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Volume 148, Issue 8

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing

cThe GenBank accession numbers for the sequences reported in this paper are AY080911–AY080961.

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Abstract

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 CHOH or CH. Distinct C-labelled DNA (C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the C-DNA revealed that bacteria related to the genera , , and had assimilated the C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CHOH 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 . Analysis of AOB-selective 16S rDNA amplification products identified and sequences in the C-DNA fractions, suggesting certain AOB assimilated a significant proportion of CO, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the C-DNA were related to the 16S rDNA sequences of members of the division, the β- and the order , 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 CHOH and CH SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations

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Keyword(s): 13C , AOB, ammonia-oxidizing bacteria , community structure , culture-independent techniques , DGGE, denaturing gradient gel electrophoresis , functional genes , g.d.w., g dry weight , methanotrophs , OTU, operational taxonomic unit and SIP, stable-isotope probing
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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.
Microbiology 148, 2331 (2002); https://doi.org/10.1099/00221287-148-8-2331
/content/journal/micro/10.1099/00221287-148-8-2331
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