Microbiology (2002), 148, 2331–2342 Printed in Great Britain Identification of active methylotroph populations in an acidic forest soil by stable- isotope probing Stefan Radajewski,1 Gordon Webster,2† David S. Reay,3‡ Samantha A. Morris,1 Philip Ineson,4 David B. Nedwell,3 James I. Prosser2 and J. Colin Murrell1 Author for correspondence: J. Colin Murrell. Tel: j44 24 7652 2553. Fax: j44 24 7652 3568. e-mail: cmurrell!bio.warwick.ac.uk 1 Department of Biological Stable-isotope probing (SIP) is a culture-independent technique that enables Sciences, University of the isolation of DNA from micro-organisms that are actively involved in a Warwick, Coventry CV4 7AL, UK specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 35) microcosms that were exposed 2 Department of Molecular 13 13 13 13 and Cell Biology, to CH3OH or CH4. Distinct C-labelled DNA ( C-DNA) fractions were resolved University of Aberdeen, from total community DNA by CsCl density-gradient centrifugation. Analysis of Institute of Medical 16S rDNA sequences amplified from the 13C-DNA revealed that bacteria related Sciences, Foresterhill, Aberdeen AB25 2ZD, UK to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13C-labelled substrates, which suggested that moderately 3 Department of Biological Sciences, University of acidophilic methylotroph populations were active in the microcosms. Essex, Wivenhoe Park, Enrichments targeted towards the active proteobacterial CH3OH utilizers were Colchester, Essex CO4 3SQ, successful, although none of these bacteria were isolated into pure culture. A UK parallel analysis of genes encoding the key enzymes methanol dehydrogenase 4 Department of Biology, and particulate methane monooxygenase reflected the 16S rDNA analysis, but University of York, PO Box 373, YO10 5YW, UK unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the β-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the 13C-DNA fractions, suggesting 13 certain AOB assimilated a significant proportion of CO2, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the 13C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the β-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by- 13 products of methylotrophic carbon metabolism. Results from the CH3OH and 13 CH4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ. "$ Keywords: methanotrophs, community structure, culture-independent techniques, C, functional genes ................................................................................................................................................. INTRODUCTION † Present address: Cardiff School of Biosciences, Cardiff University, Main Building, Cardiff CF10 3TL, UK. Methylotrophs are micro-organisms that can use re- ‡ Present address: Institute of Ecology and Resource Management, duced one-carbon compounds as a sole source of carbon University of Edinburgh, Darwin Building, Edinburgh EH9 3JU, UK. and energy (Lidstrom, 1992). Two important methylo- Abbreviations: AOB, ammonia-oxidizing bacteria; DGGE, denaturing gradient gel electrophoresis; g.d.w., g dry weight; OTU, operational trophic substrates in the terrestrial environment are taxonomic unit; SIP, stable-isotope probing. methanol (CH$OH) and methane (CH%). CH$OH is The GenBank accession numbers for the sequences reported in this paper produced during the decomposition of plant polymers are AY080911–AY080961. and is probably excreted from living leaves and by CH%- 0002-5503 # 2002 SGM 2331 S. Radajewski and others oxidizing bacteria (Anthony, 1982), whereas CH% is Difficulties associated with the isolation of the vast mainly produced by methanogenic Archaea in anoxic majority of micro-organisms into laboratory culture environments. Methylotrophs that grow on CH$OH have hampered the identification of microbial popula- and CH% have been isolated from many environments tions involved in a specific process (Gray & Head, 2001; and the characterization of pure cultures has provided Hugenholtz et al., 1998). Several recently described information on the taxonomy, physiology, genetics and techniques can, however, establish this link between ecology of these physiologically specialized micro- identity and function under conditions approaching organisms. those in situ and without prior knowledge of the micro- organisms involved (Boschker et al., 1998; Lee et al., Most aerobic methylotrophs that are in laboratory 1999; Orphan et al., 2001; Ouverney & Fuhrman, 1999; culture belong to the class Proteobacteria. All of these Radajewski et al., 