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.

Figure 1. Phylogeny of Ammonia Oxidizing Archaea gene sequences.

Neighbor-joining phylogenetic tree of (A) archaeal amoA gene sequences and (B) archaeal 16S rRNA gene sequences. Grey boxes highlight the putative low-salinity group. Significant bootstrap values (≥50) from 1000 replicates are shown in italics at branch nodes. Neighbor-joining bootstrap values are above the line and parsimony values are below the line.

Figure 2. Assembly of the N. limnia datasets.

Rarefaction analysis of the N. limnia sequence data showing assembly of the consensus dataset and as a function of the number of included single-cells.

Figure 3. Circular representation of the N. limnia genome.

From the outer ring to the inner ring: Scaffold breakpoints are indicated by the inside tick marks, predicted genes on the forward strand, predicted genes the reverse strand, G+C content, GC skew, and GGGT skew. On the outer two rings, protein coding sequences are colored gray, tRNA genes in blue, rRNA genes in red, and noncoding RNA genes in green.

Figure 4. Comparison of three AOA genomes.

(A) The top panel shows nucleotide identity of blast hits between the reference genomes and the N. limnia genome. Hits for N. maritimus and C. symbiosum A are colored according to the position in the reference (query) genome. The arrow direction indicates the hit direction. On the right, box plots summarize the distribution of hits from each reference genome to the N. limnia genome. (B) The positions of the two largest consensus scaffolds are shown. (C) The sequence novelty index, defined as 2 minus the sum of nucleotide identity of blast hits to N. maritimus and C. symbiosum A at each position is plotted. A histogram of the values is present at right. (D) The alignment depth in the final assembly is shown. A histogram of the values is present at right.