Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis

Abstract

Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ∼1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ∼70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles.

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

“…This variation may be attributed to incomplete cell lysis restricting the access of the Phi29 enzyme to the DNA, partial degradation of the template DNA before MDA and/or bias of the amplification reaction. Performing combined assemblies of closely related single cells allows estimated genome recoveries approaching completeness 1,8,10 . coMpetInG FInancIal Interests The authors declare no competing financial interests.…”

“…For example, we demonstrated that single-cell genomes from uncultivated phyla substantially improve the phylogenetic anchoring of metagenomic reads 1 . Mapping of metagenome sequences to single-cell genomes has not only been applied to validating metagenome assembly and binning 7 but also to subsequently improving the single-cell assemblies, as has been shown for SR1 bacteria, atribacteria and ammoniaoxidizing archaea [8][9][10] . Other studies successfully used fragment recruitment by single-cell genomes to investigate biogeographic distribution of uncultivated, marine taxa 11,12 .…”

Genome-enabled transcriptomics reveals archaeal populations that drive nitrification in a deep-sea hydrothermal plume

“…Both come from shallow waters, the sponge symbiont Cenarchaeum symbiosum (Hallam et al, 2006) and the aquarium isolate N. maritimus (Walker et al, 2010). Recently, draft genome sequence has been obtained from single cells and San Francisco Bay sediment enrichments of Nitrosoarchaeum limnia, recovered from an estuary in San Francisco bay (Blainey et al, 2011) and from a soil isolate, Nitrososphaera viennensis (Tourna et al, 2011). Characterization of these genomes suggests they use a modified 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon fixation, and have a copper-dependent system for ammonia oxidation and electron-transfer that is distinct from AOB.…”

A Method for Cell Culture and Maintenance of Ammonia-Oxidizing Archaea in Agar Stab

“…Among them, nitrifying organisms that were previously thought to be restricted to a few groups within the b-and c-proteobacteria are highly abundant and seem to be mainly related to the non-extremophilic Crenarchaeota (ammonia-oxidizing archaea, AOA) [3]. This result was further supported by the discovery of archaeal amoA genes encoding key enzymes of the ammonia oxidation from environmental metagenome studies [4], although the transcription of these genes was relatively less active than those of bacteria [5]. Consequently, much evidence indicates that AOA are also plausible contributors with ammonia-oxidizing bacteria to nitrification of the microcosm.…”