GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

Abstract

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2-79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 10⁵ 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45-77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O₂, suggesting a microaerophilic lifestyle.

Keywords: Acidobacteria; Acidobacteriota; GAL08; hot spring; single-cell genomics; thermophile; uncultivated bacterium.

FIGURE 1

Relative abundances of DChs_GAL08 based on 16S rRNA gene amplicon sequencing (A) and qPCR counts of DChs_GAL08 16S rRNA gene copies per gram of sediment (B) in sediments along three temperature transects in the Dewar Creek hot spring.

FIGURE 2

Amplicon-based community analysis of 15 samples collected between 64.3 and 67.4°C at the Dewar Creek hot spring during three sampling trips in 2012, 2014, and 2015. Multiple samples at unique locations were collected during each sampling trip and each sample is represented as a separate bar color-coded by year. Only bacterial phyla that were at or above 3% relative abundance in at least 1 of the 15 samples are included. The Acidobacteria are split into DChs_GAL08 and all other Acidobacteria. Relative abundance data were calculated based on 16S rRNA amplicon sequencing.

FIGURE 3

(A) Pairwise average nucleotide identity (ANI) of all recovered single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) of DChs_GAL08, showing 3 distinct species-level clusters. Genomes are identified using JGI taxon IDs. The three red clusters all show >99.2% identity, while the blue clusters all have <88.1% identity. The figure was calculated and drawn using pyani (Pritchard et al., 2016). (B) Venn diagram based on the presence or absence of COGs in the three species.

FIGURE 4

Maximum likelihood concatenated marker gene tree showing the phylogenetic placement of GAL08 within the domain Bacteria. The tree includes eleven GAL08 SAGs, five GAL08 MAGs, 68 Acidobacteria genomes, 214 genomes belonging to 6 other phyla, and 6 genomes from the Parcubacteria, serving as the outgroup. The 214 genomes were selected from a reduced set of bacterial isolate genomes available in the Integrated Microbial Genomes (IMG) database (Chen et al., 2021). The total set of (61,619) available genomes was first reduced by clustering the RNA polymerase beta-subunit gene at 65%; then, the reduced set was further minimized by screening for genomes belonging to phyla to which the GAL08 16S rRNA gene sequence shows the closest 16S rRNA gene identity (Thermotogae, Aquificae, Thermodesulfobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, at 82–84%); the final set served as a reference database for downstream tree inference.

FIGURE 5

(A) Bayesian 16S rRNA gene tree built using the ARB-Silva.de SSU rRNA gene reference database, release 138, showing the relationship of the major subgroups within the Acidobacteria. Nomenclature for the subgroups is used as suggested by Dedysh and Yilmaz (2018). The outgroup contains one 16S rRNA gene sequence from an isolate in each of 29 cultivated phyla. Confidence values, indicating posterior probabilities, are depicted as circles of different sizes. The scale bar represents 0.1 nucleotide substitutions per nucleotide position. (B) An expansion of the GAL08 within the Blastocatellia. All of the GAL08 sequences available in release 138 of the ARB-Silva database are included, as well as representative 16S rRNA gene sequences from each of the three species represented by SAGs and MAGs in this study. Two MAGs that contained 16S rRNA gene sequences from two other sites covered by the dataset of Nayfach et al. (2021) were also included. The sequences selected for Blastocatellia include all isolates and 1 to 2 sequences from each major clade available in release 138 of the ARB-Silva database. A posterior probability of 1.00 strongly supports the separation of GAL08 from the other members of the order Blastocatellales. The scale bar represents 0.1 nucleotide substitutions per nucleotide position. The list of accession numbers used in the construction of this tree is provided in Supplementary Table 1.

FIGURE 6

Cartoon depicting putative metabolism of GAL08 based on SAG analysis. Predicted metabolic components include an oxidative phosphorylation pathway, an incomplete denitrification pathway (absence of nirKS), acetate fermentation, fatty acid degradation, a small number of ABC transporters (shown in orange on the left of the figure), a PTS system for mannose, several major facilitator superfamily transporters, Sec-SRP plus TolC and Type II secretion systems, a TonB-dependent outer membrane transport system possibly acting in siderophore transport, and potential fermentation pathways. The symbol “?” and dashed lines both indicate a speculative metabolic component or product. A list of accession numbers for the genes shown is provided in Supplementary Table 4. A list of amino acid auxotrophies is shown in Supplementary Table 6.