WARNING TO THE GRAM (+) AND GRAM (-) BACTERIAL COMMUNITIES: Be on the lookout for Myxococcus xanthus! This dangerous predator is on the hunt, and bad news is, it’s not picky. M. xanthus itself is a gram negative, rod shaped, predatory bacterium. A bacteria is gram positive (+) if it has a thick peptidoglycan layer and lacks an outer membrane, while a gram negative (-) bacterium has a thin peptidoglycan layer with an outer membrane. This peptidoglycan layer comprises the cell wall and protects the cell from extreme conditions, lysis, and contributes to the maintenance of the cell shape. M. xanthus’ process of predation generally begins with the invasion of a prey colony which is enabled by its special ability to glide along a surface. M. xanthus then secretes chemicals that rupture the membrane of its prey (aka lysis), where it is finally able to consume the released biomass of its prey. Below is a cool drawing that draws comparisons between bacterial predation and predation in the animal kingdom.
Image by Victor Leshyk, Center for Ecosystem Science and Society, Northern Arizona University
A recent study by Adrend et al. in 2021 sought to uncover more of the mystery about Myxococcus xanthus’ methods of predation and how such methods might be similar or different depending on whether the prey bacteria are gram-positive or gram-negative. They looked at two Gram (+) species, Micrococcus luteus and Bacillus subtilis, and two Gram (-) species, Escherichia coli and Agrobacterium tumefaciens. These bacterium were chosen so that the authors could observe the impact of M. xanthus’ predation on Gram (-) and (+) prey. Click here for a short video of M. xanthus preying on Escherichia coli but beware – it's about as gruesome as it gets in the bacterial world!
In Figure 1A, Arent et al. looked to observe M. xanthus’ predation at a macroscopic level. They inoculated both M. xanthus and each of the four prey bacteria on four different agar plates. M. xanthus behaved similarly toward both gram positive and negative prey. It spread radially from the inoculation spot and invaded the prey colony and cleared away the prey bacteria. In Figure 1B they prepared different protein fractions of M. xanthus and observed their effect on the prey bacteria to answer the question, how is prey lysis mediated by isolated proteins? We can see that isolated secreted proteins kill and lyse gram positive prey, but not gram negative prey.
Figure 1: Myoxococcus xanthus predation behavior. A. M. xanthus against gram positive species M. luteus and B. subtilis and gram negative species E. coli and A. tumefaciens. B. Bacteriolytic activity of M. xanthus protein fractions.
After observing the destruction of prey bacteria, the researchers wanted to ensure that the cell death was a result of the secreted proteins’ role in predation, and not the result of growth inhibition. To do so, they used fluorescent dyes that bind to DNA of dead cells (PI, which stains magenta, and SYTOX, which stains green) and dyes that bind to the peptidoglycan cell wall (NADA-green, which stains white) and the outer cell membrane (a lipophilic red). Proteins secreted from M. xanthus were then added to the stained live bacteria on slides and observed for an hour. Figure 2 of the article shows how the isolated secreted proteins only lysed the Gram (+) bacteria (M. luteus and B. subtilis). In only Figures 2A and 2B, we can see the magenta DNA staining, which indicates that only the interaction with Gram (+) bacteria is resulting in cell death and DNA leakage (91% of M. luteus cells killed, and 88% of B. subtilis cells killed). This is in comparison to Figures 2C and 2D where no DNA leakage or death is seen in the Gram (-) bacteria. Further support for this argument can be seen in Figures 2E and 2F, which continue to show the leakage of DNA and the blistering of the membrane, in the Gram (+) cells respectively. Figure 2G shows how both Gram (-) bacteria (E. coli and A. tumefaciens) were unaffected by the secreted proteins.
Figure 2: Effects of the addition of secreted proteins of M. xanthus on the lysis of Gram positive bacteria M. luteus (A) and B. subtilis (B) and Gram negative bacteria E. coli (C) and A. tumefaciens (D). Cells indicated magenta indicate death, white staining indicates the peptidoglycan layer, and SYTOX staining (specified with yellow and white triangle for septums and cell poles of the cell respectively) (E). Cell blistering (yellow triangles) observed in B. subtilis after interaction with M. xanthus secreted proteins (F).
