Respiratory processes of early-evolved hyperthermophiles in sulfidic and low-oxygen geothermal microbial communities

Abstract

Thermophilic microbial communities growing in low-oxygen environments often contain early-evolved archaea and bacteria, which hold clues regarding mechanisms of cellular respiration relevant to early life. Here, we conducted replicate metagenomic, metatranscriptomic, microscopic, and geochemical analyses on two hyperthermophilic (82-84 °C) filamentous microbial communities (Conch and Octopus Springs, Yellowstone National Park, WY) to understand the role of oxygen, sulfur, and arsenic in energy conservation and community composition. We report that hyperthermophiles within the Aquificota (Thermocrinis), Pyropristinus (Caldipriscus), and Thermoproteota (Pyrobaculum) are abundant in both communities; however, higher oxygen results in a greater diversity of aerobic heterotrophs. Metatranscriptomics revealed major shifts in respiratory pathways of keystone chemolithotrophs due to differences in oxygen versus sulfide. Specifically, early-evolved hyperthermophiles express high levels of high-affinity cytochrome bd and CydAA' oxidases in suboxic sulfidic environments and low-affinity heme Cu oxidases under microaerobic conditions. These energy-conservation mechanisms using cytochrome oxidases in high-temperature, low-oxygen habitats likely played a crucial role in the early evolution of microbial life.

Fig. 1. Two filamentous streamer communities at similar pH (8–9) and temperature (82–84 °C) exhibit differences in population structure and function due to geochemical differences in dissolved sulfide (DS) versus oxygen (DO) (DS:DO = ratio of DS to DO).

A Conch Spring B Octopus Spring (Lower Geyser Basin, YNP). Scanning electron micrographs show filamentous organisms and extensive extracellular matrix (high-resolution insets) that dominate the physical fabric of both communities. Micrographs were chosen from a large collection of over 30 replicates from 3 different sample years and a minimum of 10 replicate images per year.

Fig. 2. Concentrations of DS and DO (μM) in Conch and Octopus Springs.

Filamentous communities were sampled down-gradient of spring discharge at transect position B, C (82–84 °C) for metagenomic and transcriptomic analyses (DS was <1 μM in Octopus Spring and DO <1 μM in Conch Spring). Error bars are standard deviations of 4–6 replicates, where absent, error bars fall within symbol. Source data are provided as a Source Data file.

Fig. 3. Relative abundances of microbial populations present in Conch and Octopus Springs sampled in 2011 and 2012.

Organisms common to both sites include Thermocrinis, Pyrobaculum, and Caldipriscus (Pyropristinus) (hatched). Other populations (clockwise, in order of abundance) include Thermodesulfobacteria and 2 members of the Desulfurococcales at Conch Spring (yellow patterns), and Thermoproauctor, Calescibacterium (Calescamantes), Armatimonadota T1, Calditenuis aerorhuemensis , Acidilobaceae, Armatimonadota T2, Thermoflexus, and several others <2% at Octopus Spring (blue patterns) (Table 2 and Supplementary Table 1 show complete list of phylotypes). Abundances were calculated based on the fraction of mapped reads from random metagenome sequence (CheckM). Taxonomic references are based on nucleotide identity (>95%) at either the phylum, order, family, or genus/species level. Source data are provided as a Source Data file.

Fig. 4. Phylogenomics of early-evolved bacteria in Conch and Octopus Springs.

Bayesian phylogenetic trees of Bacteria using A a concatenation of 16 ribosomal proteins (2447 residues) or B the 16S rRNA gene (sequences > 1000 bp only). Members of the Pyropristinus lineage include Caldipriscus and Thermoproauctor spp. from circumneutral (pH 7–9) hyperthermal (>75 °C) geothermal springs [OCT Octopus Spring, CON Conch Spring, BCH Bechler, FF Fairy Falls, PS Perpetual Spouter; *16S rRNA clones EM3 and EM19 from Octopus Spring ; ** lineages contain populations from Octopus Spring (e.g., Patescibacteria within Candidate Phyla Radiation (CPR) (2 types, OCT 2012), Thermus aquaticus OCT 2011, 2012, Thermoflexus hugenholtzii OCT 2011, 2012) or Conch Spring (Thermodesulfobacteria); Uncollapsed versions of these phylogenetic trees are provided in Supplementary Figs. 5 and 6.

