Culture Independent Genomic Comparisons Reveal Environmental Adaptations for Altiarchaeales

Abstract

The recently proposed candidatus order Altiarchaeales remains an uncultured archaeal lineage composed of genetically diverse, globally widespread organisms frequently observed in anoxic subsurface environments. In spite of 15 years of studies on the psychrophilic biofilm-producing Candidatus Altiarchaeum hamiconexum and its close relatives, very little is known about the phylogenetic and functional diversity of the widespread free-living marine members of this taxon. From methanogenic sediments in the White Oak River Estuary, NC, USA, we sequenced a single cell amplified genome (SAG), WOR_SM1_SCG, and used it to identify and refine two high-quality genomes from metagenomes, WOR_SM1_79 and WOR_SM1_86-2, from the same site. These three genomic reconstructions form a monophyletic group, which also includes three previously published genomes from metagenomes from terrestrial springs and a SAG from Sakinaw Lake in a group previously designated as pMC2A384. A synapomorphic mutation in the Altiarchaeales tRNA synthetase β subunit, pheT, caused the protein to be encoded as two subunits at non-adjacent loci. Consistent with the terrestrial spring clades, our estuarine genomes contained a near-complete autotrophic metabolism, H2 or CO as potential electron donors, a reductive acetyl-CoA pathway for carbon fixation, and methylotroph-like NADP(H)-dependent dehydrogenase. Phylogenies based on 16S rRNA genes and concatenated conserved proteins identified two distinct sub-clades of Altiarchaeales, Alti-1 populated by organisms from actively flowing springs, and Alti-2 which was more widespread, diverse, and not associated with visible mats. The core Alti-1 genome suggested Alti-1 is adapted for the stream environment with lipopolysaccharide production capacity and extracellular hami structures. The core Alti-2 genome suggested members of this clade are free-living with distinct mechanisms for energy maintenance, motility, osmoregulation, and sulfur redox reactions. These data suggested that the hamus structures found in Candidatus Altiarchaeum hamiconexum are not present outside of stream-adapted Altiarchaeales. Homologs to a Na(+) transporter and membrane bound coenzyme A disulfide reductase that were unique to the brackish sediment Alti-2 genomes, could indicate adaptations to the estuarine, sulfur-rich environment.

Keywords: autotrophy; comparative genomics; ecophysiology; marine sediment; metagenomics; single cell genomics; uncultured archaea.

FIGURE 1

Global distribution of Altiarchaeales 16S rRNA gene sequences present in the NCBI database.

FIGURE 2

Aqueous porewater concentrations of sulfate (triangles) and methane (circles) in replicate sediment cores (open/filled-in) taken from White Oak river estuary Station H in October 2012, with a red arrow indicating the depth at which samples for single-cell sorting were taken.

FIGURE 3

Phylogenetic placement of Altiarchaeales based on (A) a maximum likelihood tree constructed from full length (>1300 bp) 16S rRNA gene sequences; smaller sequences (>900) within the Altiarchaeales group were added to the tree using maximum parsimony (ARB); filled and open circles indicate bootstrap support at greater than 90 and 70%, respectively, (B) a Phylobayes tree constructed with 10 conserved universal proteins from 94 archaeal genomes. The 2220 amino acid positions which were conserved at 30% across the alignment were compared. pMC2A384 was added via parsimony (ARB). Percentages at nodes indicate proportions of trees which agreed with the displayed branching pattern. Sequences were derived from terrestrial springs (red) and estuarine/lacustrine (blue) samples. Scale bars show 10% difference. Gray trapezoids indicate collapsed clades with the number of individuals indicated.

FIGURE 4

The amino acids of phenylalanine tRNA-synthetase beta subunit (pheT) in six proposed Altiarchaeales genomes, were aligned using ClustalO to 104 pheT sequences from one representative of each archaeal genus with draft or finished genomes above 50% completeness, but only pheT from Pyrococcus horikoshii is shown. Each aligned gene is labeled by genome name, and the length scale is in amino acids. Colored boxes placed over the aligned sequences represent protein domains associated with pheT (InterProScan5). Black and gray boxes denote the presence of conserved residues found in 100 or 90% of archaeal pheT, respectively. The histogram tracks the relative conservation of residues across all aligned archaeal pheT.

FIGURE 5

Comparative analysis of protein clusters within all known Altiarchaeales genomes. The inner tree was constructed from matrix of protein abundances across the distantly related genomes in this study. Black bars at the tips of inner tree represent the presence or absence of a protein within a genome. Colored bins across the edge highlight co-occurrence patterns across the genomes: core Altiarchaeales proteins (red), core Alti-1 (black), core Alti-2 (blue), and homologous proteins between WOR genomes from metagenomes (green). GC content (bright green), predicted completeness (orange), and total predicted coding sequences (purple) are displayed to the right. Branch lengths in the tree above the histograms correspond to dissimilarities between predicted coding sequences in the genomes.