Saccharibacteria as Organic Carbon Sinks in Hydrocarbon-Fueled Communities

Abstract

Organisms of the candidate phylum Saccharibacteria have frequently been detected as active members of hydrocarbon degrading communities, yet their actual role in hydrocarbon degradation remained unclear. Here, we analyzed three enrichment cultures of hydrocarbon-amended groundwater samples using genome-resolved metagenomics to unravel the metabolic potential of indigenous Saccharibacteria. Community profiling based on ribosomal proteins revealed high variation in the enrichment cultures suggesting little reproducibility although identical cultivation conditions were applied. Only 17.5 and 12.5% of the community members were shared between the three enrichment cultures based on ribosomal protein clustering and read mapping of reconstructed genomes, respectively. In one enrichment, two Saccharibacteria strains dominated the community with 16.6% in relative abundance and we were able to recover near-complete genomes for each of them. A detailed analysis of their limited metabolism revealed the capacity for peptide degradation, lactate fermentation from various hexoses, and suggests a scavenging lifestyle with external retrieval of molecular building blocks. In contrast to previous studies suggesting that Saccharibacteria are directly involved in hydrocarbon degradation, our analyses provide evidence that these organisms can be highly abundant scavengers acting rather as organic carbon sinks than hydrocarbon degraders in these communities.

Keywords: enrichment cultures; genome-resolved metagenomics; groundwater; hydrocarbon degradation; symbionts.

FIGURE 1

Community profile of diesel-amended enrichment cultures AER1, AER2, and AER3. (A) Diversity of the microbial community in the enrichments based on representative rpS3 sequences of each cluster. Bar charts show the abundance from normalized coverage of the scaffolds carrying the rpS3 gene. The different bar colors correspond to which sample (AER1, AER2, or AER3) the respective organism was detected in. Circled numbers correlate to recovered genomes shown in panel (C). Full tree of rpS3 sequences is provided as Supplementary File 1. (B) Venn-Diagram shows the unique and shared community profile amongst the three samples. (C) Breadth of coverage for the de-replicated set of recovered draft genomes, higher breadth values translate to a greater recovery of the respective draft genome in each sample, with Sac_1 and Sac_2 highlighted with red asterisks. The dendrogram is based on ANI values obtained with dRep. Output of the clustering of the recovered genomes with dRep is shown in the middle columns. First number in the clustering column is the output of MASH clustering. If genomes are found to be closely related in MASH, further ANI clustering is performed, denoted in the number after the underscore. Zero in this second number means no ANI clustering was performed. The different colored names denote the different clusters obtained. Circled numbers denote the recovered genomes. Please note that the breadth of genomes that were grouped by dRep into the same cluster was also highly similar, indicating that dRep and read-mapping based analyses agree well for species delineation. Potential for hydrocarbon degradation of the recovered genomes is shown, each colored circle denotes a different pathway, more information about it can be found in Supplementary Table 8.

FIGURE 2

Phylogenomic tree of Saccharibacteria and their genome completeness. (A) Phylogenetic placement of Sac_1 and Sac_2 based on concatenation of 16 ribosomal proteins, highlighting Sac_1 and Sac_2, as well as TM7× and Teamsevenales, the cultivated representative with a circular complete genome and the strain identified by Starr et al. (2018), respectively. Tree was pruned from a full tree including all three domains of life which is available as Supplementary File 2. (B) Completeness of the Saccharibacteria genomes Sac_1 and Sac_2 based on presence/absence of SCGs, with TM7× and Teamsevenales for comparison. Scaffolds 7 and 8, that make up the genomes of Sac_1 and Sac_2, respectively, are shown to compensate for absence of SCGs in Scaffold 1. Each bullet represents a different SCG per line, and each colored outline groups the SCGs found in each strain. The complete list of SCGs is provided as Supplementary Table 7. (C) Circos plot comparing the two Saccharibacteria genomes of Sac_1 and Sac_2. While Scaffold 1 is identical, Scaffold 8 and 7 also show high similarity based on blastn (cutoff 1e^–50).

FIGURE 3

Metabolic reconstruction of Sac_1 And Sac_2. The Saccharibacteria strains show minimal metabolic capacity. No TCA cycle was detected but the strains encode for an almost complete glycolysis as well as a complete PPP. Please note that we detected several transporters with unknown function, which might aid in the uptake of, e.g., nucleotides. Supplementary Tables 2–5 give the complete lists of the annotated pathways and transporters found for Sac_1 and Sac_2.