Abstract

Nitrogen is integral to all living systems, but its diverse forms are interconverted dynamically through reductions and oxidations that constitute the nitrogen cycle. Ecosystems gain combined nitrogen by bacterial and archaeal reduction in atmospheric N2, while combined nitrogen is lost to the atmosphere as nitrogenous gases through denitrification. We sought to characterize the diversity and activity of free-living nitrogen-fixing bacteria or diazotrophs in natural prairie grasslands and the activity of the other steps of the nitrogen cycle through metatranscriptomics. DNA and mRNA were obtained from prairie sites that did not contain any leguminous plants to avoid symbiotic nitrogen fixation. Both 16S rRNA gene and nifH amplicon pools were sequenced to characterize the diversity of diazotrophs, and the expression levels of N-cycle genes were quantified by RNAseq. Nitrogen fixation without and with added carbohydrates was quantified by measuring the incorporation of 15N2. The soil samples contained a diversity of diazotrophs, as reflected both by 16S rRNA gene and nifH gene sequences. Carbohydrate amendment of soil samples led to substantial 15N2 incorporation, showing that members of the resident microbiota were able to fix nitrogen. However, we did not detect 15N2 incorporation in unamended soil samples, pointing to a lack of in situ fixation. The very low levels of nitrogenase gene transcripts supported this finding. In contrast, transcripts for nitrification and denitrification genes were expressed, pointing to a gap in the nitrogen cycle. The prevalence of diazotrophs with undetectable nitrogen fixing activity suggested the absence of available carbohydrates to provide the energy needed. The apparent imbalance in the nitrogen cycle would not be sustainable, so other possible mechanisms of acquiring combined nitrogen should be explored.

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