ABSTRACT: Source waters sampled from Perpetual Spouter hot spring (pH 7.03, 86.4°C), Yellowstone National Park, WY, have low concentrations of total ammonia, nitrite, and nitrate, suggesting nitrogen (N) limitation and/or tight coupling of N cycling processes. Dominant small-subunit rRNA sequences in Perpetual Spouter source sediments are closely affiliated with the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii" and the putatively nitrogen-fixing (diazotrophic) bacterium Thermocrinis albus, respectively, suggesting that these populations may interact at the level of the bioavailable N pool, specifically, ammonia. This hypothesis was evaluated by using a combination of geochemical, physiological, and transcriptomic analyses of sediment microcosms. Amendment of microcosms with allylthiourea, an inhibitor of ammonia oxidation, decreased rates of acetylene reduction (a proxy for N2 fixation) and nitrite production (a proxy for ammonia oxidation) and decreased transcript levels of structural genes involved in both nitrogen fixation (nifH) and ammonia oxidation (amoA). In contrast, amendment of microcosms with ammonia stimulated nitrite production and increased amoA transcript levels while it suppressed rates of acetylene reduction and decreased nifH transcript levels. Sequencing of amplified nifH and amoA transcripts from native sediments, as well as microcosms, at 2 and 4 h postamendment, indicates that the dominant and responsive populations involved in ammonia oxidation and N2 fixation are closely affiliated with Ca. Nitrosocaldus yellowstonii and T. albus, respectively. Collectively, these results suggest that ammonia-oxidizing archaea, such as Ca. Nitrosocaldus yellowstonii, have an apparent affinity for ammonia that is higher than that of the diazotrophs present in this ecosystem. Depletion of the bioavailable N pool through the activity of ammonia-oxidizing archaea likely represents a strong selective pressure for the inclusion of organisms capable of nitrogen fixation in geothermal communities. These observations help to explain the strong pattern in the codistribution of ammonia-oxidizing archaea and diazotrophs in circumneutral-to-alkaline geothermal springs.
