Domestication of the wild B. subtilis

By: Gabriella Stone, '21 Marissa Tousey-Pfarrer '21, and Jackie Rich '21

If you are a fan of genetics or are fascinated by the history and processes of animal domestication, it’s likely you’ve heard about Dmitry Belyaev. Belyaev, a Russian geneticist, is famous for conducting breeding experiments on silver foxes in the early 1950s to assess how laboratory domestication influenced various changes in the foxes’ behavior and physical appearance. Belyaev found that after years of selective breeding, the foxes became more sociable, friendly, and tame towards humans; he had successfully domesticated the silver fox!

Although Belyaev’s domestication of silver foxes was controlled, sometimes domestication happens unintentionally in laboratory settings, especially for microbes who have fast generation times. So how are Belyaev’s Russian fox domestication experiments similar to microbe domestication? Well, it turns out that Bacillus subtilis, a gram-positive soil-dwelling bacterium, similarly demonstrates changes in social, behavioral, and other phenotypic qualities in laboratory settings and thus demonstrates the ability to be ‘domesticated,’ much like that of the Russian silver fox.

Belyaev with some of his domesticated silver foxes. Image from New York Times, originally from Belyaev lab in Siberia.

An image of G7 which is a strain of B. subtilis. The top left image shows the G7 colonies and the top right image depicts stained spores of G7. The bottom image is an electron micrograph of G7. Image from Gu et al. (2019).

B. subtilis is a rod-shaped and non-pathogenic bacterium commonly found in the soil as well as the digestive tracts of humans and other animals. B. subtilis is commonly used as a model microbe in bacterial genetics research due to its easy cultivation and maintenance in the laboratory. B. subtilis is particularly sought after as an organism for study due to it being a spore former microbe, meaning that in times of environmental stress it can upregulate genes responsible for creating metabolically inactive cells or spores that can withstand harsh environmental conditions or stressors. It was this sporulation ability of B. subtilis that attracted the interest of researchers from the University of Lisbon, Portugal to investigate how laboratory conditions affect the bacterium. Barreto et al. (2020) were drawn to the results of previous studies that determined that B. subtilis sporulation ability and proficiency is altered by laboratory domestication, and wanted to further investigate what other factors and behaviors of B. subtilis were altered during laboratory domestication. The researchers aimed to see how laboratory domestication altered social traits of B. subtilis including bacterial cell motility and biofilm formation and aimed to determine which particular genes were the targets for the observed alterations.

Simplified diagram of B. subtilis spore formation. Image by author Gabriella Stone.

Barreto et al.'s (2020) research demonstrates that there are changes in the colony morphology with domestication in 5 different populations, which resulted in 3 morphotypes, a, b, and c. Their work also shows the locations of the mutation within the degU region of the chromosome, the resulting protein, and the effect of each mutation on the biofilm structure compared to that of the Ancestral population, which is the natural isolate of B. subtilis. Furthermore, their work illustrates that there are differences in swarming motility and colony architecture and that degUEvo is responsible for changes in biofilm complexity.

Figure 3 from Barreto et al. (2020) showing (a) images of LB plates showing the extent of swarming motility and (b) and (c) electron micrograph images showing biofilm architecture.

Figure 3 illustrates that degUEvo is responsible for changing both swarming motility (fig 3a) and affects colony architecture (Fig 3b,c). During domestication, they took 5 populations derived from a B. subtilis natural isolate (which they now named Ancestral) and they were passaged daily for 16 days through dilution in a rich medium with aeration and agitation. The plates used in Fig 3a were agar plates that were fortified with 0.7% of agar and were inoculated and incubated for 16 hrs at 28℃. The strains used in Fig 3b were grown at 28℃, while strains in Fig 3c were grown on MSgg agar plates that were incubated for 96 hrs at 28℃. Daily plating showed that two new colony morphotypes had emerged and they labeled them to be type a, type b, and type c. The strain they denoted to be "Evolved" was the type b colony from the first population on day 8, and their results suggested that Evolved increased traits associated with growth within laboratory conditions. To test swarming motility, they used the laboratory strain PY79, which they refer to as Lab, and is a control since it carries mutations that inhibit swarming motility. Swarming motility assays indicate that Ancestral and degUAnc are able to swarm, however swarming ability in Evolved, Lab, and degUEvo is inhibited (Fig 3a). Additionally, Ancestral and degUAnc showed they had many more wrinkles after 24 hrs of incubation and that as time went on, the more complex the colony architectures became (Fig 3b). In contrast, their results showed that Evolved and degUEvo formed flatter colonies that had fewer wrinkles (Fig 3b,c).

For Belyaev’s foxes, domestication resulted in fundamental changes in the natural social behaviors of these animals. Similarly, Barreto et al. (2020) demonstrated that domestication of B. subtilis results in social behavior changes; specifically, B. subtilis experiences changes in motility and biofilm formation and morphology. The smooth and flat biofilm morphology of B. subtilis observed during domestication is similar to changes in biofilm morphology observed in other bacteria during laboratory domestication. This shows that different species of bacteria may exhibit similar phenotypic changes during laboratory domestication. These researchers determined that genetic mutations occur quickly, after approximately 2 weeks, during laboratory domestication of B. subtilis. They also determined that many of the mutations occurred in the degU gene which appears to be a gene-targeted for mutations during laboratory domestication. This finding can be applied in future studies to determine if similar target mutation genes occur in other bacteria species.

Although this study focused on B. subtilis, these findings have broader implications for the field of microbiology. As biologists, we often find inspiration for our research in considering the interactions between an organism and its environment. Perhaps these types of questions led Belyaev to consider domesticating silver foxes. However, sometimes our experimental techniques necessitate altering the natural environment of our study organism, which can impact our experimental results. Microbiologists generally work in controlled laboratory environments which are vastly different from the natural environment of bacterial cells. As shown in this study, culturing cells in a laboratory environment can lead to evolutionary changes which results in the cells possessing traits that differ from those of cells in their natural environment. B. subtilis is just one of many strains of bacteria that have been cultured in laboratories for many years, during which time these strains have become genetically distinct from strains found in natural environments. The researchers point to microbiological research techniques, such as selecting and culturing specific bacterial colonies, which can lead to loss of traits. The findings of this study suggest that it is essential for all microbiologists to consider and evaluate the impacts of their experimental techniques on their study system to fully understand the implications and potential limitations of their experiments.


About the authors:

Image of the 3 authors; Gabriella Stone (left), Marissa Tousey-Pfarrer (middle), and Jackie Rich (right).

Marissa: Hi, I'm Marissa and I am a senior at Mount Holyoke College. I am currently working with Professor Woodard to research how various stressors, including heat shock and oxidative stress, contribute to stress resistance in Drosophila melanogaster. In the future, I will be studying epidemiology at the University of Iceland.

Jackie: Hi everyone! My name is Jackie and I use she/her pronouns. I am a senior at Mount Holyoke College majoring in biology with a concentration in marine science. I have been a member of Dr. Renae Brodie’s lab for two years studying climate change impacts on the fiddler crab Minuca pugnax. I am currently researching the effects of various environmental variables on behavioral thermoregulation in M. pugnax.

Gabriella: Hello readers! My name is Gabriella (she/they) and I am also a senior and biology major. After graduation, I will be working in a lab at Dana Farber Cancer Institute. In my spare time, I love to take my cat for walks and try new vegan ice cream flavors!