Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered as a two-step process catalyzed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes both the pathways for ammonia and nitrite oxidation, which are concomitantly expressed during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Summary paragraphNitrification, the oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalyzed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira1,2, which alone convert ammonia to nitrate, raised questions about the ecological niches where comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolated the first pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats based on a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, not all AOA are most competitive in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analyzed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM13. These results suggest a role of comammox organisms for nitrification under oligotrophic and dynamic conditions.

Ammonia-and nitrite-oxidizers are collectively responsible for the aerobic oxidation of ammonia via nitrite to nitrate and play essential roles for the global biogeochemical nitrogen cycle. The physiology of these nitrifying microbes has been intensively studied since the first experiments of Sergei Winogradsky more than a century ago. Urea and ammonia are the only recognized energy sources that promote the aerobic growth of ammonia-oxidizing bacteria and archaea. Here we report the aerobic growth of a pure culture of the ammonia-oxidizing thaumarchaeote Nitrososphaera gargensis 1 on cyanate as the sole source of energy and reductant, the first organism known to do so. Cyanate, which is a potentially important source of reduced nitrogen in aquatic and terrestrial ecosystems 2 , is converted to ammonium and CO 2 by this archaeon using a cyanase that is induced upon addition of this compound. Within the cyanase gene family, this cyanase is a member of a distinct clade that also contains cyanases of nitrite-oxidizing bacteria of the genus Nitrospira. We demonstrate by co-culture experiments that these nitrite-oxidizers supply

Proteolytic processing of amyloid precursor protein generates ␤-amyloid (A␤) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in A␤ production, the intramembranous ␥-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in ␥-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric ␥-secretase complex. Using kinetic labeling studies we show that in PS1 ؊/؊ /PS2؊/؊ cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1 ؊/؊ fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing ␥-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the ␥-secretase complex.Alzheimer's disease is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides, termed ␤-amyloid (A␤), 1 which are generated by proteolytic processing of amyloid precursor protein (APP) (1). Mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) account for the majority of the cases of familial early-onset Alzheimer's disease (2). Biochemical and genetic evidence demonstrate that PS1 and PS2 facilitate the final step in A␤ production, the intramembranous ␥-secretase cleavage of APP (3-5). Moreover, it has been proposed that PS1 deficiency alters the trafficking of select membrane proteins (4). Familial Alzheimer's diseaselinked mutant PS1 and PS2 elevate the levels of highly amyloidogenic A␤42 peptides, thus promoting A␤ deposition in senile plaques (6, 7). However, the specific mechanism(s) involved in the selective elevation of A␤42 production by familial Alzheimer's disease-linked PS variants has not been clearly understood.The precise role played by PS1 and PS2 in ␥-secretase cleavage of APP and Notch has been under intense scrutiny. Biochemical fractionation and ␥-secretase inhibitor studies have suggested a catalytic role for PS1 in the intramembraneous cleavage of substrates (5, 8 -10). On the contrary, biochemical evidence also suggests an indirect role for PS1 in facilitating ␥-secretase cleavage of substrates (11). It is unlikely that this apparent controversy can be resolved by biochemical analysis, since PS-derived N-terminal (NTF) and C-terminal (CTF) fragments are components of high molecular weight multimeric protein complexes (12). One of the more recent members of the g...

Release of A␤ peptides from ␤-amyloid precursor protein (APP) requires sequential cleavage by two endopeptidases, ␤-and ␥-secretases. ␤-Secretase was recently identified as a novel membrane-bound aspartyl protease, named BACE1, Asp2, or memapsin 2. Employing confocal microscopy and subcellular fractionation, we have found that BACE1 is largely situated in the distal Golgi membrane with a minor presence in the endoplasmic reticulum, endosomes, and plasma membrane in human neuroblastoma SHEP cells and in mouse Neuro-2a cell lines expressing either endogenous mouse BACE1 or additional exogenous human BACE1. The major cellular ␤-secretase activity is located in the late Golgi apparatus, consistent with its cellular localization. Furthermore, we demonstrate that the single transmembrane domain of BACE1 alone determines the retention of BACE1 to the Golgi compartments, through examination of recombinant proteins of various BACE1 fragments fused to a reporter green fluorescence protein. In addition, we show that the transmembrane domain of BACE1 is required for the access of BACE1 enzymatic activity to the cellular APP substrate and hence for the optimal generation of the C-terminal fragment of APP (CTF99). The results suggest a molecular and cell biological mechanism for the regulation of ␤-secretase activity in vivo.The pathological hallmarks of Alzheimer's disease are neuritic plaques and neurofibrillary tangles (see reviews in Refs. 1-3). The neuritic plaques, also known as senile plaques, are predominantly composed of A␤, a cluster of aggregated and heterogeneous peptides of 39 -43 amino acids (4). The most pathogenic A␤ peptide is the less soluble 42-amino acid peptide (A␤ 42 ), although the concentration of A␤ 40 is generally much higher than A␤ 42 . Excision of A␤ peptides from their amyloid precursor protein requires sequential proteolytic cleavages by the ␤-and ␥-secretases, respectively. ␤-secretase cleaves APP 1 to produce two major components: secreted ectodomain sAPP ␤ and the C-terminal fragment CTF99. The latter can be further cleaved by ␥-secretase to release A␤. Genetic linkage analysis suggests that increased total A␤ levels or an increase in the ratio of A␤ 42 /A␤ 40 is associated with the severity of AD dementia (reviewed in Refs.

