Dataset Information

Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.

ABSTRACT: Genomic analysis of the methanotrophic verrucomicrobium "Methylacidiphilum infernorum" strain V4 has shown that most pathways conferring its methanotrophic lifestyle are similar to those found in proteobacterial methanotrophs. However, due to the large sequence divergence of its methane monooxygenase-encoding genes (pmo), "universal" pmoA polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, "Methylacidiphilum" fixes carbon autotrophically, and uses methane only for energy generation. As a result, techniques used to detect methanotrophs in the environment such as (13)CH(4)-stable isotope probing (SIP) and pmoA-targeted PCR do not detect verrucomicrobial methanotrophs, and they may have been overlooked in previous environmental studies. We developed a modified SIP technique to identify active methanotrophic Verrucomicrobia in the environment by labeling with (13)CO(2) and (13)CH(4), individually and in combination. Testing the protocol in "M. infernorum" strain V4 resulted in assimilation of (13)CO(2) but not (13)CH(4), verifying its autotrophic lifestyle. To specifically detect methanotrophs (as opposed to other autotrophs) via (13)CO(2)-SIP, a quantitative PCR (qPCR) assay specific for verrucomicrobial-pmoA genes was developed and used in combination with SIP. Incubation of an acidic, high-temperature geothermal soil with (13)CH(4) + (12)CO(2) caused little shift in the density distribution of verrucomicrobial-pmoA genes relative to controls. However, labeling with (13)CO(2) in combination with (12)CH(4) or (13)CH(4) induced a strong shift in the distribution of verrucomicrobial-pmoA genes towards the heavy DNA fractions. The modified SIP technique demonstrated that the primary methanotrophs active in the soil were autotrophs and belonged to the Verrucomicrobia. This is the first demonstration of autotrophic, non-proteobacterial methanotrophy in situ, and provides a tool to detect verrucomicrobial methanotrophs in other ecosystems.

SUBMITTER: Sharp CE

PROVIDER: S-EPMC3421453 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.

Sharp Christine E CE Stott Matthew B MB Dunfield Peter F PF

Frontiers in microbiology 20120817

Genomic analysis of the methanotrophic verrucomicrobium "Methylacidiphilum infernorum" strain V4 has shown that most pathways conferring its methanotrophic lifestyle are similar to those found in proteobacterial methanotrophs. However, due to the large sequence divergence of its methane monooxygenase-encoding genes (pmo), "universal" pmoA polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, "Methylacidiphilum" fixes carbon autotrophically, a ...[more]

PMID: 22912630

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Project description:Benthic environments harbor highly diverse and complex microbial communities that control carbon fluxes, but the role of specific uncultivated microbial groups in organic matter turnover is poorly understood. In this study, quantitative DNA stable isotope probing (DNA-qSIP) was used for the first time to link uncultivated populations of bacteria and archaea to carbon turnover in lacustrine surface sediments. After 1-week incubations in the dark with [13C]bicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural 13C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with [13C]bicarbonate, followed by extraction and concentration of high-molecular-weight (HMW) (>50-kDa) organic matter. qSIP showed that the labeled organic matter was turned over within 1 week by 823 microbial populations (operational taxonomic units [OTUs]) affiliated primarily with heterotrophic Proteobacteria, Chloroflexi, Verrucomicrobia, and Bacteroidetes However, several OTUs affiliated with the candidate microbial taxa Latescibacteria, Omnitrophica, Aminicentantes, Cloacimonates, AC1, Bathyarchaeota, and Woesearchaeota, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated with Proteobacteria) also became labeled in 1-week incubations with [13C]bicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with 13C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within the Firmicutes, Bacteroidetes, Verrucomicrobia, and Chloroflexi, highlighting their ecological importance. Our study helps define the role of several poorly understood, uncultivated microbial groups in the turnover of benthic carbon derived from "dark" primary production.IMPORTANCE Little is known about the ecological role of uncultivated microbial populations in carbon turnover in benthic environments. To better understand this, we used quantitative stable isotope probing (qSIP) to quantify the abundance of diverse, specific groups of uncultivated bacteria and archaea involved in autotrophy and heterotrophy in a benthic lacustrine habitat. Our results provide quantitative evidence for active heterotrophic and autotrophic metabolism of several poorly understood microbial groups, thus demonstrating their relevance for carbon turnover in benthic settings. Archaeal ammonia oxidizers were significant drivers of in situ "dark" primary production supporting the growth of heterotrophic bacteria. These findings expand our understanding of the microbial populations within benthic food webs and the role of uncultivated microbes in benthic carbon turnover.

Dataset Information

Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.

Publications

Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.

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