Sacrificing for the Team: Sibling Killing in Myxococcus xanthus

By: Erin Cadora

Myxococcus xanthus is a species of Myxobacteria that engage in social behaviors that are shockingly complex for a single-celled organism. M. xanthus behaves in a way that can sometimes be more akin to multicellular behavior, exhibiting intricate interactions between cells that allow for smart survival strategies. When environmental resources are abundant, M. xanthus cells exhibit social swarming behavior and can hunt for food in a cooperative group in the form of a biofilm. If you were competing on a soccer team, you would want a team full of people with various skills in order to be competitive as possible. Similarly, hunting in a group of cells allows the bacteria to take advantage of a wider array of genetics. This way, they have a greater chance of being able to produce all the necessary digestive enzymes.

When resources are scarce, M. xanthus exhibits a different kind of social behavior called fruiting body formation. Starving conditions cause M. xanthus cells to pile together and aggregate into a mound. Eventually the mound builds up to a bulb-like structure called a fruiting body. The final purpose of the fruiting is to turn select cells into spores that can withstand the starving conditions. Within the fruiting body, cells are able to send signals to each other and different cells are assigned different roles. About one out of ten cells will transform into a spore, three out of ten cells explore the area around the fruiting body in search of food sources, and the rest of the cells will purposefully die in order to provide nutrients for the formation of spores. M. xanthus cells in the fruiting body behave less like individual organisms and more like players on a soccer team, performing different tasks that work towards a unified goal.

Diagram of M. xanthus life cycle and fruiting body formation (top) and picture of fruiting bodies (bottom).

In order for Myxobacteria to participate in these kinds of social behaviors, it needs to be able to differentiate between cells of its own kind and cells of other kinds of bacteria. During your soccer game, you would want to be able to tell who is on your team so you could pass the ball to the right person. Like teammates recognize each other by the color of their uniforms, Myxobacteria cells recognize each other through receptor proteins on their surfaces called TraA receptors. These receptors allows for outer membrane exchange (OME) between the cells. OME can allow for M. xanthus to participate in cooperative interactions, like the sharing of cellular resources, as well as competitive interactions. Both these behaviors are involved in fruiting body formation. M. xanthus cells move using two different systems- S (social) motility, which requires contact between cells and allows for swarming, and A (adventurous) motility, which allows for the movement of individual cells. A cell that is an (A− S−) mutant is nonmotile. It has been found that, through OME, nonmotile strains of M. xanthus can prevent motile cells from swarming. When resources are scarce and M. xanthus needs to form fruiting bodies, it stops its swarming behavior. If things aren’t going well in your soccer game and the other team is coming towards the goal with the ball, you would your teammates to stop playing offensively and fall back to defend. A study performed in 2016 by Dey et al. explored how swarm inhibition caused by nonmotile strains works and what the implications of that could be.

First the authors of the study established that the way nonmotile strains were causing swarm inhibition was by killing their motile siblings. They came to this conclusion after they plated a mixture of motile and nonmotile M. xanthus cells and observed phase-contrast micrographs of the cells taken at 24 hours after inoculation and 48 hours after inoculation. At 24 hours after inoculation, some motile cells were visible as having moved past the inoculation point. At 48 hours, those motile cells were no longer visible. The authors concluded that the motile cells that had moved past the inoculation had died and lysed as a result of them having had contact with the nonmotile cells.

Figure 1B. Motile cells that had come into contact with nonmotile cells died and lysed. Motile cells visible as having moved away from the inoculation point after 24 hours were no longer visible after 48 hours.

The authors then wanted to test whether blocking outer membrane exchange would allow motile cells to survive despite coming into contact with nonmotile cells. If a person on your soccer team were to put on a uniform from a different team, you might not recognize them during the game and might not pass the ball to them. Similarly, M. xanthus cells without the proper TraA receptors may not be recognized by other M. xanthus cells and wouldn’t be able to participate in OME. The authors made a mixture of nonmotile cells and motile cells that had mutations on their TraA surface receptors. Unlike the motile cells without the TraA mutations, the motile TraA mutants survived as well as the nonmotile cells, confirming that sibling killing is TraA dependent.

