by Veronica W. Rowlett
Predator-prey relationships exist all over the world of living things, from microbes to animals and humans. In defense against predators, animal prey use varied strategies such as hiding from, distracting, escaping, or fighting predators. Humans build walls and ramparts to protect themselves within cities from potential animal or human aggressors (Fig. 1). But how do bacteria defend themselves against predators? Although thought to be simple organisms, they have evolved complex responses to the challenges they face. A popular example is adopting group behavior through quorum sensing. When acting as a group, bacteria can form single or multispecies biofilms and some, like Bacillus subtilis, can sporulate when nutrients are limiting, which keeps them in a dormant state until conditions become favorable (Figure 2a). Some species of bacteria, like Myxococcus xanthus, are predators that feed on other microbes, including other bacteria and yeast. M. xanthus forms single species biofilms when nutrients are abundant, and builds fruiting bodies when starving (Fig. 2b).
The impressive list of predator-avoidance behaviors used by bacteria include changes in motility, morphology, making biofilms, and sporulation. Which do they choose to defend themselves against predator bacteria like M. xanthus ? One natural prey to this species is B. subtilis. Both M. xanthus and B. subtilis are soil dwelling. Studies of their interactions tell us that B. subtilis protects itself by forming spores and producing the antibiotic bacillaene. It turns out that's not all that B. subtilis can do. Recently, studies have shown that B. subtilis forms large cellular assemblies or "megastructures" when being cultured with M. xanthus. How about these structures? Are they fortifications used for defense against the predator?
Megastructures arise after a few days, similar in timing to colony biofilm formation. They are very large (500 µm in width and 150 ‒ 200 µm in height), tree-like in appearance (Fig. 3a, b), and full of spores. Fruiting bodies of M. xanthus are frequently found close to them, suggesting that the predators did not get sufficient nutrients from the B. subtilis cells. Since the fruiting bodies of M. xanthus formed after the B. subtilis megastructures started to form, it looks like M. xanthus first attacks B. subtilis, which in turn resists predation by producing bacillaene and forming the spore-filled megastructures. Depleted of nutrients, M. xanthus forms fruiting bodies.
Are the megastructures genetically similar to biofilms? B. subtilis mutants with altered biofilm formation were tested for their ability to form megastructures. Strains defective in biofilm formation were still able to form megastructures in the presence of M. xanthus, indicating that these are genetically different from biofilms. A spoIVA mutant of B. subtilis defective in spore production was unable to maintain megastructure integrity over a few days (Fig. 3d), suggesting that B. subtilis spore formation is important for the integrity of the structures over time.
The authors hypothesized that a mutant of B. subtilis unable to produce bacillaene would be unable to form megastructures, given that it would not have transient protection against M. xanthus. Interestingly, B. subtilis cells unable to produce bacillaene (Fig. 3c) can still sporulate, form megastructures faster than those that can produce bacillaene, suggesting that they can sense and respond sooner to predation. The spores contained in 6 week old megastructures can still germinate under favorable growth conditions, suggesting that they are stable within the megastructure for long periods of time.
Megastructures do not form in the absence of M. xanthus, which suggests that their formation is a response to predation. Since predation is enhanced by motility, the authors hypothesized that B. subtilis megastructures would be attenuated in the presence of M. xanthus cells that cannot coordinate motility. While inhibition of M. xanthus S-motility or A-motility does not fully abolish B. subtilis megastructure formation, inhibiting both motility types does, meaning that an efficient predatory attack by M. xanthus is required for megastructure formation. If M. xanthus motility is impaired, it is possible for the prey cells to evade predation by growing over the agar surface faster than M. xanthus.
Megastructure formation of B. subtilis in response to M. xanthus predation is a newly found defense mechanism against a bacterial predator, further highlighting the sophisticated ways bacteria respond to challenging environments. Megastructures are likely important for safekeeping of spores until conditions become favorable for growth. Thus, the story brings to mind what happened in human history when walled cities were besieged by invaders. By protecting their food supply, the inhabitants made it scarce for the attacking hordes. Thus, they safeguarded their city's ability to survive and later thrive.
Future work will be required to determine if megastructures structures are made as a specific response to M. xanthus predation or if they form under other environmental stresses. Also, it is unknown if similar structures can be formed by other Bacillus family members found in soil environments. More research is required to further understand the relevance and function of megastructures in mixed soil communities. M. xanthus induces the production of antibiotics in other bacterial species like Streptomyces coelicolor. By influencing antibiotic formation, this story may have clinical relevance in combating the problem of antibiotic resistance.
Veronica is a Ph.D. candidate in the Microbiology and Molecular Genetics program at The University of Texas Health Science Center at Houston. She is a member of William Margolin's lab.