Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three aquificales lineages

Abstract

The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal "filamentous streamer" communities (∼40 Mbp per site), which targeted three different groups of Aquificales found in Yellowstone National Park (YNP). Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae) populations, whereas the circum-neutral pH (6.5-7.8) sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae). Thermocrinis (Aquificaceae) populations were found primarily in the circum-neutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse-TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl). The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl-CoA synthetase (Ccs), and citryl-CoA lyase (Ccl). All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I) involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2) have resulted in niche specialization among members of the Aquificales.

Keywords: autotrophic processes; functional genomics; phylogeny; sulfide oxidation; thermophiles.

Figure 1

Site photographs of Aquificales “streamer” communities sampled from Yellowstone National Park. The sites represent diverse geochemical environments as noted from the different mineralogy apparent visually [DS_9 (elemental sulfur); OSP_14 (Fe-oxides); MHS_10 (calcium carbonate); CS_12 (pyrite, amorphous Fe-sulfides); OS_11 (none); BCH_13 (none, Fe(III)-staining on silica)].

Figure 2

Frequency plots of G + C content (%) of random shotgun sequence reads obtained from six Aquificales habitats in Yellowstone National Park (YNP). Phylogenetic classification of each sequence read (∼800 bp) was performed using MEGAN (based on “blastx”), and provides one method for visualizing the predominant phylotypes that contribute to metagenome sequence in these environments (gray = total reads; orange = Hydrogenobaculum; red = Metallosphaera; green = Sulfurihydrogenibium; violet = Thermus; blue = Aquificaceae; light-blue = Hydrogenivirga; light-orange = Domain Bacteria; light-green = Thermoproteaceae).

Figure 3

Nucleotide word frequency PCA plots of metagenome assemblies from six Aquificales streamer communities in YNP. (A) Data colored by site (black circles): DS_9 = yellow and OSP_14 = red; MHS_10 = green and CS_12 = violet; OS_11 = dark-blue and BCH_13 = light-blue. (B) Identical PCA orientation with phylogenetic analysis to the closest reference genomes (white-dashed circles): orange = Hydrogenobaculum, green = Sulfurihydrogenibium, dark-blue = Aquificaceae (no reference genomes adequately describe the similar Thermocrinis-like Aquificales phylotypes observed in OS_11 and BCH_13).

Figure 4

Phylogenetic tree of Aquificales 16S rRNA gene sequences observed within assembled metagenome sequence from six YNP geothermal outflow channels [DS_9 (orange), OSP_14 (red), MHS_10 (green), CS_12 (violet), OS_11 (dark-blue), BCH_13 (light-blue) [neighbor-joining tree with 1000 bootstraps].

Figure 5

Principal components analysis (PCA) of relative gene abundances across six Aquificales streamer communities. (A) All TIGRFAMs grouped into functional categories, and (B) Only those TIGRFAMs associated with the role category “Electron Transport.” Site colors: DS_9 (yellow), OSP_14 (red), MHS_10 (green), CS_12 (violet), OS_11 (dark-blue), BCH_13 (light-blue). Circled sites represent “replicates” that contain one of three dominant Aquificales lineages (Hydrogenobaculum, Sulfurihydrogenibium, or Thermocrinis).

Figure 6

Hierarchical cluster analysis of relative gene abundances in the TIGRFAM role category “Electron Transport” across six Aquificales streamer communities. TIGRFAMs with low variation across the sites were removed before the clustering to retain ∼50 of the most variable families. Subunits of the protein complexes were only represented by one representative TIGRFAM family. Pearson correlation was used as the distance measure for average linkage agglomerative clustering. Sites cluster consistent with pH and the presence or absence of sulfide.

Figure 7

Deduced protein tree of ATP citrate lyase (AclB), a key marker for CO₂ fixation via the reverse-TCA cycle found in Sulfurihydrogenibium sp. in MHS_10 and CS_12 (neighbor-joining tree with 1000 bootstraps).

Figure 8

Deduced protein tree of (A) citryl-coA synthetase (CcsA), and (B) citryl-CoA lyase (Ccl) showing metagenome entries from Aquificales sites (neighbor-joining tree with 1000 bootstraps).