Thaumarchaeota are responsible for a significant fraction of ammonia oxidation in the oceans and in soils that range from alkaline to acidic. However, the adaptive mechanisms underpinning their habitat expansion remain poorly understood. Here we show that expansion into acidic soils and the high pressures of the hadopelagic zone of the oceans is tightly linked to the acquisition of a variant of the energy-yielding ATPases via horizontal transfer. Whereas the ATPase genealogy of neutrophilic Thaumarchaeota is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically distinct clades of acidophilic/acid-tolerant and piezophilic/piezotolerant species. A presumptive function of pumping cytoplasmic protons at low pH is consistent with the experimentally observed increased expression of the V-ATPase in an acid-tolerant thaumarchaeote at low pH. Consistently, heterologous expression of the thaumarchaeotal V-ATPase significantly increased the growth rate of E. coli at low pH. Its adaptive significance to growth in ocean trenches may relate to pressure-related changes in membrane structure in which this complex molecular machine must function. Together, our findings reveal that the habitat expansion of Thaumarchaeota is tightly correlated with extensive horizontal transfer of atp operons.

Abiotic processes involving the reactive ammonia-oxidation intermediates nitric oxide (NO) or hydroxylamine (NHOH) for NO production have been indicated recently. The latter process would require the availability of substantial amounts of free NHOH for chemical reactions during ammonia (NH) oxidation, but little is known about extracellular NHOH formation by the different clades of ammonia-oxidizing microbes. Here we determined extracellular NHOH concentrations in culture media of several ammonia-oxidizing bacteria (AOB) and archaea (AOA), as well as one complete ammonia oxidizer (comammox) enrichment (Ca. Nitrospira inopinata) during incubation under standard cultivation conditions. NHOH was measurable in the incubation media of Nitrosomonas europaea, Nitrosospira multiformis, Nitrososphaera gargensis, and Ca. Nitrosotenuis uzonensis, but not in media of the other tested AOB and AOA. NHOH was also formed by the comammox enrichment during NH oxidation. This enrichment exhibited the largest NHOH:final product ratio (1.92%), followed by N. multiformis (0.56%) and N. gargensis (0.46%). The maximum proportions of NH converted to NO via extracellular NHOH during incubation, estimated on the basis of NHOH abiotic conversion rates, were 0.12%, 0.08%, and 0.14% for AOB, AOA, and Ca. Nitrospira inopinata, respectively, and were consistent with published NH:NO conversion ratios for AOB and AOA.

The biotransformation of some micropollutants has previously been observed to be positively associated with ammonia oxidation activities and the transcript abundance of the archaeal ammonia monooxygenase gene (amoA) in nitrifying activated sludge. Given the increasing interest in and potential importance of ammonia-oxidizing archaea (AOA), we investigated the capabilities of an AOA pure culture, Nitrososphaera gargensis, to biotransform ten micropollutants belonging to three structurally similar groups (i.e., phenylureas, tertiary amides, and tertiary amines). N. gargensis was able to biotransform two of the tertiary amines, mianserin (MIA) and ranitidine (RAN), exhibiting similar compound specificity as two ammonia-oxidizing bacteria (AOB) strains that were tested for comparison. The same MIA and RAN biotransformation reactions were carried out by both the AOA and AOB strains. The major transformation product (TP) of MIA, α-oxo MIA was likely formed via a two-step oxidation reaction. The first hydroxylation step is typically catalyzed by monooxygenases. Three RAN TP candidates were identified from nontarget analysis. Their tentative structures and possible biotransformation pathways were proposed. The biotransformation of MIA and RAN only occurred when ammonia oxidation was active, suggesting cometabolic transformations. Consistently, a comparative proteomic analysis revealed no significant differential expression of any protein-encoding gene in N. gargensis grown on ammonium with MIA or RAN compared with standard cultivation on ammonium only. Taken together, this study provides first important insights regarding the roles played by AOA in micropollutant biotransformation.