Bacteria and archaea make up most of the living world, but the vast majority of species, including some that are intimately associated with humans, have never been isolated or cultured.
Sequencing of DNA from natural microbe populations has allowed the identification of previously unknown taxa and in some cases provided detailed genomic information about the organisms. But having sequence data is “like having the parts list” for a machine, says microbiologist Karsten Zengler of the University of California, San Diego. This alone “does not tell you what this machine will do.”
For a better understanding of a microbe’s physiology and functions, researchers need to study living specimens, or at least whole cells. To that end, microbial geneticist Mircea Podar of Oak Ridge National Laboratory and colleagues are examining the sequence data of uncultured microbes to design tools with which to capture the bugs.
Focusing on bacteria present in the human mouth, Podar’s team compared available sequence data from uncultured organisms therein with sequences of previously cultured bacteria to identify codes for potential cell-surface proteins with regions unique to the uncultured organisms’ DNA. The researchers then generated peptides from these candidate codes, injected them into rabbits to create antibodies against them, and labeled the antibodies with fluorescent markers. Adding these fluorescent antibodies to microbe samples from human saliva enabled the detection of bacteria with corresponding surface proteins and their isolation via fluorescence-activated cell sorting (FACS).
The team isolated two different taxa of previously uncultured oral bacteria—TM7 and SR1—both of which the team was able to cultivate using carefully selected media. In addition to enriching for the target organisms themselves, the antibody-driven technique pulled out specific types of bacteria with which the TM7 and SR1 organisms were physically associated. In fact, the success of subsequent cultivation of TM7 and SR1 may be due in part to the co-isolation of microbes required for their growth.
While in principle the technique could be applied to any bacteria, it will be necessary with each new target strain to carefully design the peptides used for antibody generation, says Podar. The design is a careful balance between having similarities to known surface proteins in other microbes and being specific for the desired organism. So “it’s never going to be a kit,” he says. Rather, “it’s a guided approach that enhances your chance of success.”
Zengler, who was not involved in the research, says the technique “will help us to get more organisms in culture and to learn more about organisms that we only know from their genomic fingerprint.” (Nat Biotechnol, 37:1314–21, 2019)
|Studying uncultured bacteria||Isolating cells||Advantages||Disadvantages||Culturing Potential|
|Single-cell sequencing||Individual cells from a mixed population are sorted into droplets for genomic amplification and sequencing.||The approach can be high-throughput and uses the increasingly commonplace techniques of microfluidics and single-cell sequencing.||Yields no morphological or direct physiological information. Difficult to gather data from especially rare organisms. Little or no information on interspecies interactions.||None|
|Reverse genomics–enabled isolation||Bacterial DNA sequences are used to generate peptides for antibody production. The antibodies are then used to isolate the bacteria of interest from a mixed population.||Target organisms are greatly enriched, facilitating genomic, morphological, and physiological characterization. Associated organisms may also be isolated, enabling insights into relationships.||Choosing peptides for antibody generation requires a tricky balancing act between homology and specificity.||Culturing involves some trial and error, but an abundance of the target organism and isolation of associated microbes improves chances of success.|