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. (1), which is accomplished using multiple displacement amplification (MDA) (2-5) of genomic DNA to obtain sufficient template. We applied a high-throughput strategy to capture and sequence genomes of bacteria from a biofilm in a hospital sink including pathogens, such as the oral periodontal pathogen (Porphyromonas gingivalis) (6) and uncultivated members (this study). Despite the fact that a typical person spends ∼90% of their time indoors (7), our knowledge of the microbial diversity of the indoor environment has only recently begun to be explored using culture-independent methods (8, 9). Biofilms within water distribution systems in particular are thought to be diverse microbial communities and potential reservoirs of disease-causing organisms in the indoor environment. Several pathogens including Escherichia coli, Legionella pneumophila (10-13), Vibrio cholerae (14), and Helicobacter pylori (15, 16) have been detected in biofilms within water distribution systems. A recent 16S rRNA gene (abbreviated henceforth as 16S unless otherwise stated) molecular survey also revealed significant loads of Mycobacterium avium in showerhead biofilms (17). Based on these findings, indoor environments can clearly serve as significant reservoirs of pathogenic bacteria, and therefore there is great interest in investigating the rare and abundant bacterial species within biofilms in these environments.One approach to capture uncultivated bacteria is to isolate single bacterial cells by fluorescent activat...

Diverse uncultivated ultra-small bacterial cells in groundwater

“…Features expected in symbionts include small genome size, AT bias and loss of biosynthetic pathways 29,30 . Recently, McLean et al 31 reported a similarly small genome for a member of the TM6 CP and suggested that the organism is a symbiont, and Gong et al 32 showed that an OD1 bacterium occurs intracellularly in a protist.…”

Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle

“…XH001 alone and XH001/TM7x coculture were used to infect J2 BMM cells, and quantitative PCR was conducted to monitor their TNF-α induction. Furthermore, XH001/ TM7x cocultures grown in the SHI medium were shotgun-sequenced and assembled using SPAdes, followed by contig binning and annotation as described by McLean et al (31) to assemble the genomes independently. Genome completeness for all TM7x and published TM7 assemblies was estimated based on 111 universal, single-copy genes.…”

Insights into the metabolism, lifestyle and putative evolutionary history of the novel archaeal phylum ‘Diapherotrites’

“…Finally, it is plausible that Cand. IA might also employ a modified electron transport chain, similar to that recently suggested for candidate division TM6 (McLean et al, 2013). The chain will involve disulfide bond oxidoreductase D (DsbD), a thioredoxin-disulfide (NADPH) reductase (EC.…”