Pathways and key intermediates required for obligate aerobic ammonia-dependent chemolithotrophy in bacteria and Thaumarchaeota

Abstract

Chemolithotrophic ammonia-oxidizing bacteria and Thaumarchaeota are central players in the global nitrogen cycle. Obligate ammonia chemolithotrophy has been characterized for bacteria; however, large gaps remain in the Thaumarchaeotal pathway. Using batch growth experiments and instantaneous microrespirometry measurements of resting biomass, we show that the terrestrial Thaumarchaeon Nitrososphaera viennensis EN76 T exhibits tight control over production and consumption of nitric oxide (NO) during ammonia catabolism, unlike the ammonia-oxidizing bacterium Nitrosospira multiformis ATCC 25196 T . In particular, pulses of hydroxylamine into a microelectrode chamber as the sole substrate for N. viennensis resulted in iterative production and consumption of NO followed by conversion of hydroxylamine to nitrite. In support of these observations, oxidation of ammonia in growing cultures of N. viennensis, but not of N. multiformis, was inhibited by the NO-scavenger PTIO. When based on the marginal nitrous oxide (N 2 O) levels detected in cell-free media controls, the higher levels produced by N. multiformis were explained by enzyme activity, whereas N 2 O in N. viennensis cultures was attributed to abiotic reactions of released N-oxide intermediates with media components. Our results are conceptualized in a pathway for ammonia-dependent chemolithotrophy in Thaumarchaea, which identifies NO as an essential intermediate in the pathway and implements known biochemistry to be executed by a proposed but still elusive copper enzyme. Taken together, this work identifies differences in ammonia-dependent chemolithotrophy between bacteria and the Thaumarchaeota, advances a central catabolic role of NO only in the Thaumarchaeotal pathway and reveals stark differences in how the two microbial cohorts contribute to N 2 O emissions.

Vertical segregation among pathways mediating nitrogen loss (N<sub>2</sub> and N<sub>2</sub>O production) across the oxygen gradient in a coastal upwelling ecosystem

“…Ammonium oxidizing archaea may also participate in N 2 O production (Martens-Habbena et al, 2009;Santoro et al, 2010Santoro et al, , 2011Jung et al, 2014). In this sense, the large dominance of 45 N 2 O over 46 N 2 O observed could be explained by the recently described "hybrid" pathway (Kozlowski et al, 2016), in which labelled hydroxylamine ( 15 NH 2 OH) produced from AO might be combined with NO reduced from the NO − 2 present in the native pool of the incubations. The coupling between AO and NiR after the formation of NO − 2 is less plausible, because any labelled NO − 2 that is produced would be diluted by the existing pool of NO − 2 in the environment.…”

“…4c, d). The O 2 -limited incubations carried out during September, relative to in situ O 2 conditions during the January experiments, could, together with a greater availability of particles occurring at the sampling depth, generate an O 2 -depleted environment, in which the yield of N 2 O formed through nitrifier denitrification Goreau et al, 1980;Lipschultz et al, 1981;de Wilde and de Bie, 2000) or through the "hybrid" pathway (Kozlowski et al, 2016) will increase. There is ample evidence that N 2 O production via nitrification in aquatic systems is increased under conditions of reduced oxygen and abundant ammonium availability McElroy et al, 1978;de Wilde and de Bie, 2000;Löscher et al, 2012).…”

“…N 2 O can also be produced during ammonium (NH + 4 ) oxidation, a chemolithoautotrophic process by which NH + 4 is oxidized aerobically either by bacteria or archaea. However, the pathways of N 2 O production appear to differ between ammonium oxidizers from these two domains (Blainey et al, 2011;Kim et al, 2011;Spang et al, 2012;Kozlowski et al, 2016). While N 2 O is a minor product in ammonium oxidation (up to 10 % relative to NO − 2 production; Goreau et al, 1980), the yield of N 2 O produced by nitrifiers relative to NO − 2 increases ∼ 20 times as O 2 saturation in the atmosphere diminishes to 1 % (Kester et al, 1977).…”

“…Conversely, and despite several reports suggesting that archaeal ammonium oxidizers are an important source of N 2 O based on their ubiquity, abundance, activity and high NH + 4 affinity (Könneke et al, 2005;Wuchter et al, 2006;MartensHabbena et al, 2009;Santoro et al, 2011;Jung et al, 2014), the pathway through which the N 2 O is produced by this group is still not fully understood. Nonetheless, it was recently demonstrated that archaea involved in ammonium oxidation do not have the enzymatic capacity to reduce NO to N 2 O through nitrifier denitrification, and N 2 O formation was suggested to occur through a hybrid pathway pairing nitrogen from NH 2 OH and NO (Stieglmeier et al, 2014;Kozlowski et al, 2016).…”

Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream

“…Such experiments provide key insights into the instantaneous rate of ammonia oxidation (or nitrification) but still have the following potential defects: (i) potential stimulation of nitrification by added tracers, (ii) underestimation of the ammonia oxidation rate by isotope dilution of nitrite arising from the concurrency of nitrification and denitrification, and (iii) potential change in microbial community composition during incubation. Experiments involving incubation with selective inhibitors of AOB or AOA activity (e.g., allylthiourea, 1-octyne, and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) could be useful to assess bacterial and archaeal contributions to total nitrification (15)(16)(17), but it is still uncertain to what extent the ammonia oxidation rates measured in the inhibition experiments represent bacterial and archaeal ammonia oxidation rates in nature, as the minimum concentrations of the inhibitors to stop ammonia oxidation activity in natural environments with complex physical and ecological characteristics (e.g., permeability and cell density in the local environment) are largely unknown (16,18).…”