Chlamydial contribution to anaerobic metabolism during eukaryotic evolution

Abstract

The origin of eukaryotes is a major open question in evolutionary biology. Multiple hypotheses posit that eukaryotes likely evolved from a syntrophic relationship between an archaeon and an alphaproteobacterium based on H₂ exchange. However, there are no strong indications that modern eukaryotic H₂ metabolism originated from archaea or alphaproteobacteria. Here, we present evidence for the origin of H₂ metabolism genes in eukaryotes from an ancestor of the Anoxychlamydiales-a group of anaerobic chlamydiae, newly described here, from marine sediments. Among Chlamydiae, these bacteria uniquely encode genes for H₂ metabolism and other anaerobiosis-associated pathways. Phylogenetic analyses of several components of H₂ metabolism reveal that Anoxychlamydiales homologs are the closest relatives to eukaryotic sequences. We propose that an ancestor of the Anoxychlamydiales contributed these key genes during the evolution of eukaryotes, supporting a mosaic evolutionary origin of eukaryotic metabolism.

Fig. 1. Anoxychlamydiales and anaerobic eukaryotes use similar anoxic metabolic strategies.

The presence of genes encoding components of the indicated energy metabolism pathways (left), encoded in the genomes of Chlamydiae (gray), Anoxychlamydiales (orange), and select anaerobic eukaryotes (blue), is indicated by a filled circle. For eukaryotic representatives, the subcellular location of each gene product or pathway is indicated by a filled blue circle (cytoplasm) or a filled circle with an outline (organelles). Outlined blue, green, and purple filled circles indicate the mitochondrion or MRO, the plastid (green), and the vacuole (purple), respectively (see legend). Eukaryotic representatives A. castellanii and Blastocystis sp. have hydrogen-producing mitochondria, Pygsuia biforma and T. vaginalis have hydrogenosomes, and Chlamydomonas reinhardtii has anaerobically functioning mitochondria with plastidal hydrogen production. The number of genomes obtained from the indicated environmental sources for each Chlamydiae clade is shown in the top right. The boxplot on the lower right shows the normalized coverage of contigs from the corresponding Anoxychlamydiales MAGs obtained from Loki’s Castle marine sediments (data S1). HYDA-G, hydrogenase subunits A to G. ADI, arginine deiminase; OTC, ornithine transcarbamoylase; CK, carbamate kinase; PFO, pyruvate:ferredoxin oxidoreductase; PDC, pyruvate dehydrogenase complex; FEO, ferrous iron transport; ACK, acetate kinase; PTA, phosphotransacetylase; NaH, Na⁺:H⁺ antiporter. The Chlamydiae species tree is a representation of species relationships derived from Dharamshi et al. (28).

Fig. 2. Anoxychlamydiales genes are the closest prokaryotic relatives of eukaryotic genes encoding anaerobic metabolism.

(A to D) Maximum likelihood (ML) phylogenies for H₂ metabolism proteins. Circles [nonparametric (NP)] and squares [ultrafast (UF)] summarize bootstrap support values (BP, boostrap percentage) for each bipartition mapped onto the best-scoring ML phylogeny. BP_UF, BP_NP, and transfer bootstrap expectation (TBE) for monophyly of eukaryotes (α) and eukaryotes with Anoxychlamydiales (β) or Anoxychlamydiales and other taxa (β′) are indicated. Eukaryotes and Anoxychlamydiales are shaded blue and orange, respectively (see data S1 for model parameters and alignment features and data S2 for full phylogenies). (E) The origin of mitochondrial metabolism in eukaryotes is an evolutionary mosaic. In aerobic mitochondria (solid lines), pyruvate is oxidized by PDC to acetyl-CoA, which is fed into the TCA cycle to produce reducing equivalents for the ETC that fuels ATP synthesis by oxidative phosphorylation. In MROs (dashed lines), pyruvate is oxidized by PFO (1) to acetyl-CoA, which is used for ATP synthesis by substrate-level phosphorylation (12). Hydrogen is produced by a trimeric confurcating hydrogenase (5 to 7) using electrons from Fd⁻ and NADH. The 4Fe-4S cluster of HYDA (5) is assembled by maturases (2 to 4). In this hypothetical cell, colors represent the proposed origin of each component [data S2 and figs. S5 and S6; (–23)]. 1 to -5 function in the plastids of some algae (15). (F and G) Scenarios for the timing of acquisition of hydrogen metabolism relative to major events in eukaryotic evolution. Relative timings of gene acquisitions from an Anoxychlamydiales ancestor (orange arrows) (F) before or immediately after the emergence of last eukaryotic common ancestor (LECA) or (G) after the radiation of eukaryotes, mediated by HGT into and between eukaryotes (blue arrows). Uncertainty regarding the timing of mitochondrial integration is depicted with a purple triangle. Gene losses are shown with an “X.” Ancestral Asgard archaeon (As; gray) and the alphaproteobacterial ancestor of the mitochondrion (α; purple).