The ammonia-oxidizing archaea have recently been recognized as a significant component of many microbial communities in the biosphere. Although the overall stoichiometry of archaeal chemoautotrophic growth via ammonia (NH 3 ) oxidation to nitrite (NO 2 − ) is superficially similar to the ammonia-oxidizing bacteria, genome sequence analyses point to a completely unique biochemistry. The only genomic signature linking the bacterial and archaeal biochemistries of NH 3 oxidation is a highly divergent homolog of the ammonia monooxygenase (AMO). Although the presumptive product of the putative AMO is hydroxylamine (NH 2 OH), the absence of genes encoding a recognizable ammonia-oxidizing bacteria-like hydroxylamine oxidoreductase complex necessitates either a novel enzyme for the oxidation of NH 2 OH or an initial oxidation product other than NH 2 OH. We now show through combined physiological and stable isotope tracer analyses that NH 2 OH is both produced and consumed during the oxidation of NH 3 to NO 2 − by Nitrosopumilus maritimus, that consumption is coupled to energy conversion, and that NH 2 OH is the most probable product of the archaeal AMO homolog. Thus, despite their deep phylogenetic divergence, initial oxidation of NH 3 by bacteria and archaea appears mechanistically similar. They however diverge biochemically at the point of oxidation of NH 2 OH, the archaea possibly catalyzing NH 2 OH oxidation using a novel enzyme complex.M icrobial oxidation of ammonia (NH 3 ) to nitrite (NO 2 − ), the first step in nitrification, plays a central role in the global cycling of nitrogen. Recent studies have established that marine and terrestrial representatives of an abundant group of archaea, now classified as Thaumarchaeota, are autotrophic NH 3 oxidizers (1-5). Despite increasing evidence that ammonia-oxidizing archaea (AOA) generally outnumber ammonia-oxidizing bacteria (AOB), and likely nitrify in most natural environments, very little is known about their physiology or supporting biochemistry (6, 7). Genome sequence analyses have pointed to a unique pathway for NH 3 oxidation, likely using copper as a major redox active metal, and coupled to a variant of the hydroxypropionate/ hydroxybutyrate cycle (8). However, the only genome sequence feature that associates the archaeal pathway for NH 3 oxidation with that of the better characterized AOB is a divergent variant of the ammonia monooxygenase (AMO), which may or may not be a functional equivalent of the bacterial AMO. Thus, the supporting biochemistry of a biogeochemically significant group of microorganisms remains unresolved (8,9).Among the AOB, as represented by the model organism Nitrosomonas europaea, NH 3 is first oxidized to hydroxylamine (NH 2 OH) by AMO, an enzyme composed of three subunits encoded by amoC, amoA, and amoB genes (7). NH 2 OH is subsequently oxidized to NO 2 − by the hydroxylamine oxidoreductase (HAO) (7), a heme-rich enzyme encoded by the hao gene (7). Of the four electrons released from the oxidation of NH 2 OH to NO 2 − , two are transfe...

Ammonia (NH 3 )-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH 3 oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to <20 M concentrations of 1-alkynes C 8 and C 9 . In contrast, NH 3 oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 M C 8 (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH 4 ؉ -stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH 4 ؉ -stimulated net nitrification rates of 2 and 7 g N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 M) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyneresistant fraction of NH 4 ؉ -saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification. For about a century, most ammonia (NH 3 ) oxidation in soils was thought to be carried out by chemolithoautotrophic ammonia-oxidizing bacteria (AOB). In 2005, the nitrification paradigm changed with the discovery of another type of microorganism from the phylum Thaumarchaeota that performs NH 3 oxidation (1). Molecular techniques have shown that ammoniaoxidizing Thaumarchaeota (AOA) are widely distributed in soils throughout the world (2, 3). AOA are usually more numerous in soil than AOB, and in some soils, AOB are present at levels below the detection limit of quantitative PCR (qPCR) (4, 5). This has led to speculation about the extent to which AOA contribute to soil nitrification (6, 7). AOA may be more metabolically versatile than AOB, with some cultured AOA growing at acid pH (8), scavenging NH 4 ϩ at low concentrations (9), and showing mixotrophic growth on a combination of pyruvate and NH 4 ϩ (10), and an AOA soil population has been shown to convert organic N sources to NO 3 Ϫ (11). The evidence for AOA contributing to soil nitrification has arisen from enrichment approaches involving long incubations (4 to 6 weeks) of soil in the laboratory, where NH 3 oxidation was accompanied either by the incorp...

High representation by ammonia-oxidizing archaea (AOA) in marine systems is consistent with their high affinity for ammonia, efficient carbon fixation, and copper (Cu)-centric respiratory system. However, little is known about their response to nutrient stress. We therefore used global transcriptional and proteomic analyses to characterize the response of a model AOA, Nitrosopumilus maritimus SCM1, to ammonia starvation, Cu limitation and Cu excess. Most predicted protein-coding genes were transcribed in exponentially growing cells, and of ~74% detected in the proteome, ~6% were modified by N-terminal acetylation. The general response to ammonia starvation and Cu stress was downregulation of genes for energy generation and biosynthesis. Cells rapidly depleted transcripts for the A and B subunits of ammonia monooxygenase (AMO) in response to ammonia starvation, yet retained relatively high levels of transcripts for the C subunit. Thus, similar to ammonia-oxidizing bacteria, selective retention of amoC transcripts during starvation appears important for subsequent recovery, and also suggests that AMO subunit transcript ratios could be used to assess the physiological status of marine populations. Unexpectedly, cobalamin biosynthesis was upregulated in response to both ammonia starvation and Cu stress, indicating the importance of this cofactor in retaining functional integrity during times of stress.

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 microM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH3- and NH2OH-dependent O2 consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.

BackgroundIn response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family.ResultsOf the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 μM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.ConclusionsOur studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.