2000). Certain phospholipid-fatty- methylotrophs use the enzyme methanol dehydrogenase acid- and DNA-labelling techniques have been devel- to oxidize CH OH to formaldehyde, which is the central $ oped using one-carbon substrates enriched with radio- intermediate of one-carbon metabolism. Formaldehyde "% "$ active-carbon ( C) or stable-carbon ( C) isotopes; can subsequently be assimilated into cell carbon (Lid- these have been subsequently applied to identify the strom, 1992) or can be oxidized further to formate and active methylotroph communities in environmental CO to gain energy and to regenerate reducing equiva- # samples directly (Boschker et al., 1998; Radajewski et lents (reviewed by Vorholt et al., 1999). CH -oxidizing % al., 2000; Roslev & Iversen, 1999). bacteria (methanotrophs) are a subgroup of methylo- trophs that have been divided into types I and II (γ- and In a preliminary report, we described the development α-Proteobacteria, respectively) based on phenotypic and and application of stable-isotope probing (SIP) (Rada- phylogenetic characteristics (Hanson & Hanson, 1996). jewski et al., 2000). This technique exploits the fact that "$ "& Methanotrophs oxidize CH% to CH$OH using the certain stable isotopes (e.g. C and N) occur at low enzyme methane monooxygenase, which occurs in two abundance in nature but can be highly enriched (i.e. distinct forms that have been reviewed in detail by "99%) in commercially available compounds. As Murrell et al. (2000b). A particulate, membrane-bound demonstrated by Meselson & Stahl (1958) with Escheri- "& + methane monooxygenase has been reported in all chia coli grown on NH%, DNA synthesized during methanotrophs except Methylocella palustris (Dedysh microbial growth on a substrate enriched with a heavy, et al., 2000), whereas only certain strains contain a stable isotope becomes labelled sufficiently to be re- soluble, cytoplasmic methane monooxygenase. The solved from unlabelled DNA by density-gradient centri- particulate methane monooxygenase has several simi- fugation. This principle has been demonstrated with "$ larities to the ammonia monooxygenase of ammonia- methylotroph and AOB cultures grown on CH OH "$ $ oxidizing bacteria (AOB), and it has been suggested that (Radajewski et al., 2000) and CO# (Whitby et al., 2001) these two enzymes may be evolutionarily related as a carbon source, respectively. Although the buoyant (Holmes et al., 1995). Although some AOB can oxidize density of DNA varies with its GjC content, the CH , none are known to use it as an energy source incorporation of a high proportion of a naturally rare, % # "& "$ (Be! dard & Knowles, 1989). stable isotope ( H, Nor C) into DNA enhances Functional genes encoding key enzymes in the methylo- greatly the density difference between labelled and trophic pathways of several Proteobacteria have been unlabelled DNA fractions (Rolfe & Meselson, 1959; identified. The large subunit of methanol dehydro- Vinograd, 1963). Indeed, SIP has been applied suc- cessfully to identify active CH OH utilizers in an oak genase, which contains the active site of the enzyme, is $ "$ encoded by mxaF (Lidstrom et al., 1994; Nunn & forest soil (Radajewski et al., 2000). Isolation of the C- Lidstrom, 1986). The putative active site subunit of labelled DNA fraction simultaneously established the particulate methane monooxygenase is encoded by function and activity of the micro-organisms and pmoA, with the homologous subunit in ammonia sequence analysis subsequently determined their iden- monooxygenase encoded by amoA (reviewed by Murrell tity. The aim of the present study was to characterize the active aerobic methylotroph populations in an acidic et al., 2000a). Conserved domains within the amino-acid "$ "$ sequences have enabled the design of PCR primers that oak forest soil exposed to CH$OH or CH% and to isolate certain methylotrophs detected in our prelimin- specifically amplify methane monooxygenase or meth- "$ anol dehydrogenase genes from methylotroph, enrich- ary study using CH$OH. ment culture and environmental DNA samples (Dedysh et al., 2002; Dunfield et al., 1999; Henckel et al., 1999; METHODS Holmes et al., 1999; McDonald & Murrell, 1997). In cultivated strains, the phylogenetic relationships be- Soil and microcosm experimental conditions. Microcosm tween the derived amino-acid sequences of pmoA and experiments were used to characterize the active methylotroph amoA, and to a lesser extent mxaF, reflect those obtained communities in an oak (Quercus petraea)