AUTHOR=Sharp Christine , Stott Matthew , Dunfield Peter 

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

JOURNAL=Frontiers in Microbiology

VOLUME=3

YEAR=2012

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2012.00303

DOI=10.3389/fmicb.2012.00303

ISSN=1664-302X

ABSTRACT=<p>Genomic analysis of the methanotrophic verrucomicrobium “<italic>Methylacidiphilum infernorum</italic>” 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 (<italic>pmo</italic>), “universal” <italic>pmoA</italic> polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, “<italic>Methylacidiphilum</italic>” fixes carbon autotrophically, and uses methane only for energy generation. As a result, techniques used to detect methanotrophs in the environment such as <sup>13</sup>CH<sub>4</sub>-stable isotope probing (SIP) and <italic>pmoA</italic>-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 <italic>Verrucomicrobia</italic> in the environment by labeling with <sup>13</sup>CO<sub>2</sub> and <sup>13</sup>CH<sub>4</sub>, individually and in combination. Testing the protocol in “<italic>M. infernorum</italic>” strain V4 resulted in assimilation of <sup>13</sup>CO<sub>2</sub> but not <sup>13</sup>CH<sub>4</sub>, verifying its autotrophic lifestyle. To specifically detect methanotrophs (as opposed to other autotrophs) via <sup>13</sup>CO<sub>2</sub>-SIP, a quantitative PCR (qPCR) assay specific for verrucomicrobial-<italic>pmoA</italic> genes was developed and used in combination with SIP. Incubation of an acidic, high-temperature geothermal soil with <sup>13</sup>CH<sub>4</sub> + <sup>12</sup>CO<sub>2</sub> caused little shift in the density distribution of verrucomicrobial-<italic>pmoA</italic> genes relative to controls. However, labeling with <sup>13</sup>CO<sub>2</sub> in combination with <sup>12</sup>CH<sub>4</sub> or <sup>13</sup>CH<sub>4</sub> induced a strong shift in the distribution of verrucomicrobial-<italic>pmoA</italic> 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 <italic>Verrucomicrobia</italic>. This is the first demonstration of autotrophic, non-proteobacterial methanotrophy <italic>in situ</italic>, and provides a tool to detect verrucomicrobial methanotrophs in other ecosystems.</p>