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.