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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Sampling and geochemical measurements from acetate amendment field experiment conducted in aquifer well CD-01 at the Rifle IFRC site. (472 KB)
a, b, Samples were collected for metagenomics and metatranscriptomics at six time points (A–F) spanning several redox transitions during acetate stimulation of groundwater microbial communities. a, Groundwater was pumped from the alluvial aquifer and filtered through serial 1.2, 0.2 and 0.1 μm filters. DNA was extracted and sequenced from both the 0.2 and 0.1 μm filters, and RNA extracted and sequenced from the 0.2 μm filters (aerial image provided by S. M. Stoller for the US DOE under contract DE-AM01-07LM00060). b, Geochemical measurements were taken throughout the time series, showing a transition from dominant iron reduction to sulfate reduction through to methane production in the sampling environment.
- Extended Data Figure 2: Validation of 20 draft-quality genomes by ESOM clustering of genome fragments based on tetranucleotide sequence composition. (1,007 KB)
For validation, 20 draft genomes from a sample with a high proportion of CPR genomes (GWA2) were chosen at random. Each data point represents a 5–10 kb genome fragment. The ESOM was trained for 100 epochs with normalized tetranucleotide frequencies. Dark lines between data points indicate strong separation between regions. Data points are coloured based on the genome the fragment originated from. The ESOM shows well-delineated clusters for most of the 20 draft genomes, with few sequence fragments falling outside of these clusters. Two genomes from the same Microgenomates (OP11) phylum were not well delineated in the tetranucleotide-based ESOM (genomes 18 and 19). This shows how the method we used for binning, which takes into account abundance patterns in addition to sequence signatures, provides more accurate genome reconstructions. The white box distinguishes a single period on the repeating map. Genomes split into multiple clusters are labelled in red.
- Extended Data Figure 3: Relative abundance of bacterial community members during acetate amendment. (124 KB)
a, b, Relative abundance was calculated based on stringent mapping of paired-read sequences from each sample to 16S rRNA gene sequences assembled from all samples. Relative abundance of cells from 0.2 μm filters (a) and from 0.1 μm (b) filters. Enrichment of CPR organisms in the 0.2 μm filtrate indicates that these organisms have ultra-small cell sizes.
- Extended Data Figure 4: Features of insertion sequences encoded within 16S rRNA genes from the Silva database. (175 KB)
The non-redundant Silva 16S rRNA gene database (v. 115) was analysed to assess the prevalence of insertions. Only 761 of the 418,498 16S rRNA gene sequences from bacteria encode insertions. While many small insertions were identified, unlike the 16S rRNA gene sequences assembled from groundwater, these sequences (1) rarely encode large insertions, (2) do not contain both ORFs and introns, (3) do not encode ORFs that could be assigned to Pfam families, and (4) may be found in one of multiple copies of the 16S rRNA gene.
- Extended Data Figure 5: 16S rRNA gene copy number estimations for genomes reconstructed from groundwater metagenomics. (138 KB)
a, b, 16S rRNA gene copy number was estimated for all draft CPR genomes and genome bins for organisms outside the CPR. This was achieved by comparing the coverage of 16S rRNA gene regions to the coverage of the rest of the genome. Importantly, coverage was calculated only with stringently mapped reads (no mismatches were allowed) to improve the accuracy of coverage calculations. a, Histogram of the number of 16S rRNA gene sequence copies estimated for each genome by calculating (16S rRNA gene coverage)/(genome coverage). Several WWE3 genomes were estimated to have high 16S rRNA gene copy number (Supplementary Table 7), but it was later determined that these estimates were skewed by the presence of a highly abundant closely related strain. The complete WWE3 genome assembled previously3 has an identical 16S rRNA gene and confirms that it is found in only one copy for this genotype. Thus, we removed these estimates from subsequent copy number analysis. b, Density plot comparing estimated copy number of genomes for organisms found within and outside the CPR, where the longer tail for non-CPR genomes depicts the propensity for multiple 16S rRNA copies, a trait absent from the CPR.
- Extended Data Figure 6: Features of insertion sequences encoded within 23S rRNA genes recovered from groundwater-associated bacteria. (223 KB)
Bacteria associated with the CPR encode insertions within their 23S rRNA genes (Supplementary Table 5). These insertions share many features with those identified in 16S rRNA gene sequences from CPR bacteria. Taxonomy was determined by inclusion in a genome with an established phylogeny.
- Extended Data Figure 7: Analysis of the ability of PCR primers 515F and 806R to bind to recovered groundwater-associated 16S rRNA gene sequences. (183 KB)
a, b, PrimerProspector was used to assess the ability of primers 515F and 806R to bind a non-redundant set of assembled near-complete 16S rRNA gene sequences (clustered at 97% sequence identity). The percentage of sequences that would be amplified by these primers is shown on the left axis, the total number of sequences analysed is on the top of each bar, and the number of sequences these primers would not bind to is indicated by the shading. Many assembled groundwater-associated 16S rRNA gene sequences would evade amplification by PCR primers 515F and 806R. Results of the analysis are shown at the domain (a) and superphylum or phylum (b) levels.
- Extended Data Figure 8: Metabolic potential and ribosomal protein analysis of genomes from CPR and TM6 organisms. (1,010 KB)
Assembled genomes were analysed using ggKbase (Supplementary Data 4). Shown here is a non-redundant set of complete and near-complete genomes (≥75% of single copy genes, ≤1.125 copies) organized based on a subset of a maximum-likelihood 16S rRNA gene phylogeny (Supplementary Fig. 1). CPR organisms have partial tricarboxylic acid (TCA) cycles and lack electron transport chain (ETC) complexes. In addition, they have incomplete biosynthetic pathways for nucleotides and amino acids. The Peregrinibacteria are a notable exception to some of these limitations. Several Parcubacteria exhibit a complete ubiquinol (cytochrome bo) oxidase operon, as previously seen in Saccharibacteria3. However, lack of NADH dehydrogenase and other ETC components suggests that this enzyme is involved in oxygen scavenging/detoxification rather than energy production. AA Syn., amino acid synthesis; PP, pentose phosphate pathway.
Extended Data Tables
- Supplementary Information (129 KB)
This file contains a guide to Supplementary Figure 1, Supplementary Tables 1-10 and the Supplementary Data (see separate files).
- Supplementary Figure (1.3 MB)
This file contains Supplementary Figure 1 (see the Supplementary Information file for details).
- Supplementary Tables (3.1 MB)
This file contains Supplementary Tables 1-10 (see the Supplementary Information file for details).
- Supplementary Data (13.7 MB)
This zipped file contains the Supplementary Data (see the Supplementary Information file for details).