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...

Root-deposited photosynthate (rhizodeposition) is an important source of readily available carbon (C) for microbes in the vicinity of growing roots. Plant nutrient availability is controlled, to a large extent, by the cycling of this and other organic materials through the soil microbial community. Currently, our understanding of microbial community dynamics associated with rhizodeposition is limited. We used a 13 C pulse-chase labeling procedure to examine the incorporation of rhizodeposition into individual phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of greenhouse-grown annual ryegrass (Lolium multiflorum Lam. var. Gulf). Labeling took place during a growth stage in transition between active root growth and rapid shoot growth on one set of plants (labeling period 1) and 9 days later during the rapid shoot growth stage on another set of plants (labeling period 2). Temporal differences in microbial community composition were more apparent than spatial differences, with a greater relative abundance of PLFAs from gram-positive organisms (i15:0 and a15:0) in the second labeling period. Although more abundant, gram-positive organisms appeared to be less actively utilizing rhizodeposited C in labeling period 2 than in labeling period 1. Gram-negative bacteria associated with the 16:15 PLFA were more active in utilizing 13 C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:26,9 was the most highly labeled. These results demonstrate the effectiveness of using 13 C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in ␣-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.

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...

The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196 T ) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.Nitrification is a key process in the nitrogen cycle of terrestrial, wastewater, and marine systems. The first step in the aerobic process is the oxidation of ammonia, mediated by ammonia-oxidizing bacteria (AOB) or ammonia-oxidizing archaea. Because we are particularly interested in the genetic complement adaptive for ammonia-based chemolithotrophy in the soil environment, we completed the genome sequence of the soil AOB Nitrosospira multiformis (ATCC 25196 T ). Obtaining the N. multiformis genome sequence offers a unique opportunity for comparison to the available genomes of other betaproteobacterial AOB (beta-AOB), Nitrosomonas europaea (16), and Nitrosomonas eutropha (63). The AOB isolated or detected by noncultural methods in aerobic surface soils all have been members of the Betaproteobacteria (order Nitrosomonadales, family Nitrosomonadaceae). Recent evidence suggests that Crenarchaeota may also contribute to ammonia oxidation in soils (43).The sequenced AOB, Nitrosospira multiformis ATCC 25196 T , was isolated from soil near Paramaribo, Surinam, by enrichment culturing, followed by serial dilution to extinction (71). Originally, this isolate was the type strain for Nitrosolobus multiformis, wit...