Identification of active methylotroph populations in an acidic forest soil by stableisotope probing

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 (CH3)-C-13 OH or (CH4)-C-13- Distinct C-13-labelled DNA (13 C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13 C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the C-13-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 beta-subclass of the Proteobacteria. Analysis of AOB-selective 165 rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the C-13-DNA fractions, suggesting certain AOB assimilated a significant proportion of (CO2)-C-13, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the C-13-DNA were related to the 165 rDNA sequences of members of the Acidobacterium division, the beta-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 (CH3OH)-C-13 and (CH4)-C-13 SIP experiments thus provide a rational basis for further investigations 4 into the ecology of methylotroph populations in situ.