Subgroup level differences of physiological activities in marine Lokiarchaeota

Abstract

Asgard is a recently discovered archaeal superphylum, closely linked to the emergence of eukaryotes. Among Asgard archaea, Lokiarchaeota are abundant in marine sediments, but their in situ activities are largely unknown except for Candidatus 'Prometheoarchaeum syntrophicum'. Here, we tracked the activity of Lokiarchaeota in incubations with Helgoland mud area sediments (North Sea) by stable isotope probing (SIP) with organic polymers, ¹³C-labelled inorganic carbon, fermentation intermediates and proteins. Within the active archaea, we detected members of the Lokiarchaeota class Loki-3, which appeared to mixotrophically participate in the degradation of lignin and humic acids while assimilating CO₂, or heterotrophically used lactate. In contrast, members of the Lokiarchaeota class Loki-2 utilized protein and inorganic carbon, and degraded bacterial biomass formed in incubations. Metagenomic analysis revealed pathways for lactate degradation, and involvement in aromatic compound degradation in Loki-3, while the less globally distributed Loki-2 instead rely on protein degradation. We conclude that Lokiarchaeotal subgroups vary in their metabolic capabilities despite overlaps in their genomic equipment, and suggest that these subgroups occupy different ecologic niches in marine sediments.

Fig. 1. Development of δ¹³C values of TOC in SIP incubations.

All incubations were amended with ¹³C-DIC (n = 3, error bar = SD).

Fig. 2. Total sum scaling charts of Lokiarchaeota abundances of archaeal 16S rRNA gene sequences from selected “light” and “heavy” gradient fractions.

a Long-term RNA-SIP samples amended with ¹³C-DIC, b Long-term DNA-SIP incubations amended with ¹³C-DIC, c Short-term RNA-SIP samples from lactate and protein incubations. Differences in x-axis scales between RNA and DNA-SIP are due to the different gradient media used (CsTFA vs CsCl, respectively). For RNA-SIP, pairs of fractions (fractions 4 and 5, 6 and 7, 8 and 9, 10 and 11) were combined for Illumina sequencing, whereas individual fractions were used for DNA-SIP. Density was indicated as the average density of combined fractions for RNA-SIP samples. Due to density differences between RNA and DNA, the threshold density fractions to delineate ¹³C-labelled nucleic acids differ between RNA (>1.797 g/ml) and DNA (>1.702 g/ml). “X” indicates that cDNA synthesis failed because of insufficient amount of RNA in these fractions. For the Loki-2 OTUs which were not detectable in controls, label incorporation activity was detected by their presence in heavy fractions. An asterisk indicates inter-gradient increase of Loki-3 OTUs (see Fig. S3 for intra-gradient assessment; both approaches were in agreement). DNA with densities >1.71 g/ml was not obtained from DIC incubations. Lep lepidocrocite. DIC dissolved inorganic carbon, i.e. bicarbonate.

Fig. 3. Maximum-likelihood phylogeny of Lokiarchaeota.

a Maximum-likelihood tree of Lokiarchaeotal 16S rRNA genes. 16S rRNA sequences and OTUs obtained in the present study were marked in bold, and those extracted from MAGs were marked in red. SF: clones from incubation amended with sulfur and lepidocrocite; HumF: clones from incubation amended with humic acid and lepidocrocite; LigF: clones from incubation amended with lignin and lepidocrocite. Clone library construction is described in the Supplementary Methods. MK-D1: Lokiarchaeota MAG obtained from enrichment [11]. L15, L04, L11: 16S rRNA genes from near-complete Lokiarchaeota MAGs obtained from a previous study [26]. An asterisk indicates the same reference sequences used for 16S rRNA gene tree construction with a previous study using Namibian sediments [9]. b Maximum-likelihood tree of Lokiarchaeotal MAGs inferred from a concatenated alignment of 122 archaeal marker genes and re-rooted with superphylum TACK. MAGs Hel238.bin13, Hel238.bin105 and Hel238.bin90 were obtained from Helgoland Mud sediment; MAG HMA.SIP.bin2 was obtained from heavy DNA of SIP incubations amended with sulfur, lepidocrocite and ¹³C-DIC; MAGs SZ_4 and DZG were retrieved from original sediment of South China Sea (Table S1). Information of all Lokiarchaeota MAGs was described in Table S1.

Fig. 4. Key genes and metabolic pathways in Lokiarchaeota.

a Number of gene homologues in Lokiarchaeota MAGs. Lokiarchaeota MAGs marked with green indicate Loki-3 obtained from Helgoland sediment and sediment incubations. MAGs marked with blue and cyan indicate Loki-2b and Loki-2a, respectively (see Table S1 for detail MAG information). MK-D1: Candidatus ‘Prometheoarchaeum syntrophicum’ strain MK-D1 [11]. Symbol “-“ indicates absence of gene in MAGs. b Proposed active pathway in Lokiarchaeota (Loki-3). Pathways were constructed based on Lokiarchaeotal MAGs obtained from this study (Table S3). EMP Embden–Meyerhof–Parnas pathway, WL tetrahydromethanopterin-dependent Wood-Ljungdahl pathway. Incomplete pathways were indicated by dashed line. Pathway names associated with the colours: yellow: β-oxidation; pink: WL; light blue: EMP; purple: amino acid degradation.

Fig. 5. Carbon assimilation patterns into nuleic acid by Lokiarchaeota.

a Inorganic carbon assimilation into nucleic acids; b lactate utilization for nucleic acid synthesis in Loki-3. All genes involved in the biosynthetic pathways of nucleic acids were present in Loki-3 MAGs (Table S3). Labelling levels for each intermediate in (a) were based on previous studies [16, 74, 75].

Fig. 6. Carbon utilization pattern by Lokiarchaeota in marine sediments.

a Organic polymer (lignin and humic acids) degradation and potential ecological roles of Loki-3. b Carbon utilization of Loki-2 indicated from SIP incubations.