Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea Neeraja Vajralaa,1, Willm Martens-Habbenab,1, Luis A. Sayavedra-Sotoa, Andrew Schauerc, Peter J. Bottomleyd, David A. Stahlb, and Daniel J. Arpa,2 Departments of aBotany and Plant Pathology and dMicrobiology, Oregon State University, Corvallis, OR 97331; and Departments of bCivil and Environmental Engineering and cEarth and Space Science, University of Washington, Seattle, WA 98195 Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved December 7, 2012 (received for review August 17, 2012) The ammonia-oxidizing archaea have recently been recognized as or cytochromes c554 and cM552 critical for energy conversion in a significant component of many microbial communities in the AOB (8–15). Thus, either the product of NH3 oxidation is not biosphere. Although the overall stoichiometry of archaeal chemo- NH2OH or, alternatively, these phylogenetically deeply branch- − autotrophic growth via ammonia (NH3) oxidation to nitrite (NO2 ) ing thaumarchaea use a novel biochemistry for NH2OH oxida- is superficially similar to the ammonia-oxidizing bacteria, genome tion and electron transfer (8). sequence analyses point to a completely unique biochemistry. The In an attempt to gain further insights into the biochemistry and only genomic signature linking the bacterial and archaeal bioche- physiology of these unique archaeal nitrifiers, we here investigated Nitrosopumilus maritimus mistries of NH3 oxidation is a highly divergent homolog of the the role of NH2OH in metabolism. ammonia monooxygenase (AMO). Although the presumptive These studies were complicated by the extremely oligotrophic product of the putative AMO is hydroxylamine (NH2OH), the ab- character of this organism contributing to very low cell densities sence of genes encoding a recognizable ammonia-oxidizing bacte- in culture (16). To overcome the challenge of working with low ria-like hydroxylamine oxidoreductase complex necessitates either cell density cultures of N. maritimus, we established a method to a novel enzyme for the oxidation of NH2OH or an initial oxidation concentrate cells on nylon membrane filters such that the cells − product other than NH2OH. We now show through combined remained competent for NH3-dependent NO2 formation and physiological and stable isotope tracer analyses that NH2OH is oxygen (O2) uptake. This method enabled us to carry out relatively SCIENCES both produced and consumed during the oxidation of NH3 to short duration physiological studies and stable isotope tracer ENVIRONMENTAL − NO2 by Nitrosopumilus maritimus, that consumption is coupled experiments directed at determining if N. maritimus can oxidize − to energy conversion, and that NH2OH is the most probable prod- exogenous NH2OH to NO2 while consuming O2 and producing uct of the archaeal AMO homolog. Thus, despite their deep phy- ATP, and if NH2OH is an intermediate in NH3 oxidation pathway logenetic divergence, initial oxidation of NH3 by bacteria and of N. maritimus. archaea appears mechanistically similar. They however diverge bio- chemically at the point of oxidation of NH2OH, the archaea possibly Results − catalyzing NH2OH oxidation using a novel enzyme complex. N. maritimus Oxidizes NH2OH to NO2 . The apparent lack of genes encoding a recognizable AOB-like HAO complex in the genome − N. maritimus icrobial oxidation of ammonia (NH ) to nitrite (NO ), the of has generated considerable interest in the 3 2 fi Mfirst step in nitrification, plays a central role in the global pathway of NH3 oxidation in AOA (8). Because puri cation and cycling of nitrogen. Recent studies have established that marine direct biochemical characterization of AMO enzymes has thus far been unsuccessful, we aimed to determine if N. maritimus and terrestrial representatives of an abundant group of archaea, − could convert NH2OH to NO2 . To determine if N. maritimus now classified as Thaumarchaeota, are autotrophic NH3 oxidizers − – can oxidize NH2OH to NO2 , it was necessary to expose the (1 5). Despite increasing evidence that ammonia-oxidizing N. maritimus + archaea (AOA) generally outnumber ammonia-oxidizing bacte- cells to NH2OH in the absence of the NH4 used to grow the culture. Hence, a technique was developed to con- ria (AOB), and likely nitrify in most natural environments, very μ fi little is known about their physiology or supporting biochemistry centrate the cells onto 0.2- m pore size nylon membrane lters (6, 7). Genome sequence analyses have pointed to a unique using a vacuum manifold system. The manifold system allowed simultaneous filtration of several liters of culture. In addition to pathway for NH3 oxidation, likely using copper as a major redox + active metal, and coupled to a variant of the hydroxypropionate/ concentrating the cells and separating them from residual NH4 in the medium, the nylon membranes served as a solid support hydroxybutyrate cycle (8). However, the only genome sequence for convenient handling of concentrated cells in subsequent in- feature that associates the archaeal pathway for NH oxidation 3 cubation assays. The membrane-associated N. maritimus cells ac- with that of the better characterized AOB is a divergent variant tively oxidized NH for several hours. of the ammonia monooxygenase (AMO), which may or may not 3 To investigate whether NH2OH can be oxidized by N. maritimus, be a functional equivalent of the bacterial AMO. Thus, the − a series of short-term NO accumulation assays was conducted, supporting biochemistry of a biogeochemically significant group 2 in which membrane-associated N. maritimus cells were exposed of microorganisms remains unresolved (8, 9). to various concentrations of NH OH. Irrespective of the initial Among the AOB, as represented by the model organism 2 Nitrosomonas europaea,NH3 is first oxidized to hydroxylamine (NH OH) by AMO, an enzyme composed of three subunits 2 Author contributions: N.V., W.M.-H., L.A.S.-S., P.J.B., D.A.S., and D.J.A. designed research; encoded by amoC, amoA, and amoB genes (7). NH2OH is sub- − N.V., W.M.-H., L.A.S.-S., and D.A.S. performed research; A.S. contributed new reagents/ sequently oxidized to NO2 by the hydroxylamine oxidoreductase analytic tools; N.V., W.M.-H., L.A.S.-S., A.S., P.J.B., D.A.S., and D.J.A. analyzed data; and (HAO) (7), a heme-rich enzyme encoded by the hao gene (7). Of N.V., W.M.-H., L.A.S.-S., and D.A.S. wrote the paper. fl the four electrons released from the oxidation of NH2OH to The authors declare no con ict of interest. − NO2 , two are transferred to the terminal oxidase for respiratory This article is a PNAS Direct Submission. purposes and two are transferred to AMO for further oxidation 1N.V. and W.M.-H. contributed equally to this work. of NH3 (7). Although all available genome sequences for the 2To whom correspondence should be addressed. E-mail: [email protected]. amoB amoC AOA contain homologs of the bacterial AMO ( , , This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and amoA), there are no obvious homologs of AOB-like HAO, 1073/pnas.1214272110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1214272110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 concentration of NH2OH (50–200 μM), N. maritimus oxidized − ∼50 μMNH2OH to NO2 over 20 h (Fig. 1). During the first 30 min of exposure to 200 μMNH2OH, the rate of NH2OH- − dependent NO2 production was equal to the rate of NH3-de- − pendent NO2 production [0.19 ± 0.01 μmol/(min × mg protein)]. With longer incubation times, the rate of NH2OH oxidation de- creased gradually and finally stopped after accumulation of − ∼50 μMofNO2 (Fig. 1). Addition of 1 mM NH2OH led to a − complete inhibition of the NO2 formation (Fig. 1), reflecting a toxicity at higher concentrations also observed for AOB (17). Attempts to grow N. maritimus on NH2OH as sole energy source failed as previously observed in N. europaea (18). Acetylene (C2H2) (19) and allylthiourea (ATU) (20) are specific inhibitors of NH3 oxidation by AMO in AOB and AOA (19–21). In the present work, the ability of N. maritimus to oxidize NH3 or NH2OH in presence of either C2H2 or ATU was analyzed − by monitoring NO2 accumulation. C2H2 (0.1%) was added to the headspace of sealed bottles containing membrane-attached N. maritimus cells in synthetic crenarchaeota medium (SCM) con- + taining either NH4 or NH2OH. C2H2 completely inhibited NH3- − dependent NO2 accumulation but had no effect on NH2OH- − dependent NO2 production (Fig. S1 A and B)aspreviously observed in N. europaea (19). Similarly, the addition of ATU also − inhibited NH3-dependent NO2 production but did not inhibit − NH2OH-dependent NO2 production (Fig. S1 A and B)(20). These results clearly indicate that N. maritimus can oxidize Fig. 2. Stoichiometry of NH2OH oxidation in N. maritimus.O2 uptake (solid − NH2OH and its oxidation is independent of the AMO activity squares), NH2OH consumed (open circles), NO2 produced (gray triangles); (Fig. S1 A and B). Data are means of triplicates, with variation of less than 10%. Incubations were carried out for 8 min while the conditions were at optimum (i.e., the NH2OH Oxidation Is Coupled to O2 Uptake in N. maritimus. Further unavoidable degradation of NH2OH did not interfere with the stoichiome- experiments were conducted to determine if oxidation of NH2OH try). The experiment was repeated at least three times and produced similar − results. Error bars represent SEM. to NO2 by N. maritimus is accompanied by consumption of a stoichiometric amount of O2. To test this, NH2OH consumption, − O2 uptake, and NO2 accumulation were independently deter- 1NH3:1.5 O2 previously reported for both the bacterial and ar- mined (Fig. 2). The rate of NH2OH-dependent O2 consumption − by N. maritimus remained constant for 8 min at a rate of 0.20 ± chaeal oxidation of NH3 to NO2 (16, 23).