplify pmoA genes using non-restrictive Verrucomicrobia phylum, of which most members remain uncul- conditions (annealing temperature lowered from 56 uC to 48 uC; tivated6, is coupled to a geochemically relevant reaction. no false-positive clones obtained), pointing to the presence of Significant amounts of geological methane, produced within the pmoA genes with low similarity to known sequences. This is sup- Earth’s crust, are currently released naturally into the atmosphere3,7,8. ported by the phylogenetic analyses of the pmoA sequences, which The preliminary global estimate of these methane emissions indicates show that the Solfatara pmoA sequences group into two clusters: one that there are probably more than enough sources to provide the represents a completely new, deep branch within the pmoA/amoA amount of methane required to account for the suspected missing phylogenetic tree, sharing very low homology to known sequences source of global methane8. Recent findings from the Haakon Mosby (Fig. 1); the other cluster groups with the Gammaproteobacterial and Carpatian mud volcanoes showed that these systems may also act methanotrophs. as sinks for this geological methane4,5,9. At these sites with moderate Intrigued by the new pmoA sequences, we used mud and mixed- environmental conditions (2–25 uC and a neutral pH), 16S rRNA soil samples from this site to start enrichment cultures at 50 uC and genes of both aerobic and anaerobic methane-oxidizing micro- pH 2 with methane as the sole source of energy and carbon. After organisms were present. In contrast, fumaroles such as those located 3 weeks, methane consumption was observed in both soil and mud in the Solfatara at Pozzuoli near Naples (southern Italy), which also incubations. Non-restrictive PCR amplification of pmoA sequences, emit significant amounts of methane (73 tonnes of CH4 per km2 per with DNA from the enrichment as a template, resulted in five clones year)2, are characterized by soils with a low pH (down to 1.0) and (from two different enrichments) with sequences almost identical to elevated temperatures (up to 70 uC). The H2S-rich sulphurous fumes the distant group within the environmental clones (Fig. 1). Repeated 1 Department of Microbiology, IWWR, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands. 2Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy. 874 ©2007 Nature Publishing Group NATURE | Vol 450 | 6 December 2007 LETTERS serial transfers of the mud culture into fresh medium (see Methods) amino acids of PmoA were present, whereas the signature amino and finally diluting the culture onto floating polycarbonate filters17 acids of AmoA were absent19. Of all 42 highly conserved amino acids resulted in a pure culture, named strain SolV. Tiny whitish colonies in all bacterial PmoA/AmoA proteins19, 6 to 8 were not shared by one appeared on the filters after 1 week and microscopic observation or more of the pmoA genes from strain SolV (Supplementary Fig. 2). revealed only one rod-shaped morphotype. When exponentially Phylogenetic analysis of the pmoA genes showed that pmoA1 and growing cells of SolV were tested, the sMMO activity test (conversion pmoA2 are highly similar to the environmental sequences from the of naphthalene to naphthol) was negative, but pMMO activity (par- Solfatara and the enrichments (Fig. 1, and see above). The pmoA3 ticulate MMO; using propylene) could easily be measured (50 nmol - gene represents another completely new, deep branch. Together these per min per mg of protein). Genomic DNA from SolV was extracted three new pmoA sequences indicate that methanotrophic bacteria are and subjected to pyrosequencing18. From these data, we could phylogenetically much more diverse than currently assumed. Recent identify many genes of C1 metabolism (Table 1), indicating that genomic data have shown that two either identical or distantly related strain SolV may use a new combination of the serine, tetrahydrofo- pmoA genes can be present in one Alpha- or