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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.

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