RT Journal Article SR Electronic(1) A1 Radajewski, Stefan A1 Webster, Gordon A1 Reay, David S. A1 Morris, Samantha A. A1 Ineson, Philip A1 Nedwell, David B. A1 Prosser, James I. A1 Murrell, J. ColinYR 2002 T1 Identification of active methylotroph populations in an acidic forest soil by stable-isotope probingccThe GenBank accession numbers for the sequences reported in this paper are AY080911–AY080961. JF Microbiology, VO 148 IS 8 SP 2331 OP 2342 DO https://doi.org/10.1099/00221287-148-8-2331 PB Microbiology Society, SN 1465-2080, AB Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3·5) microcosms that were exposed to 13CH3OH or 13CH4. Distinct 13C-labelled DNA (13C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but 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 certain AOB assimilated a significant proportion of 13CO2, 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-products of methylotrophic carbon metabolism. Results from the 13CH3OH and 13CH4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ. , UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-148-8-2331