Gamma-proteobacterial late and ribulose-1,5 bisphoshosphate pathways for carbon assimila- methanotroph20–22. Expression of pmoA1 and pmoA2 messenger tion. The diagnostic genes of the ribulose-monophosphate pathway RNA was confirmed by RT–PCR on mRNA extracted from meth- seem to be absent (Table 1). Conversion of formaldehyde seems to be ane-grown SolV cells using specific primers (see Methods). The mediated by a tetrahydrofolate-dependent pathway or directly by stacked membrane structures characteristic for methanotrophs formaldehyde dehydrogenase (activity 110 nmol per min per mg of expressing pMMO were not observed in SolV by transmission elec- protein). The methanol dehydrogenase activity was 60 nmol per - tron microscopy (Supplementary Fig. 3). Instead, circular bodies of min per mg of protein and the mxaF gene showed 50% identity to about 50–70 nm were observed after fixation with glutaraldehyde or mxaF of Methylococcus capsulatus. None of the subunits of sMMO cryofixation. These bodies may be reminiscent of the vesicles was found. However, two complete pmoCAB operons and one observed in the acidiphilic methanotroph Methylocella palustris23. pmoCAB cluster with a partial pmoC were identified. Several (two Growth of strain SolV occurred between pH 0.8 and 5.8 (Fig. 2). to nine) mismatches with pmoA primers A189/A682 were found The temperature optimum is 55 uC, with only minor growth observed (Supplementary Fig. 1), explaining the low recovery in PCR below 40 uC and above 65 uC. The maximum-specific-growth rate on amplification from environmental DNA. However, all signature methane was 0.07 h21 (doubling time 10 h). Carbon dioxide and the Soil Pmo3 87 Fangaia Pmo6/soil Pmo4 Fangaia Pmo1/Pmo5 95 Methylomicrobium sp. NI 63 Fangaia Pmo3 89 Fangaia Pmo4 100 Soil Pmo1 Gamma- 82 Clonothrix fusca proteobacteria PmoA Thermophilic methanotroph HB 78 Methylococcus capsulatus Bath 50 Methylocaldum szegediense 86 98 Methylocaldum tepidum 93 Methylocaldum gracile 76 Nitrosococcus oceanus Gamma- 100 Nitrosococcus sp. C113 proteobacteria 99 Methylocystis sp. SC2 PmoA2 AmoA Methylosinus sporium SC8 PmoA2 63 92 Methylocapsa acidiphila Alpha- proteobacteria 68 Methylocystis sp. SC2 PmoA1 PmoA 100 Methylosinus sporium SC8 PmoA1 Acidimethylosilex fumarolicum PmoA2 Acidimethylosilex fumarolicum PmoA1 99 Soil Pmo8 99 45 Soil Pmo5 92 Fangaia Pmo8 Enrichment B3 Enrichment A2, A3, B2, B1 95 Nitrosolobus multiformis Beta- 92 Nitrosospira briensis proteobacteria Nitrosomonas europaea AmoA 59 Arctic methanotrophs (PmoA) Gamma- 74 Crenothrix polyspora strains (PmoA) proteobacteria 96 Acidimethylosilex fumarolicum PmoA3 PmoA Crenarchaeota (AmoA) 100 0.5 Figure 1 | Phylogenetic relationship among deduced PmoA and AmoA fangaia Pmo and soil Pmo prefix refer to environmental clones from DNA proteins. The neighbour-joining tree calculated with the PAM Dayhoff extracted from the central mudpot and bare, acid soil, respectively. matrix is shown with bootstraps values of 500 replicates at the branches. The Enrichment refers to clones obtained from DNA extracted from two bar represents a 50% estimated-sequence divergence. Application of different enrichments (A and B). different methods of compiling trees revealed congruent tree topologies. The 875 ©2007 Nature Publishing Group LETTERS NATURE | Vol 450 | 6 December 2007 Table 1 | Genes of C1 metabolism of Acidimethylosilex fumarolicum SolV Enzyme Enzyme Commission Gene BLASTP search against Methylococcus capsulatus (EC) number Identity (%) Similarity (%) Expected (E)-value GenBank 280 Methane mono-oxygenase 1.14.13.25 pmoA1 53 71 4.6 3 10 mca1797 pmoA2 57 74 5.5 3 10281 mca1797 pmoA3 41 62 1.2 3 10251 mca1797 pmoB1 39 57 6.8 3 10275 mca2853 pmoB2 40 58 1.6 3 10276 mca2853 pmoB3 38 56 8.7 3 10275 mca2853 pmoC1* pmoC2 58 72 5.9 3 10277 mca0295 pmoC3 43 60 8.0 3 10250 mca0295 mmoX Not present in SolV Methanol dehydrogenase 1.1.99.8 mxaF 50 64 2.4 3 102169 mca0299 mxaJ 36 55 1.7 3 10236 mca0300 mxaG 34 51 2.0 3 1028 mca0781 Formaldehyde dehydrogenase 1.2.99.3 adhP 41 58 8.5 3 10268 mca0775 Formaldehyde-activating enzyme 4.3.