15N2–DNA–stable isotope probing of diazotrophic methanotrophs in soil

Nucleic acid stable isotope probing (SIP) is a powerful tool that can identify and characterize the microorganisms that mediate specific soil processes and explore the flow of C and N through functional groups in the soil food web. While 13C–SIP has been used successfully in a range of applications, methodological constraints have limited the applicability of 15N-labelled compounds in nucleic acid SIP. However, 15N–DNA–SIP can now be achieved and this method when used with 15N2 provides a powerful new tool for characterizing free-living diazotrophs in natural ecosystems. A diverse array of non-cultivated diazotrophs have been observed in soil and yet the characteristics of these organisms and their environmental significance remain almost completely unknown. 15N2–DNA–SIP can identify those diazotrophs that are active in situ while providing access to gene sequences and genome fragments that can yield insights on their evolutionary history and functional capacities. Further insights on the ecology of free-living diazotrophs in soil can be provided by performing 15N2–DNA–SIP on microcosms in which the response of the diazotrophic community is determined in relation to experimental manipulation. We describe the use of 15N2–DNA–SIP to explore linkages between different C sources and N-fixation by specific diazotroph populations in soil. Methane addition to soil was observed to stimulate N-fixation and the organisms that were found to be responsible for this activity were Type II methanotrophs most closely related to the genus Methylocystis. This report provides insights on the use of nucleic acid SIP to identify and characterize microorganisms that mediate specific soil processes and represents the first time that a specific group of methanotrophs has been shown to mediate N-fixation while in the soil environment.