An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales

Abstract

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Keywords: archaea; evolution; horizontal gene transfer; hydrogenase; marine benthic group d; metagenomics.

Fig. 1.

Phylogenomic tree of 146 Thermoprofundales genomes. Different colors covering the tree branches indicate the five Thermoprofundales subgroups. The filled or hollow star at each genome ID indicates the full or partial MBH subunit genes, respectively. The colors of the inner ring indicate the availability of light. The colors of the middle ring indicate the temperature range. The colors of the outer ring indicate the environmental source of each genome. Ultrafast bootstrapping was used to estimate the reliability of each branch with 1,000 times resampling, and the nodes with a bootstrap value >80 are marked with black dots.

Fig. 2.

Reconstructed metabolic pathways of Thermoprofundales. A full list of genes labeled with different letters is provided in supplementary table S4, Supplementary Material online.

Fig. 3.

Ancestral genome content reconstruction and HGT donor predictions of Thermoprofundales. The solid or open circles outside the tree represent the presence or absence of the genes, respectively. Node 1 and Node 2 indicate the ancestral node of all Thermoprofundales genomes and Subgroup E, respectively. The values in parentheses indicate the number of gained (+) and lost (−) genes, and full lists of the genes are provided in supplementary table S5 and S6, Supplementary Material online. The pie charts show the ratios of HGT donors. The phylogenomic tree based on 55 conserved single-copy ubiquitous archaeal genes was constructed using IQ-TREE with ModelFinder. Ultrafast bootstrapping was used to estimate the reliability of each branch with 1,000 times resampling, and the nodes with a bootstrap value >80 are marked with black dots.

Fig. 4.

(a) Phylogenetic tree of Thermoprofundales based on the MbhL gene. The shaded area indicates the position of the MbhL gene, and the red branches indicate the MbhL genes of Thermoprofundales. The label at each clade indicates a hydrogenase group. (b) H₂ production by the recombinant MbhL protein from Thermoprofundales FT_bin5.232. Hydrogenase activity was measured in a reaction medium containing the recombinant MbhL protein or inactivated MbhL protein. A blank assay was run without any protein. The dashed line marks the H₂ peak by gas chromatography.

Fig. 5.

(a) Gene arrangement of the MBH complex in Thermoprofundales. ARC, the proposed common ARC. (b) Illustration of the MBH complex. (c–e) Growth performances of Escherichia coli KNabc strains cultured in 0, 0.1, and 0.2 M NaCl, respectively. KNabc/pET-22b-MbhA-M, a KNabc strain containing a gene cluster MbhABCDEFGHIM inserted into a pET-22b(+) plasmid. KNabc/pET-22b, a KNabc strain containing an empty pET-22b(+) plasmid. Three replicates are performed for each treatment. (f) Relative transcriptional abundances of the peripheral and membrane arms in four MAGs from the FT mangrove sediments. The numbers at the right of each row indicate sediment depth intervals of the FT metatranscriptomes.

Fig. 6.

(a) Phylogenomic tree of the MBH-containing archaeal genomes in the NCBI GenBank database. The tree based on 55 archaeal marker genes is constructed using IQ-TREE with ModelFinder. (b) Phylogenetic tree of the concatenated 14-subunits MBH gene in the NCBI GenBank database. The colors covering the tree branches indicate different archaeal lineages. The nodes with a bootstrap value >80 are marked with black dots.

Fig. 7.

Evolutionary timeline of the MBH-containing archaeal lineages. The values in parentheses are the posterior 95% confidence intervals.