-.- fae Not present in SolV Formate dehydrogenase fdhA 50 67 3.1 3 10228 mca1393 fdhB 62 78 6.7 3 102172 mca1392 fdhC 68 81 0 mca1391 fdhD 48 67 3.0 3 10212 mca1389 Serine–glyoxylate aminotransferase 2.6.1.45 agxt/spt 31 50 6.1 3 10240 mca1406 Hydroxypyruvate dehydrogenase 1.1.1.29 hprA 32 52 5.0 3 10223 mca1407 Formate–tetrahydrofolate ligase 6.3.4.3 fhs 53 70 1.5 3 102165 mca2219 Serinehydroxymethyl transferase 2.1.2.1 glyA 57 75 2.6 3 102135 mca1660 5-formyltetrahydrofolate cycloligase 6.3.3.2 mthfs 29 45 5.7 3 10211 mca2773 Methylenetetrahydrofolate dehydrogenase/ 1.5.1.5/3.5.4.9 folD Not present in M. capsulatus { methenyltetrahydrofolate cyclohydrolase Hexulose-6-phosphate synthase 4.1.2.- hspA Not present in SolV Hexulose-6-phosphate isomerase 5.-.-.- sgbU Not present in SolV { Ribulose bisphosphate carboxylase 4.1.1.39 cbbL 60 75 4.9 3 102165 mca2743 cbbS 41 62 4.5 3 10218 mca2744 Phosphoribulokinase 2.7.1.19 cbbP 64 80 6.6 3 102108 mca3051 Phosphoglycerate kinase 2.7.2.3 cbbK 40 61 1.5 3 10276 mca2021 Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.13 cbbG 49 64 1.4 3 10272 mca2598 Genes of C1 metabolism were identified in an assembly of the genome after pyrosequencing. The assembly was produced from 88.9 Mb of sequence information and resulted in a 35-fold coverage, based on an estimated genome size of 2.5 Mb. Translated protein sequences, based on genes identified, were used for a BLAST search in the Methylococcus capsulatus genome (http://pedant.gsf.de/). *partial gene (48 amino acids); {best NCBI BLAST hit with folD from Prosthecochloris aestuarii (identity 51%; similarity 71%; E-value 1.0 3 10273); {best NCBI BLAST hit with gutQ (sugar phosphate isomerase family) from Burkholderia phytofirmans, (identity 45%; similarity 66%; E-value 2.0 3 10278). inorganic fraction of mud water stimulated growth. Methane was converted to carbon dioxide according to a stoichiometry that is a 9 2.0 typical for methanotrophs: CH4 1 1.6 O2 R 0.65 CO2 1 1.55 H2O 1 0.35 CH20 (biomass) with a yield of 6.4 g of dry weight per mol of 8 methane. Optical density (600 nm) 7 1.6 Acetate, malate, succinate, formate, formaldehyde and yeast 6 extract (all at 1 g l21) completely inhibited growth of SolV on meth- CH4(mmol) 1.2 5 ane at pH 2. The bacterium apparently is very sensitive towards 4 uncoupling by small organic acids at low pH values, because at 0.8 3 pH 5 formate (pKa 3.75) did not inhibit growth. No growth took y = 0.01e0.072x place above 100 mM NaCl or in media containing glucose. In addi- 2 0.4 tion to methane, hydrogen gas was also oxidized. Strain SolV grew 1 well on methanol, but the added methanol completely repressed 0 0.0 methane consumption. After methanol was depleted, methane con- 0 20 40 60 80 Time (h) sumption and growth started only after 4 h. Ethane inhibited growth b 0.07 although it was converted simultaneously with methane as a com- petitive substrate at virtually the same rate. Acetylene (0.1% v/v) 0.06 instantaneously caused a complete inhibition of methane consump- tion, an observation that supports pMMO being the primary meth- 0.05 ane-oxidizing system. SolV could use both ammonium and nitrate as Growth rate (h–1) a nitrogen source. No growth occurred on ammonium without 0.04 methane. Nitrogen fixation and anoxic nitrate-dependent methanol oxidation was not observed. 