Archaea dominate oxic subseafloor communities over multimillion-year time scales

Abstract

Ammonia-oxidizing archaea (AOA) dominate microbial communities throughout oxic subseafloor sediment deposited over millions of years in the North Atlantic Ocean. Rates of nitrification correlated with the abundance of these dominant AOA populations, whose metabolism is characterized by ammonia oxidation, mixotrophic utilization of organic nitrogen, deamination, and the energetically efficient chemolithoautotrophic hydroxypropionate/hydroxybutyrate carbon fixation cycle. These AOA thus have the potential to couple mixotrophic and chemolithoautotrophic metabolism via mixotrophic deamination of organic nitrogen, followed by oxidation of the regenerated ammonia for additional energy to fuel carbon fixation. This metabolic feature likely reduces energy loss and improves AOA fitness under energy-starved, oxic conditions, thereby allowing them to outcompete other taxa for millions of years.

Fig. 1. Microbial diversity, abundance, and nitrification in the North Atlantic abyssal subseafloor.

(A) Left: Dissolved nitrate, quantitative polymerase chain reaction (qPCR) of 16S rRNA and AOA amoA genes, and qPCR-normalized abundance of bacterial and archaeal 16S rRNA genes. Right: Diversity of 16S rRNA genes. Note that two biological replicates were sequenced at site 11, depth 2.8 mbsf. Arrows show the depths for ¹⁸O-labeling incubations. (B) Nonmetric multidimensional scaling analysis of 16S rRNA genes, the size of the points (samples) are normalized by the number of 16S rRNA gene copies per sample (qPCR), the numbers above each point indicate the depth in meters below the seafloor, and the shading indicates the ratio of archaeal to bacterial sequences. The ANOSIM analyses were performed on sample groupings as displayed by the dashed lines. Note that each meter in (A) represents ca. 1 million years of sediment deposition, and the communities in the deepest sample at site 11 have been subsisting for ca. 15 million years.

Fig. 2. Utilization of eDNA by living microbes, and a lack of detectable eDNA added to autoclaved sediments.

Samples derived from 2.8 mbsf, the depth used for the ¹⁸O-SIP experiment from site 11. The lack of DNA detection in autoclaved sediments, where eDNA was added after autoclaving (open circles) indicates that eDNA is tightly bound to sediment particles such as clay minerals and that the DNA extracted with our protocol should target intact living cells, and is unlikely to be biased by eDNA from dead microbes. Error bars are the range across three technical replicates.

Fig. 3. In situ distributions of the most abundant populations and ¹⁸O-labeling.

Shown are the most abundant populations in situ, indicating those that were viable (¹⁸O-labeled) in H₂¹⁸O incubations. Asterisks show the depths for ¹⁸O-labeling incubations. Note that a single viable population of AOA (Thaumarchaeota), OTU3, dominated the microbial communities at both sites and that each meter represents ca. 1 million years of sediment deposition (thus, the deepest sample is ca. 15 million years old).

Fig. 4. Diversity of 16S rRNA and amoA genes from AOA.

(A) Histograms showing the relative abundance of AOA OTUs based on 16S rRNA gene data. Asterisks mark the OTUs that were ¹⁸O-labeled in the long-term incubations. (B) Phylogenetic analysis (PhyML) of amoA encoding genes from AOA at sites 11 and 12. Note that amoA genes from sites 11 and 12 form two separate bootstrap-supported clades consisting primarily of deeper (6.6 and 12 mbsf) sequences, which likely derive from the highly abundant OTU3 in these deeper intervals [in (A)]. Tree is based on an alignment of 655 nucleotides under a general time reversible (GTR) model of evolution with four rate categories. Bootstraps were calculated from 100 replications.

Fig. 5. Taxonomic representation and metabolic potential in metagenomes.

(A) The number of ORFs as a function of depth at sites 11 and 12. (B) Relative abundance of taxonomic groups represented in the metagenomes. (C) Relative abundance of metabolic functions in the metagenomes. The amoA and HP/HB cycle genes were only encoded by Thaumarchaeota (AOA). In both (B) and (C), “percent reads mapped” refers to the percentage of raw reads mapped to ORFs encoding contigs.

Fig. 6. Metabolic potential for mixotrophic deamination is dominated by Thaumarchaeota.

(A) Top shows the relative abundance of ORFs in either site 11 (white bars) or site 12 (black bars), corresponding to transporters of organic matter, deaminating (ammonia lyases and deaminases), and proteolytic (proteases) enzymes. Bottom shows the taxonomic affiliation of the same ORFs. (B) Conceptual model of how AOA proteolysis and deamination could provide carbon sources for mixotrophic growth and additional energy (ATP) for chemolithoautotrophic growth. The outer lines represent the archaeal cell membrane, which is less permeable and may help to reduce loss of intracellular regenerated ammonia. The “H⁺” represents protons derived from oxidation of ammonia that can be used to produce additional energy (ATP) required to fix carbon via the HP/HB cycle. pmf, proton motive force; CPR, Candidate Phyla Radiation.