by Sophia Swartz
figures by Jasmin Joseph-Chazan

If you put all of the living things on Earth in a box–from humans to anteaters to teeny-tiny tardigrades–and then plucked one of these organisms out at random, it is very, very likely that you just found yourself a microbe. Microbes, although too small to be seen with the naked eye, are some of the most common forms of life. Scientists estimate that there are approximately one trillion microbial species in, on, and around us. For example, in one gram of dental plaque–the white, gooey stuff the dentist scrapes off your teeth–there will be approximately 10¹¹ (or 100,000,000,000) individual bacteria, about the same number of humans that have ever lived (Figure 1).

Dental plaque is particularly interesting because the vast numbers of microbes that make it up are not just acting alone: instead, they team up and form dense communities called biofilms. In plaque, microbes collaborate by dividing up tasks: some produce acidic compounds to dissolve tooth enamel, while their neighbors process the dissolved enamel products. This division of labor is possible largely because of the degree of heterogeneity–or diversity– in the behaviors of individual microbes within these sprawling biofilm communities. But where does this heterogeneity come from?

Microbes with the same name play a different game

The different behaviors that microbes in dental plaque display, called phenotypes, are specified by a molecular instruction manual buried within them: the microbes’ genome. Different microbial species have different genomes and are therefore able to perform different tasks based on the information their genomes contain. However, even microbes of the same species, which have the same genome, may behave very differently. That is because each microbe can interact differently with external factors, like the environment in which it lives and the chemicals to which it is exposed. Such interactions can affect what information from the microbe’s genome will be activated and turned into observable behavior. The phenomenon of genomically-identical microbes in the same environment showing different phenotypes is called phenotypic heterogeneity, and it plays a very important role in biofilms.

When microbes grow together in a shared space to form a biofilm, the microbial community achieves specific properties through the phenotypic heterogeneity of its microbial cells. Like patches in a quilt, different partitions of a microbial community perform unique functions that are dictated by the structure of the biofilm (Figure 2). Where and with whom the microbial cells live plays a key role, with minute changes in microbial genomes sometimes leading to extensive phenotypic heterogeneity. However, it is unclear how the biofilm structure affects this “division of labor” in a microbial community.

Putting microbial cities on the map 

To resolve the question of how different subsets of a microbial community obtain different functions, a research team led by Dr. Dianne Newman at the California Institute of Technology developed a new method called parallel-sequential fluorescence in situ hybridization (par-seqFISH). par-seqFISH is a technique that allows researchers to zoom into microscopic microbial communities and image a bird’s-eye view of both who lives where and who does what. In complex biofilms–the microscopic equivalent of human cities–par-seqFISH is able to parse out distinct neighborhoods and match single bacterial cells with specific jobs (Figure 3).

Specifically, Newman’s team studied how the availability of oxygen determined how microbes break down different nutrients. The bacteria they studied, Pseudomonas aeruginosa, is capable of living in environments both rich and poor in oxygen. However, to survive in oxygen-poor environments, P. aeruginosa must activate extra internal processes that are needed for fermentation, which is how bacteria can extract energy from their food in the absence of oxygen. Activating these different processes allows us to distinguish the P. aeruginosa that can survive without oxygen from P. aeruginosa cells that have plenty of available oxygen and do not need to activate fermentation.  

Using par-seqFISH, Newman’s team observed a biofilm that grew for 10 hours straight and mapped zones of bacteria that had or had not activated fermentation. Within the span of extremely small distances, different clusters of microbes showed different phenotypes based on local oxygen availability. Perhaps more surprisingly, bacteria that could and could not ferment were found to coexist in the same patch of biofilm.

Their findings established a way to explore the phenotypic heterogeneity of a dense community on a cell-by-cell basis, almost akin to taking a census. Scientists are now able to interrogate individual microbes in a biofilm and study their specific responses to different environments or stimuli, whether that be oxygen availability, antibiotic resistance, or some other phenotype of interest. By using this technique on different phenotypes, scientists can begin to understand how biofilm structure and phenotypic heterogeneity interact to generate the rich functional diversity that allows biofilms to thrive.

Future outlook for spatially-resolved biofilm research

Microbial communities consist of a complex mixture of different bacterial cells displaying different phenotypes able to perform different tasks. Even in a biofilm consisting entirely of the same species of bacteria, one bacterial cell can differ hugely from another, all within the scale of several micrometers. The phenotypes within a biofilm are often studied at the level of the whole community, but the phenotypic heterogeneity that divides biofilms into functional zones means biofilm communities cannot be fully understood without exploration at the level of individual bacterial cells in specific spatial contexts.

Understanding biofilm communities is additionally important for human health: both the bacteria that exist in our bodies and help us survive (our microbiome) and bacteria that infect and make us sick sometimes structure themselves into biofilms. The health of many complex bacterial ecosystems (such as the human gut, for example) is strongly affected by the health of its microbiome, which consists of bacteria showing different phenotypes. Antibiotic resistance is another good example. Experiments have shown that when biofilms are exposed to powerful antibiotics in the lab, a small fraction of the biofilm will interact with the antibiotic differently and survive, while the other microbes perish. So understanding how these communities form and function will allow us to advance human health. Recent new methods, like the par-seqFISH technique described here, hold exceptional promise to resolve the spatial structure of microbial communities and answer new and exciting questions about the role of phenotypic heterogeneity in shaping microbial biogeography. As we are creatures that are surrounded by microbes both inside and out, our ability to finally study how microbes respond to their environment and build communities is an exciting and captivating scientific development.

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Sophia Swartz is a senior at Harvard College studying Molecular and Cellular Biology.

Jasmin Joseph-Chazan is a third-year graduate student at Harvard University studying Immunology.

Cover image by geralt from pixabay

For more information:

• Check out this article for a discussion on how science is working to redefine the way in which we study microbes and the kinds of limits scientists apply to individual microbes versus microbial communities. 
• If you want to learn more about the complex behaviors and community phenotypes displayed by microbial biofilms and slime molds in how they navigate their environments, visit here.
• Listen to this podcast episode for more on the complex and beautiful relationships that form at the microscopic level between humans and their resident microbes.
• Want to dive deeper into ongoing research efforts to channel our scientific understanding of biofilms/microbiomes into usable human health therapeutics? Check this article out.