0.03 SolV has a typical Ks value for methane, namely 6 mM. However, the affinity for oxygen was exceptionally high (Ks 0.7 mM), reflecting 0.02 0.5 1.0 1.5 2 Figure 2 | Growth characteristics of strain SolV. a, Typical growth curve 0.01 showing decrease of methane (circles) and increase of optical density (triangles) at pH 2 and 55 uC. The equation is the best exponential fit 0 through the data points. b, Growth rate in relation to pH. The insert shows 0 2 4 6 8 an enlargement of the data below pH 2. Different symbols indicate pH experiments performed on different days. 876 ©2007 Nature Publishing Group NATURE | Vol 450 | 6 December 2007 LETTERS the need to compete for oxygen in its natural habitat, where microbial A. fumarolicum may be common inhabitants of these extreme oxygen consumption and a constant flux of oxygen-depleted fumar- environments. The new pmoA and 16S rRNA gene sequences may olic gases, containing mainly carbon dioxide, will cause oxygen help to identify the Planctomycetes/Verrucomicrobia/Chlamydiae concentrations to be very low. superphylum methanotrophs from less extreme habitats and to show Fluorescence in situ hybridization (FISH) analysis of the isolate how they are globally distributed. using the probe EUBIII, which is designed to mainly cover the Verrucomicrobiales24,25, showed a strong hybridization signal METHODS SUMMARY (Supplementary Fig. 4). No signal was obtained with EUBI, EUBII Enrichments were started with mud and mixed soil samples from the Solfatara or the alpha (ALF968), beta (BET42a) or gamma (GAM42a) proteo- and incubated at 50 uC and pH 2.0 with methane as the sole source of energy and bacterial probes25,26. The Verrucomicrobia-like identity was con- carbon. When methane consumption was observed, serial transfers into fresh medium were started. Finally a pure culture was obtained using the floating-filter firmed by the sequence of its 16S rRNA gene obtained from technique17. Purity was checked by FISH and plating on medium enriched with pyrosequencing (see above). A specific probe was designed on the yeast extract, without methane in the head space. DNA from environmental basis of this sequence (SolV830, see Methods) and used together with samples and genomic DNA from strain SolV was isolated as described28. The probe EUBIII to confirm the purity of the SolV culture. All cells from pmoA and 16S rRNA genes were amplified by hot start using primers A189 and an exponentially growing culture showed double hybridization A682 (ref. 16), and 616F (59-AGA GTT TGA TYM TGG CTC AG-39) and 630R (Supplementary Fig. 4). Phylogenetic analysis of the 16S rRNA (59-CAK AAA GGA GGT GAT CC-39)28, respectively. Pyrosequencing on geno- sequence of SolV indicated that the isolate represents the first mem- mic DNA was done as described18. FISH microscopy was performed as ber of a new subdivision within the Verrucomicrobia phylum (Fig. 3 described29 using the following nucleotide probes: EUBI, EUBII, EUBIII and and Supplementary Fig. 5). Pairwise distance analysis revealed ,81% SolV830 (5’-GGT CGA TTC CGC CAA CGC-39). The latter probe was designed with the ARB program30. Expression of pmoA mRNA was analysed by RT–PCR. identity with members of other subdivisions6,27. The affinity for methane was estimated using cells from a batch culture Strain SolV is the first reported extreme acidiphilic methanotrophic (OD600 5 0.24). The apparent affinity constant for oxygen was estimated by bacterium and is phylogenetically placed outside the subphyla of the measuring the methane respiration of a stirred culture in a 1 ml glass chamber Alpha- and Gammaproteobacteria containing the established metha- equipped with a micro-oxygen sensor (Unisense A/S). Enzyme activities men- notrophs, and we propose to name it: ‘Acidimethylosilex fumarolicum’, tioned in Table 1 were measured according to ref. 23. gen. nov. sp. nov. (Supplementary Information). Full Methods and any associated references are available in the online version of So far the Verrucomicrobia phylum contains only a few cultivated the paper at www.nature.com/nature. strains that are anaerobic or aerobic heterotrophs, growing on sugars in more or less complex media. However environmental clone Received 4 July; accepted 4 September 2007. libraries show that there is a large biodiversity of Verrucomicrobia Published online 14 November 2007. and they are encountered in many ecosystems (soils, peat bogs, acid 1. Dimitrov, L. Mud volcanoes — the most important pathway for degassing deeply rock drainage and landfill leachate) often in relatively