Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum

Abstract

The "dark matter of life" describes microbes and even entire divisions of bacterial phyla that have evaded cultivation and have yet to be sequenced. We present a genome from the globally distributed but elusive candidate phylum TM6 and uncover its metabolic potential. TM6 was detected in a biofilm from a sink drain within a hospital restroom by analyzing cells using a highly automated single-cell genomics platform. We developed an approach for increasing throughput and effectively improving the likelihood of sampling rare events based on forming small random pools of single-flow-sorted cells, amplifying their DNA by multiple displacement amplification and sequencing all cells in the pool, creating a "mini-metagenome." A recently developed single-cell assembler, SPAdes, in combination with contig binning methods, allowed the reconstruction of genomes from these mini-metagenomes. A total of 1.07 Mb was recovered in seven contigs for this member of TM6 (JCVI TM6SC1), estimated to represent 90% of its genome. High nucleotide identity between a total of three TM6 genome drafts generated from pools that were independently captured, amplified, and assembled provided strong confirmation of a correct genomic sequence. TM6 is likely a Gram-negative organism and possibly a symbiont of an unknown host (nonfree living) in part based on its small genome, low-GC content, and lack of biosynthesis pathways for most amino acids and vitamins. Phylogenomic analysis of conserved single-copy genes confirms that TM6SC1 is a deeply branching phylum.

Keywords: MDA; genome assembly; metagenomics; single-cell genomics; symbiotic bacteria.

Fig. 1.

Summary of genera found in the biofilm sample from single and multievent sorts. The total number of 16S rRNA gene sequences for each observed bacterial genera recovered in individual MDA-amplified wells. Data are presented for wells in which 1, 20, or 100 events were sorted from either a high- or a low-fluorescence event population. Data from the 20- and 100-event wells that were sorted from the high fluorescence population are grouped together.

Fig. 2.

Circular representation of the TM6SC1 genome as a pseudomolecule derived from the concatenated contigs for MDA2. From the inner to the outer ring: GCskew−, GCskew+, G+C content, BLASTN alignment against MDA1 contigs, BLASTN alignment against MDA3 contigs, predicted CDS, rRNA, and MDA2 contigs (contigs were ordered by length and then concatenated).

Fig. 3.

Evolutionary relationships of candidate division TM6. (A) Phylogenetic relationship of 16S rRNA gene sequences designated as members of TM6 in public databases reveal the global distribution and sequence diversity within this group. An asterisk indicates the TM6 sequences from this study. (B) Unrooted 16S rRNA gene tree based on maximum-likelihood analysis of representative candidate division TM6 and Proteobacteria sequences. One thousand bootstrapped replicate resampled datasets were analyzed. Bootstrap values are indicated as percentages and not shown if below 50%.

Fig. 4.

Phylogenetic tree illustrating the major lineages (phyla) of the domain Bacteria analyzed with AMPHORA2 and 29 protein phylogenetic markers. The TM6 gene sequences were aligned against the AMPHORA2 seed alignment consisting of sequences from over 1,000 genomes through HMM. Tree branch lengths ≤0.4 were collapsed. For the original tree, see SI Appendix, Fig. S9.

Fig. 5.

Predicted metabolic pathways of phylotype TM6SC1. Predicted ABC transporters (e.g., amino acid importers, nucleotide/nucleoside importers, divalent ion importers) (red) as well as a cellobiose importer (orange). ATP-driven transporters are indicated by the ATP hydrolysis reaction. The copper ion transporting P-type ATPase is proposed to serve as both uptake and efflux systems, which is shown by a bidirectional arrow. Several protein-secretion components belonging to the type II secretion pathway; GSP and Sec proteins were identified as well as five prepilin related domains and a type IV pilB ATPase (green). A modified electron transport chain was also identified consisting of seven thioredoxins and five thioredoxin reductases, a V-type pyrophosphatase, an F-type ATPase synthase, and a protein disulfide reductase (DsbD) (blue). Thiolperoxidases (Bcp and bacterioferretine-like), superoxide dismutases (manganese and iron), were identified as cytosolic and periplasmic enzymes protecting against oxidative stress. A β-glucosidase that catalyzes the formation of β-d-glucose from disaccharides (e.g., cellobiose) was identified as well as all enzymes involved in the pentose phosphate pathway. However, other enzymes (e.g., enzymes from the Calvin cycle or the citric acid cycle) involved in the conversion of 3-glyceraldehyde-3-P could not be identified. Enzymes for de novo synthesis of amino acids were absent, however several amino acid importing membrane proteins (ABC transporters), and 14 cytosolic and proteolytic peptidases were identified, indicating that TM6 has a high capacity for amino acid scavenging.