Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth

Abstract

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.

Fig. 1.

Single-cell genome amplification device. (A) Photograph of a single-cell isolation and genome amplification chip capable of processing eight samples in parallel. To visualize the architecture, the channels and chambers have been filled with blue food coloring, and the control lines to actuate the valves have been filled with red food coloring. (Scale bar, 5 mm.) (B) Schematic diagram of a single amplification unit. The feed line is used to bring reagents into the chambers when the V_R valve is open and to the waste when the V_w valve is open. The V_in valve allows deposition of a single bacterium into the sorting chamber. The lysis (3.5 nl), neutralization (3.5 nl), and reaction chambers (50 nl) are used in sequence and are separated by individual valves V_L, V_N, and V_R, respectively. Valve V_out allows recovery of the amplified genomic material from the chip into an individual microfuge tube. (C) After a cell is trapped in the chamber, the feed line is filled with lysis buffer. (D) The lysis buffer is used to push the cell into the lysis chamber. (E) While the lysis buffer is mixing with the cell solution by diffusion, the feed line is flushed. (F) Neutralization buffer is loaded into the feed line and used to push the cell lysate into the neutralization chamber. (G) While the neutralization reaction is mixing by diffusion, the feed line is flushed. (H) The WGA reagents are loaded into the feed line and used to push the neutralized cell lysate into the reaction chamber. (I) The amplification reaction proceeds in a closed system comprising sorting, lysis, neutralization, and reaction chambers.

Fig. 2.

Results of rod-like morphotype survey. (Left) Phylogenetic tree showing bacterial phyla based on 16S rRNA gene analysis (adapted from ref. 1). Green text indicates that at least one member of the phylum has been cultivated; different shades of blue indicate the number of genome sequencing projects in a particular phylum that were completed or in progress as of May 2006. Red numbers and percentages indicate the results of our single-cell survey of the human subgingival crevice, in which filamentous bacteria with rod-like morphotypes were isolated and lysed and their genomes were amplified. [Reprinted with permission from ref. (Copyright 2003, Annual Reviews, www.annualreviews.org).] (Right) Optical micrographs of the four TM7 cells isolated in this survey.

Fig. 3.

TM7b has much higher sequence similarity to the TM7a assembly than to the F. nucleatum or C. aurantiacus genomes. Mapping was performed by using BLAST (21); ≈100,000 individual sequence reads with average length 104 bp were mapped onto each genome. The histogram shows E-values returned by BLAST, which indicate the statistical significance with which the read can be mapped onto the genome.