Archaea within the phylum Thaumarchaeota are among the most abundant single-celled organisms in the ocean’s interior. The best characterized members of the phylum are ammonia-oxidizing archaea (AOA), which derive energy from the oxidation of ammonia and fix inorganic carbon for biosynthesis. Not all Thaumarchaeota are ammonia oxidizers though - several lineages lacking the ammonia-oxidation machinery have been described in various terrestrial systems, most of which are anaerobic heterotrophs.

In marine waters, Thaumarchaeota diversify into several depth-segregated sub-populations. We do not quite understand the specific genomic or mechanistic adaptations that allow each sub-population to succeed in their individual niche. The overarching goal of our project was to systematically characterize differences in metabolic adaptations across these sub-populations. Since pelagic Thaumarchaeota are not particularly amenable to culturing, our primary approach involved omics methods, specifically metagenome sequencing of community DNA and subsequent reconstruction of population genomes. Several ‘aged’ seawater incubations (i.e., bottles of unamended Monterey Bay seawater kept in the incubator for 8 years; Figure 1) that we had going in the lab seemed like perfect candidates for obtaining high-quality genomes of ‘naturally’ enriched Thaumarchaeota. Among the several thaumarchaeal genomes assembled from these incubation metagenomes, two stood out because they lacked any resemblance to typical AOA genomes.

Phylogenetically, the genomes affiliated within the elusive pSL12-like clade of Thaumarchaeota (Figure 2), first described by Ed DeLong’s group in 2006 based on environmental 16S rRNA gene sequences. The pSL12-like lineage was later proposed to be involved in ammonia oxidation, based on gene abundance correlations, but there was no conclusive genomic evidence to support this conjecture. Intriguingly, the genomes we assembled do not support the case for ammonia oxidization within the pSL12-like lineage. In fact, the genomes do not suggest the use of any inorganic electron donors for energy conservation. Instead, these organisms appear to be heavily dependent on membrane-bound alcohol and sugar dehydrogenases that use pyrroloquinoline quinone as a cofactor. Another unexpected finding was the presence of a ribulose 1,5-bisphosphate carboxylase (RuBisCO) gene in both genomes, which most-closely resembled the Form III-a RuBisCO in methanogenic archaea. While the AOA-specific carbon fixation pathway was not identified in these genomes, it is unlikely that the RuBisCO gene is involved in an alternative CO₂-fixation pathway - the most parsimonious metabolic inference suggests its role in reclaiming nucleosides for central carbon metabolism. Metabolic reconstructions suggest an overall heterotrophic growth strategy, with the ability to respire oxygen. This is a significant deviation from the expected growth strategy of typical pelagic Thaumarchaeota, and points to hitherto overlooked phylogenetic and metabolic diversity within this phylum in the marine environment.

Our results are yet another testament to the power of genome-resolved metagenomics in discovering the metabolic potential of uncultured microbes. For years, these archaea were presumed to be ammonia oxidizers, and verifying this has been difficult since many continue to evade cultivation. Genome reconstructions have finally helped unveil the metabolic potential of these archaea. Now, we have genomic evidence pointing to a non-ammonia-oxidizing, heterotrophic, basal lineage of Thaumarchaeota co-existing with AOA in mesopelagic waters. We believe this discovery inspires future studies re-assessing the thaumarchaeal diversification trajectory, especially as it relates to the evolution of mesophilic AOA from basal lineages. Finally, our results suggest that many exciting new lineages are waiting to be discovered in bottles and carboys of ‘aged’ seawater in cold rooms all over the world.