Figure 9

Protein tree of heme-copper oxidases (subunit I of terminal oxidase complex). Metagenome entries are highlighted by site, and labels correspond to the major types of heme-copper oxidases observed in different Aquificales “streamer” communities. Similar heme-copper oxidases are found in sites OS_11 and BCH_13, and these are significantly different than the terminal oxidases found in Hydrogenobaculum and or Sulfurihydrogenibium from sites DS_9, OSP_14, MHS_10 and CS_12 (neighbor-joining tree constructed using nitric oxide reductase (NorB) as the out group; 1000 bootstraps).

Figure A1

Recruitment of metagenome sequence reads (∼800 bp per read) from six Aquificales streamer samples to the most relevant reference genomes currently available in reference databases. Recruitment of metagenome sequence fragments across the complete genomes (x-axis) are shown from 50 to 100% nucleotide identity (y-axis). Site Colors: Yellow = Dragon Spring (DS_9), Red = One Hundred Spring Plain (OSP_14); Green = Mammoth Hot Springs (MHS_10), Violet = Calcite Springs (CS_12); Dark-Blue = Octopus Spring (OS_11), light-blue = Bechler Springs (BCH_13). Reference genomes: Hydrogenobaculum sp. Y04AAS1; Sulfurihydrogenibium sp. Y03AOP1 (with and without the 220 Mb of pyrosequence for MHS_10); Thermocrinis albus DSM 14484; Hydrogenobacter thermophilus TK-6; Thermus aquaticus HB8 (Plot constructed using JCVI bioinformatic utilities, Rusch et al., 2007).

Figure A2

Nucleotide word frequency PCA plots showing detailed analysis of Thermocrinis-like populations in Calcite and Bechler Springs compared to Thermocrinis albus reference sequence and other phyla present in these two communities. (A) Sequence data colored by site (CS_12 and BCH_13), with T. albus reference sequence (red) added for comparison., and (B) Sequence data in identical orientation now analyzed phylogenetically to reveal specific assembled sequence corresponding to particular population types within the two different sites (green = Sulfurihydrogenibium sp., blue = Aquificaceae, violet = Thermus sp.; light-green = Thermoproteaceae; unassigned = black). White circles indicate major assemblies from BCH_13, and black circles indicate major assemblies in CS_12.

Figure A3

Phylogenetic tree of all 16S rRNA genes observed in metagenome sequence (bold) combined with 16S rRNA genes amplified using PCR (up to 384 sequences per library) across six Aquificales streamer communities. Entries are colored by site: DG_9 = orange; OSP_14 = red; MHS_10 = green; CS_12 = violet; OS_11 = dark-blue; BCH_13 = light-blue (core tree established using neighbor-joining methods with maximum-likelihood correction; metagenome entries added by parsimony).

Figure A4

Distribution of PCR-amplified 16S rRNA genes observed in ribosomal panels across Aquificales streamer habitats. Universal archaeal and bacterial primers were used for each site and up to ∼384 clones were sequenced per primer set.

Figure A5

Consensus phylogenetic classification (AMPHORA; Wu and Eisen, 2008) of assembled sequence using analysis of 31 housekeeping genes. Although the Aquificales are the dominant members of each streamer sample, diverse and novel members of other bacterial and archaeal lineages are predicted to vary in abundance across sites.

Figure A6

Hierarchical cluster analysis of relative gene abundances across six Aquificales streamer communities using all TIGRFAMs grouped into functional categories. Broad TIGRFAM categories include all cellular processes such as regulatory functions, energy metabolism, central C metabolism, mobile elements, transcription, cofactors and transporters. Data was standardized by functional category before clustering to avoid biasing analysis by a few categories with high gene abundance. Pearson correlation was used as the distance measure for average linkage agglomerative clustering.

Figure A7

Functional categories with the most significant differences in relative gene abundance among the six Aquificales streamer communities [(A) biosynthesis of cofactors, prosthetic groups and carriers and (B) signal transduction: PTS; p-values = 0.0047 and 0.0057, respectively]. The relative proportion of sequences identified within each site are indicated (sites colored as before), and site pairs with similar geochemistry and Aquificales populations reveal replicate behavior in these specific TIGRFAMs.

Figure A8

Four most pH-dependent TIGRFAM families among six Aquificales streamer communities indicate a greater proportion of glutathione disulfide and thioredoxin-disulfide reductases in low-pH sites (blue bars) (sulfide present in both DS_9 and OSP_14) and a greater representation of formate dehydrogenases and NADH-plastoquinone oxidoreductases in higher-pH sites (orange bars) with (MHS_10, CS_12) or without (OS_11, BCH_13) sulfide. The relative proportion of sequences to all electron transport-associated genes is shown for each streamer community.