high numbers, buried sediments. Earth Sci. Rev. 59, 49–76 (2002). but with an unknown physiology6. It is interesting to speculate that 2. Castaldi, S. & Tedesco, D. Methane production and consumption in an active volcanic environment of Southern Italy. Chemosphere 58, 131–139 (2005). the widely distributed Verrucomicrobia phylum, from which most 3. Etiope, G. & Klusman, R. W. Geologic emissions of methane to the atmosphere. members remain uncultivated6, may be coupled to a geochemically Chemosphere 49, 777–789 (2002). relevant reaction. BLAST searches with the strain SolV 16S rRNA 4. Niemann, H. et al. Novel microbial communities of the Haakon Mosby mud gene sequence showed very high identity (98–99%) to six environ- volcano and their role as a methane sink. Nature 443, 854–858 (2006). 5. Alain, K. et al. Microbiological investigation of methane- and hydrocarbon- mental clones (Fig. 3) that were obtained during a geochemical study discharging mud volcanoes in the Carpathian Mountains, Romania. Environ. on microbial communities in acidic hot springs (Rainbow and Microbiol. 8, 574–590 (2006). Joseph’s Coat) in Yellowstone National Park (unpublished; NCBI 6. Horn, M. & Wagner, M. The Planctomycetes, Verrucomicrobia, Chlamydiae and accession numbers: AY882698, AY882699, AY882710, AY882819, sister phyla comprise a superphylum with biotechnological and medical relevance. Curr. Opin. Biotechnol. 17, 241–249 (2006). AY882820 and AY882834). This shows that bacteria similar to 7. Houghton, J. Global warming. Rep. Prog. Phys. 68, 1343–1403 (2005). 8. Houghton, J. T., et al. Climate Change 1995: The Science of Climate Change Acidimethylosilex fumarolicum Verrucomicrobia (Cambridge Univ. Press, New York, 1995). SolV + Yellowstone (subdivision 4) 9. Lösekann, T. et al. Diversity and abundance of aerobic and anaerobic methane clones oxidizers at the Haakon Mosby Mud Volcano, Barents Sea. Appl. Environ. Microbiol. 73, 3348–3362 (2007). 10. Hanson, R. S. & Hanson, T. E. Methanotrophic bacteria. Microbiol. Rev. 60, 439–471 (1996). Verrucomicrobia 11. Dedysh, S. N. et al. Methylocella tundrae sp. nov., a novel methanotrophic (subdivision 3) 97 99 Verrucomicrobia (subdivision 5) bacterium from acidic tundra peatlands. Int. J. Syst. Evol. Microbiol. 54, 151–156 62 64 93 79 (2004). 12. Lieberman, R. L. & Rosenzweig, A. C. Crystal structure of a membrane-bound 100 71 metalloenzyme that catalyses the biological oxidation of methane. Nature 434, Verrucomicrobia Lentisphaerae 177–182 (2005). (subdivision 2) 13. Trotsenko, Y. A. & Khmelenina, V. N. Biology of extremophilic and extremotolerant methanotrophs. Arch. Microbiol. 177, 123–131 (2002). 14. Dedysh, S. N. et al. Isolation of acidophilic methane-oxidizing bacteria from Verrucomicrobia Protochlamydiae northern pet wetlands. Science 282, 281–284 (1998). (subdivision 1) /Chlamydiae 15. Dedysh, S. N. et al. Methylocystis heyeri sp. nov., a novel type II methanotrophic bacterium possessing ’signature’ fatty acids of type I methanotrophs. Int. J. Syst. Evol. Microbiol. 57, 472–479 (2007). 0.05 Planctomycetes 16. Holmes, A. J., Costello, A., Lidstrom, M. E. & Murrell, J. C. Evidence that participate Anammoxacaea methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol. Lett. 132, 203–208 (1995). To outgroups 17. Visser, J. M., Stefess, G. C., Robertson, L. A. & Kuenen, J. G. Thiobacillus sp. W5, the Figure 3 | Phylogenetic relationship between the 16S rRNA gene sequence dominant autotroph oxidizing sulfide to sulfur in a reactor for aerobic treatment of sulfidic wastes. Antonie Van Leeuwenhoek 72, 127–134 (1997). of strain SolV and representatives of the Planctomycetes/ 18. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre Verrucomicrobia/Chlamydiae superphylum. The tree was calculated using reactors. Nature 437, 376–380 (2005). the neighbour-joining algorithm with Kimura 2-parameter correction. 