Filling in the Gap
“There is still a gap between the microorganisms present in the environment and the ones that we can cultivate,” points out Martin Keller, Ph.D., director of the Oak Ridge National Laboratory’s biosciences division. “How can we fill this gap and use cutting-edge sequencing technologies to help us understand what specific microorganisms are doing in the environment? This is where single-cell microbiology comes into play.”
Recently, Dr. Keller and collaborators combined fluorescence in situ hybridization and flow cytometry with whole-genome amplification and sequencing. They illustrated the possibility of obtaining a significant fraction of an uncultured bacterial genome starting with several cells selectively isolated from a heterogeneous environmental sample. For a representative of the TM7 phylum that is less than 2% abundant in the soil, the investigators achieved approximately 20% genome coverage.
Next Level of Biofuels
An important effort in Dr. Keller’s group is exploring the plant-microbe interface and focuses on specific groups of microorganisms associated with the degradation of cellulosic material and carbon sequestration. Photosynthesis uses solar energy to generate plant cellulosic material, and cellulose decomposition by biomass-degrading microorganisms generates sugars that are subsequently fermented into alcohol.
“If we understand the connection between microbes and plants, we can really make a significant impact and help solve some of our major issues in carbon and energy,” adds Dr. Keller. “This is where I strongly believe that having these new tools—microbial ecology and single-cell genomics—and all the genomics tools together, can help us understand a lot about carbon fixation and about how we can go to the next level of biofuels.”
The world’s oceans harbor some of the most diverse microbial populations. Ramunas Stepanauskas, Ph.D., senior scientist at the Bigelow Laboratory for Ocean Sciences, and collaborators, recently combined fluorescence activated cell sorting with whole-genome amplification on coastal water samples to isolate and sequence two uncultured flavobacteria from the Gulf of Maine, with estimated 91% and 78% genome recoveries.
“Recent large-scale metagenomic sequencing unveiled the enormous richness of genes encoded in environmental microbial assemblages,” explains Dr. Stepanauskas, “However, it is very difficult, if not impossible, to assemble discrete genomes and to understand entire biochemical pathways through metagenomics alone.”
The study illustrated the power of these technologies in obtaining reference genomes, and represents a major advance because cultivation-based studies so far mostly recovered “weeds” with no ecological significance in the natural environments. The annotation of the two genomes unveiled several metabolic pathways with potential in bioenergy production such as biopolymer degradation and proteorhodopsin photometabolism.
“I have tremendous expectations for single-cell genomics,” says Tanja Woyke, Ph.D., research scientist at the Lawrence Berkeley National Laboratory/DOE Joint Genome Institute and first author of the study. “Together with other technologies aimed at accessing uncultured microbes, I think it will become one of the dominant methods that will benefit microbial ecology as well as other fields such as bioprospecting.”