After establishing that swarm inhibition was caused by the killing of motile cells and that the killing of motile cells was dependent on TraA receptors, the authors wanted to explore the genetic factors involved in sibling killing. They tested a variety of different nonmotile M. xanthus strains used in labs for their ability to cause swarm inhibition. They found that a nonmotile strain called DK101 caused swarm inhibition while a nonmotile strain called DK1622 did not cause swarm inhibition. DK101 is an ancestor of DK1622; it is a strain that was cultivated in the lab earlier than DK1622 and was used to help make DK1622. This led the authors to wonder if a strain being ancestral was the key in whether motile cells would be killed. They mixed a strain derived from DK1622 with three different ancestral strains and let the mixture incubate for 48 hours, afterwards comparing the number of DK1622-derived cells with ancestral cells. All three ancestral strains were shown to vastly outcompete the DK1622-derived strain in terms of cell numbers, leading the authors to conclude that ancestral strains are capable of killing other strains.

The next step was to investigate what the key difference was about ancestral strains that makes contact with them lethal. The authors noticed that the genome of DK1622 is missing a region when compared to the genomes of two of the ancestral strains. A small part of this missing region contains “a defective prophage-like element called Mx alpha” (Dey), prophage being the genetic material of viruses that infect bacteria. The fact that Mx alpha seemed to be present only in the strains that were doing the killing led the authors to conduct an experiment exploring whether Mx alpha was necessary for cells to kill their siblings.

In the experiment the authors took DW2403, a nonmotile strain that was known to kill siblings, and deleted its Mx alpha region. They mixed it both with another nonmotile aggressor strain that still had its Mx alpha region and a non-aggressor strain that lacked an Mx alpha region. The deletion of Mx alpha in DW2403 seemed to render it unable to kill the non-aggressor strain. The deletion also made DW2403 vulnerable to being killed by the aggressor strain still containing Mx alpha. This finding led to the conclusion that the presence of Mx alpha is in fact necessary for an M. xanthus strain to be able to kill its siblings. Mx alpha also appeared to be necessary to make a strain resistant to sibling killing. Mx alpha being necessary both for killing and resistance to killing suggested that it could be acting as a toxin-antitoxin system. This would mean that through outer membrane exchange, an M. xanthus cell containing Mx alpha would transfer a toxin to the sibling cell. If the sibling cell also contained Mx alpha, it would contain an antitoxin that would allow the cell to survive. If the sibling cell didn’t have Mx alpha, it would be killed by the toxin.

Figure 7A. Results of the experiment showing Mx alpha is necessary for sibling killing. Shows the growth of DW2403 with its Mx alpha deleted when mixed with an aggressor strain containing Mx alpha (left) vs when mixed with a non-aggressor strain (right).

Figure 7B. Results of experiment showing that Mx alpha is necessary for resistance to killing. Top line shows the ratio of the cells of an aggressor strain containing Mx Alpha to the cells of DW2403 with its Mx alpha deleted. The aggressor strain greatly outcompeted the DW2403 strain, suggesting the deletion of Mx alpha made DW204 vulnerable to killing.

The major finding of this study, that Mx alpha is the key player in sibling killing, could give some insight into how M. xanthus is capable of such complex social behaviors. When food is scarce and M. xanthus forms fruiting bodies, cells take on different roles and the majority of the cells die to provide the nutrients necessary to form spores. The way that these cells are killed and the way that cells are selected for killing is currently unknown. It could be possible that Mx alpha is part of the process of directing cells in fruiting bodies to die and provide food, perhaps like a soccer coach telling players to fall back on the field and help defend their goal. Cells containing Mx alpha could also possibly be contributing something important to the genetic diversity that M. xanthus takes advantage of by acting as a social species. Mx alpha is a prophage-like element, and the presence of a prophage can make cells immune to infection by some bacteriophages- viruses that destroy bacteria.

Further studies could be done to investigate the role that Mx alpha might play in the social behaviors of M. xanthus. For example, an experiment could be conducted with two separate groups of various M. xanthus strains, one group in which aggressor strains had Mx alpha deleted and one group in which aggressor strains had Mx alpha present. These groups could be placed in an environment that didn’t have enough food, promoting the formation of fruiting bodies. We could then observe whether the presence of Mx alpha had an effect on the number of cells dying to provide food. The social capacity of M. xanthus is elaborate and much of it is not well understood, but the authors of this study may have found a piece of the puzzle in Mx alpha.