19. Stoecker, K. et al. Cohn’s Crenothrix is a filamentous methane oxidizer with an Bootstrap values of 500 replicates are shown at the nodes. The scale bar unusual methane monooxygenase. Proc. Natl Acad. Sci. USA 103, 2363–2367 represents 0.05 nucleotide changes per position. (2006). 877 ©2007 Nature Publishing Group LETTERS NATURE | Vol 450 | 6 December 2007 20. Dunfield, P. F. et al. Isolation of a Methylocystis strain containing a novel pmoA-like 29. Snaidr, J., Amann, R., Huber, I., Ludwig, W. & Schleifer, K.-H. Phylogenetic analysis gene. FEMS Microbiol. Ecol. 41, 17–26 (2002). and in situ identification of bacteria in activated sludge. Appl. Environ. Microbiol. 21. Tchawa Yimga, M., Dunfield, P. F., Ricke, P., Heyer, J. & Liesack, W. Wide 63, 2884–2896 (1997). distribution of a novel pmoA-like gene copy among type II methanotrophs, and its 30. Ludwig, W. et al. ARB: a software environment for sequence data. Nucleic Acids expression in Methylocystis strain SC2. Appl. Environ. Microbiol. 69, 5593–5602 Res. 32, 1363–1371 Æhttp://www.arb-home.deæ (2004). (2003). Supplementary Information is linked to the online version of the paper at 22. Ward, N. et al. Genomic insights into methanotrophy: The complete genome www.nature.com/nature. sequence of Methylococcus capsulatus (Bath). PLoS Biol. 2, 1616–1619 (2004). 23. Dedysh, S. N. et al. Methylocella palustris gen. nov., sp. nov., a new methane- Acknowledgements The authors thank M. Strous and S. Castaldi for critical oxidizing acidophilic bacterium from peat bogs, representing a novel subtype reading and discussion, L. van Niftrik and G.-J. Janssen for technical assistance with of serine-pathway methanotrophs. Int. J. Syst. Evol. Microbiol. 50, 955–969 electron microscopy, M. Schmid for assistance with FISH microscopy and (2000). phylogenetic analyses, and H. A. Mohammadi and M. Gerrits for technical 24. Daims, H., Bruhl, A., Amann, R., Schleifer, K.-H. & Wagner, M. The domain- assistance in cultivation. H. Lunstroo is acknowledged for allowing access to the specific probe EUB338 is insufficient for the detection of all Bacteria: development 454-sequencing technology, and G. Angarano for allowing access to the Solfatara and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22, and P. Mariani for assistance during sampling. 434–444 (1999). 25. Loy, A., Maixner, F., Wagner, M. & Horn, M. probeBase — an online resource for Author Contributions A.P. and D.T. performed the sampling; A.P. did the rRNA-targeted oligonucleotide probes: new features 2007. Nucleic Acids Res. 35, enrichment and isolation; K.H. and A.P. carried out the physiological experiments; D800–D804 (2007). K.H. and H.R.H. were responsible for the molecular analysis; A.P. and H.J.M.O.d.C. performed phylogenetic analyses, alignments and probe design. The research was 26. Manz, W., Amann, R., Ludwig, W., Wagner, M. & Schleifer, K.-H. Phylogenetic conceived by A.P., M.S.M.J. and H.J.M.O.d.C. and was based on observations made oligodeoxy-nucleotide probes for the major subclasses of proteobacteria: by D.T. A.P., M.S.M.J., D.T. and H.J.M.O.d.C. contributed to interpreting the data Problems and solutions. Syst. Appl. Microbiol. 15, 593–600 (1992). and writing the paper. 27. Hugenholtz, P., Goebel, B. M. & Pace, N. R. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, Author Information The nucleotide sequence data have been deposited in 4765–4774 (1998). GenBank under accession numbers EF591085 (pmo_1), EF591086 (pmo_2), 28. Juretschko, S. et al. Combined molecular and conventional analyses of EF591087 (pmo_3) and EF591088 (16S rRNA). Reprints and permissions nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and information is available at www.nature.com/reprints. Correspondence and Nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol. 64, requests for materials should be addressed to M.S.M.J. ([email protected]) or 3042–3051 (1998). H.J.M.O.d.C. ([email protected]). 