Bacteria are ubiquitous. They defend and define us, constituting over 90% of the cells in the human body. They wield the ability to survive in the most extreme of environments, and the chemical reactions they catalyze hold the potential for vital technological advances such as clean energy. Yet they defy domestication; scientists have been able to grow in the lab, or “culture,” less than 1% of the bacterial species on Earth .
The remaining 99% of uncultured bacterial species are not particular to a given environment. They live in the sea, in the soil beneath our feet, in our guts and in our mouths. We know that they exist because a region of their DNA, which encodes for an essential RNA molecule called 16S rRNA, is shared between species. Every bacterial species has a functional copy of this RNA, but, because DNA mutations accumulate with time, each species builds its own unique variant code for this molecule. The more closely related the bacterial species, the more similar the DNA for their 16S rRNA will be. The DNA for the16S rRNA is similar enough between species for scientists to locate it and examine it, but unique enough to distinguish different species. Perhaps surprisingly, some of the DNA sequences for 16S rRNA of uncultured bacteria are extremely similar to those of widely studied cultured species, indicating that they are closely related. However, despite the efforts of hundreds of dedicated microbiologists, we know little about these “wild” species—other than that they exist.
The cultural challenge
The basic procedure for culturing a bacterial species is pretty straightforward. Take a sample—for example ocean water, soil, or spit—and dilute it in water. Then spread a droplet of this dilution on a petri dish full of nutrients. Each individual bacterium lands in a unique spot on the dish. It replicates and divides in this spot, and its copied daughter cells replicate over and over again, forming a visible “colony” of tens of thousands of genetically identical cells that is about the size of the back end of a needle. By altering the nutrients in the dish and surveying many samples, scientists have isolated colonies of several thousand different bacterial species.
However, hundreds of thousands (perhaps millions) of species simply do not form colonies in the lab, preventing their study for fundamental science and technical and medical applications. Like disappointed teachers, many scientists gave up on studying this 99% of bacteria, deeming it “unculturable.” Recently, though, determined scientists developed techniques to isolate up to 40% of the diverse species in a given environment- a vast improvement from previous yields. For example, one research group encapsulates a sample of seawater in agar (the same gelatin-like material that is in petri dishes), covers the agar in a membrane that allows for the diffusion of seawater, and returns the seawater-agar to its natural environment. After a few weeks, the bacteria in these agar “shells” form colonies large enough to study . By growing the bacteria an environment that more closely resembles their natural one, it’s not necessary to try out all the nutrient conditions to find the right recipe for each temperamental species.
What are the components and conditions that differ between the wild and the lab? While in some cases the secret ingredient is a nutrient, recent studies point to many cases where these uncultured bacteria require other bacteria species to survive, much like we depend on expert farmers, pharmacists and builders. In the case of bacteria, their neighbors may produce important metabolic molecules, excrete pheromone-like molecules that signal growth, or remove toxins from the environment. For example, a group of scientists at Northeastern University recently discovered that many uncultured bacteria cannot capture iron on their own; they rely on their bacterial neighbors to secrete iron-capturing molecules called “siderophores.” By growing samples alongside of siderophore-producing bacteria, the group has isolated many new previously “unculturable” species.
Just as many of these species are not fit for life in the lab, one might expect for dependent bacteria to be unfit in the wild; chances of survival could be reduced if your survival depends on you and your neighbor’s! How have these bacteria weathered the tides of time and evolutionary pressures? This question remains unanswered, but one hypothesis is that the presence of well-known neighbors signals to the bacteria that they are in the proper environment for growth. By relying on their neighbors, these uncultured bacteria do not grow and adapt to environments where they are unlikely to thrive in the long-term .
Closing in on culture
While technical breakthroughs are now allowing us to study more of nature’s vast diversity, there are still many questions to be answered. Why is it so difficult to culture the remaining 60% of bacteria that these new techniques fail to capture? How will we culture them? Particularly, how can we culture the yet uncultured bacteria, both commensal and pathogenic, that live in our own bodies? The answers to these questions should lead to new cures and to further understanding of our “microbial organ”.
The practice of culturing bacteria independently masked the importance of inter-species relationship in the 20th century. Today, the study of bacterial interactions is a booming scientific field, with many unanswered questions within our grasp.
By Harvard Graduate Student Tami Lieberman
1. Schloss and Handelsman. Status of the microbial census. Microbiol Mol Biol Rev (2004) vol. 68 (4) pp. 686-91
2. Kaeberlein et al. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science (2002) vol. 296 (5570) pp. 1127-9.
3. Donofrio et al. Siderophores from Neighboring Organisms Promote the Growth of Uncultured Bacteria. Chemistry & Biology (2031) vol. 17 (3) pp. 254-264
Links of interest
This article was inspired by a more technical blog post from The American Society for Microbiology’s blog, Small Things Considered. http://schaechter.asmblog.org/schaechter/2010/07/the-uncultured-bacteria.html
See the previous SITN Flash article: Our Microbial Organ, April 30, 2010.