Approximately 5x1030 bacteria are estimated to inhabit our planet, and microorganisms colonizing our bodies outnumber our own cells by a factor of 10. Nevertheless, we know relatively little about our unicellular neighbors. A widespread approach used to explore microbial genomes is to culture the organism and analyze its genetic material, however, it is estimated that 99% of the microorganisms fail to grow in culture.
Sequence analysis of the 16S ribosomal RNA gene provides another powerful tool for the molecular identification of thousands of bacteria from complex samples. Fragmenting and sequencing total DNA extracted from an environmental sample, known as shotgun sequencing, reveals information on the many organisms present, but assembling individual discrete genomes is tremendously difficult, if possible at all.
A more recent approach involves sequencing DNA derived from one bacterial cell at a time. While these sequences derive from a single genome, amplifying the few femtograms (10-15 grams) of DNA in a cell to obtain sufficient material for sequencing presents considerable challenges.
“A key technology, multiple displacement amplification (MDA), developed almost ten years ago, was a huge advance compared to what had been done before,” says Roger Lasken, Ph.D., professor at the J. Craig Venter Institute. “MDA was a breakthrough in whole-genome amplification and, for the first time, we were able to amplify major portions of the genome from a single cell.”
MDA relies on bacteriophage Φ29 DNA polymerase, an enzyme with strong strand-displacement activity and high processivity. This approach uses random primers to synthesize amplicons with an average length of 12 kb and exhibits much less amplification bias than older PCR-based whole-genome amplification methods.
“I think MDA is going to be an important method for discovering uncultured bacteria. It is going to allow us to obtain bacterial cells from many different environments and also from human clinical specimens, and sequence their genomes without needing to develop culture methods,” continues Dr. Lasken.
Together with collaborators, Dr. Lasken tested DNA sequencing of Borrelia burgdorferi, the causative agent of Lyme disease, on single cells captured from the tick midgut by micromanipulation with a glass capillary. Alternative methods such as shotgun sequencing or PCR analysis from whole tissue would not have been informative on whether the sequences came from the same cell or from different ones.
Single-cell genomics will benefit not just unculturable microorganisms, but the ones that form colonies in culture as well.
Although bacterial colonies are derived from single cells, culturing may select fast-growing variants or counter-select certain virulence factors, and such genotypic changes might not accurately reflect the in vivo behavior of the microorganism. For example, some bacteria such as Neisseria gonorrhoeae or Helicobacter pylori lose certain virulence factors during in vitro culturing. “This is the reason, even in the cases when you can culture the pathogen, to sequence from single cells without culturing in order to investigate genotypes relating to the disease,” adds Dr. Lasken.
An essential aspect of genomics is the choice of DNA polymerases. To efficiently amplify sequences from heterogeneous microbial mixtures, an ideal enzyme should combine several characteristics such as high processivity and strand displacement activity, thermostability, and increased amplification fidelity. Thermophilic phage polymerases appear to exhibit several of these qualities and provide promising tools for single-cell applications.
“The promise of single-cell genomics will not be realized until the right DNA polymerase is developed that eliminates bias at the single molecule level, does not suffer from AT amplification bias, is compatible with heat lysis of cells, does not produce artifacts such as strand-switching chimeras or primer dimers, and can generate fewer branched molecules and more dsDNA,” says David Mead, Ph.D., CEO of Lucigen.
Recently, Thomas Schoenfeld, vp of enzyme discovery at Lucigen, examined viral populations inhabiting two hot springs, Bear Paw and Octopus, from Yellowstone National Park. From the over 200 polymerase sequences identified in the two viral metagenomes that Schoenfeld and his collaborators generated, two classes of phage enzymes emerged, with their members very divergent from each another and from other classes of polymerases. Of these, PyroPhage 3173 became the most extensively studied enzyme, and represents the first thermostable phage DNA polymerase.
A unique combination of characteristics, which includes the ability to amplify GC-rich templates, strong proofreading and superior replication fidelity, reverse transcription and remarkable strand-replacement activity, make PyroPhage 3173 a promising tool for single-cell genomics.