Fig. 5. Electron transfer and carboxylation genes in microbial populations from Conch and Octopus Springs.

Keystone microbial populations present in both springs (A), or populations present only in Conch (B) or only in Octopus (C) Springs [sox sulfur oxidation pathway, aio arsenite oxidase, sqr sulfide:quinone oxidoreductase, ttr tetrathionate reductase, psr polysulfide reductase, dsr dissimilatory sulfite/sulfate reductase, hco heme Cu oxidases, cytbd cytochrome bd ubiquinol oxidase, cydAA’ archaeal cytochrome oxygen reductase, ccl/ccs citryl-CoA lyase/citryl-coA synthetase, fdh formate dehydrogenase, acc acetyl CoA carboxylase, por pyruvate oxidoreductase].

Fig. 6. Analysis of metatranscriptomes from Conch (2016) and Octopus (2011, 2016) Spring filamentous streamer communities.

A Transcript abundance by major phylotype (Octopus 2011 solid blue, Octopus 2016 hatched blue, Conch 2016 hatched yellow). B Abundance of transcripts mapped to specific functions within Thermocrinis, Pyrobaculum, and Caldipriscus metagenome assembled genomes (MAGs), expressed as a percent of total transcripts within each MAG. [Armatimona. T1 Armatimonadota type 1, aio arsenite oxidase, sox sulfur oxidation complex, hco heme Cu oxidase complex subunits I, II and III, cytBC cytochrome bc complex, rhod rhodanese sulfur transferase, sqr sulfide:quinone oxidoreductase, cytBD cytochrome bd ubiquinol oxidase; cydAA’ = archaeal cytochrome oxidase, por pyruvate ferrodoxin oxidoreductase complex, hgl hemoglobin. Source data are provided as a Source Data file.

Fig. 7. Primary respiratory metabolism and energy generation (percent of total transcripts within each organism) in keystone populations present in both Conch and Octopus Springs.

Ratios of DS:DO were nearly 4000 times higher at sample sites in Conch versus Octopus Springs. [Gene names: sqr = sulfide:quinone oxidoreductase, cytBD = bd-type ubiquinol cytochrome oxidase; cydAA’ = archaeal cytochrome oxidase; sox = sulfur oxidation complex including subunits soxA, soxB, soxX, soxY, and soxZ; aioAB = arsenite oxidase large and small subunits; HCO = heme Cu oxidase complex subunits I, II and III; and energy generating ATPase subunits a, b, and c.].

Fig. 8. Type IV Filament Systems and Field-Emission Scanning Electron Microscopy.

Highly expressed type four filament (Tff) systems in Thermocrinis MAGs (A) likely explain the extensive network of pili-like structures (~20–25 nm diameter) commonly observed in filamentous streamer communities from Conch Spring (B) and Octopus Spring (C). [Abbreviations and Definitions: Bechler 2008 = Thermocrinis entry from Bechler Spring, YNP. Arrows in C indicate Tff structures versus cells. [pilA = Type IV pilus assembly, pilW = Type IV pilus assembly, pulG = Type II secretory pathway, fimT = Type IV fimbrial biogenesis, pilY = adhesin; nfuA = Fe-S biogenesis, pilQ = pilus secretin, recJ = ssDNA exonuclease, HCO = heme copper oxidase complex subunits I, II, and III. Source data are provided as a Source Data file. Micrographs shown in B and C were chosen from a large collection of over 30 replicates from 3 different sample years and a minimum of 10 replicate images per year. Additional FE-SEM micrographs are provided in Supplementary Fig. 14. Complete tables of observed transcripts and expression levels are provided in Supplementary Data 1–3].