878 ©2007 Nature Publishing Group doi:10.1038/nature06222 METHODS After 3 weeks, methane consumption was observed and repeated serial transfers of the mud and mixed-soil culture into fresh medium (see below) were started. Finally the culture was serially diluted and aseptically filtered through 25-mm polycarbonate filters (0.2 mm, Nucleopore), which were placed floating on med- ium in Petri dishes and incubated in closed jars under a methane atmosphere (see below)17. Tiny whitish colonies appeared on the filters after 1 week and micro- scopic observation revealed only one rod-shaped morphotype. Purity was checked by FISH and plating on medium enriched with yeast extract, without methane in the head space. No growth was observed on this medium. Culture conditions. The culture medium was based on the Fangaia mineral concentrations and composed of 0.4 mM MgCl2, 2 mM CaHPO4, 1 mM Na2SO4, 2 mM K2SO4, 2 mM (NH4)2SO4, 3% autoclaved liquid from the Fangaia mud pool at Pozzuoli; 1 ml l21 trace elements (in mg l21) ZnSO4?7H2O (4.4), MnCl2?4H20 (1.0), FeSO4?7H2O (1.0), (NH4)6MO7O24? 4H2O (0.22), CuSO4?5H2O (0.32), CoCl2?6H2O (0.32). The pH was adjusted with H2SO4 or NaOH. Bacteria were grown in 120 ml serum bottles with 10 ml of medium and 2–5% CO2 and 2–5% CH4 in the headspace. Bottles were incubated at 50–55 uC on a rotary shaker at 250 r.p.m. To determine the reaction stoichi- ometry, gas samples were taken from triplicate cultures with a gaslock syringe and methane, carbon dioxide, oxygen and hydrogen were analysed on a HP 6890 gas chromatograph with a Porapak Q column and thermal conductivity detec- tion. Yield on methane was determined by harvesting cells in the late exponential phase. Cells were centrifuged and washed with 1 mM HCl and dried under vacuum at 70 uC until constant weight. pmoA and 16S rRNA gene sequence analysis. DNA from environmental sam- ples and genomic DNA from strain SolV was isolated as described28 without the use of lytic enzymes. For pmoA PCR under non-restrictive conditions the anneal- ing temperature was lowered from 56 uC to 48 uC. The products were purified from an agarose gel with the QIAEX II gel extraction kit (Qiagen) and cloned using the TOPO TA cloning kit (Invitrogen). Plasmids were purified with FlexiPrep kit (Amersham Biosciences) and sequenced with M13R and M13F primers, which flank the cloning site of the vector. Pyrosequencing on genomic DNA was done as described18. Real-time RT–PCR analysis. Samples (50 ml at OD600 5 0.85) from methane- grown chemostat cultures were rapidly cooled and RNA was isolated using the Omega RNA extraction kit (Omega Bio-Tec) according to the manufacturer’s protocol. Transcription products of pmoA were detected using SolV-specific primers (REVp1032 59-GCAAARCTTCTCATYAGTWCC-59; FORp1034 59-GTGGATGAATCGGTATTGG-39). Reverse transcription was performed with primer REVp1032 and RevertAid M_MulV (Fermentas) Quantitative PCR was done using the iQ custom SYBR Green supermix kit (Bio-Rad), accord- ing to the manufacturer’s instructions. The PCR program on the Biorad MyiQ was 3 min 95 uC and 40 cycles 30 s at 95 uC, 30 s at 54 uC, 30 s at 72 uC. FISH microscopy. On the basis of the obtained 16S rRNA gene (see above) a new oligonucleotide probe (SolV830, 59-GGT CGA TTC CGC CAA CGC-39) was designed using the probe-design software of the ARB program30. Optimum formamide concentration for this probe was 20%. Kinetics and enzyme activities. The affinity for methane was estimated by measuring the consumption rate in a series of incubations of 10 ml samples, taken from a batch culture (OD600 5 0.24) in 100 ml bottles at 50 uC. Various amounts of methane were added and bottles were shaken vigorously at 500 r.p.m. Virtually linear rates were measured during one hour. Rates were proportional to the cell density in the range used, indicating that there was no mass-transfer limitation for methane. Five ml of the culture were preincubated at 50 uC in a closed 100 ml bottle with 30 ml of methane to ensure excess methane compared to oxygen. The oxygen-consumption rates were calculated from the decrease in oxygen concentration over time. ©2007 Nature Publishing Group