Now to get into the meat of our article, let’s look at some of the main findings of Arend et. al (2021) which can be seen in Figures 6 and 7 shown below. The overall conclusions, in conjunction with the results from Figure 2, showed that M. xanthus possesses the unique ability to kill both Gram (+) and Gram (-) prey bacteria but the methods it uses are different depending on its prey. So how do we know this?
As seen in Figure 2, isolated secreted M. xanthus proteins were shown to lyse Gram (+) bacteria only. To observe the mechanics of the predator-prey relationships, M. xanthus cells were mixed with the four different prey species cells of M. luteus, B. subtilis, E. coli, and A. tumefaciens separately on agarose. Figure 6 shows a cartoon that depicts observations after 30 minutes where we can see that the Gram (+) bacteria (M. luteus and B. subtilis) remain intact but the Gram (-) bacteria (E. coli and A. tumerfaciens) have been ruptured. Taking this with the data of Figure 2, this creates an interesting dynamic where isolated secretions of M. xanthus only lyse the Gram (+) bacteria while direct cell contact with M. xanthus appears to lyse only the Gram (-) bacteria.
Figure 6: M. xanthus cells (blue) mixed with the four prey cells (orange) on agarose pads and pictured at 30 minute intervals.
To further investigate this, we can look to Figure 7 where, similar to Figure 6, M. xanthus cells were put into direct contact with the four different prey species. What’s different with Figure 7 is that this time, the researchers used fluorescence to identify the different components of each bacterial cell. NADA-green fluorescence that appears white in Figure 7 represents the peptidoglycan layer of the cells and the magenta colored PI fluorescence highlights the dead DNA in cells. To find the M. xanthus cells, look for the yellow arrows, while the white arrows are pointing to DNA leakage. Using this information, what we see is that all four prey cell types are actually dying after contact with M. xanthus (Figures A-D). Figure 7E shows that there is significantly more death of DNA (indicating overall cell death) when bacterial cells were in contact with M. xanthus vs. not. The difference in death of each of these cells can be seen in the fact that DNA leakage can only be seen in the Gram (-) cells, indicating that only those cell membranes were lysed. This confirms the data from Figure 6 as well, but provides new insight to the fact that though they were not lysed and therefore did not leak DNA after M. xanthus contact, the Gram (+) cells were also dying. This suggests then that the M. xanthus is still killing the Gram (+) cells but through a different method than the Gram (-) cells that does not require the lysing of its membrane. Considering one of the main differences between Gram (+) and Gram (-) membranes are that Gram (+) have an inner membrane and a thick peptidoglycan layer while Gram (-) membranes have thinner peptidoglycan layers and an additional outer membrane, this may suggest that these features may play a role determining whether or not a cell can be lysed by the M. xanthus secretory proteins. This would perhaps be an avenue for further study.
Figure 7: Individual M. xanthus cells are mixed with the four prey species M. luteus (A), B. subtilis (B), E. coli (C), A. tumefaciens (D). The peptidoglycan layers fluoresce white as a result of NADA-green, while the DNA of dead cells fluoresce purple as a result of PI fluorescence. The cells are observed for one hour. The white arrows show DNA leakage in the Gram (+) prey bacterial cells (E. coli and A. tumerfaciens). Yellow arrows point to the M. xanthus cell. 7E compares the percentage of PI stained prey cells (indicating dead DNA) when cells are in contact with M. xanthus vs. not.
We know that M. xanthus is a bacteria that increases its biomass by killing other bacteria and other researchers have noted that the mechanisms it uses differ whether the prey bacteria is Gram (-) or (+). In this study, the researchers attempt to answer the question of the difference of M. xanthus predation on Gram (+) and Gram (-) bacteria by breaking down the predator bacteria in numerous ways to see what components make it successful in predation. The results of this study were particularly interesting: while M. xanthus can cause cell death to both Gram (-) and Gram (+) bacteria, the mechanisms used differ. The main mechanism used for Gram (+) bacteria comes through the usage of proteins to break the cell envelope and cause leakage. For Gram (-) bacteria, on the other hand, contact with M. xanthus is enough to cause the lysis of the cell. Despite knowing this, however, there are still larger questions at hand. One major question that this article poses is how these independent processes are dependent on the encounterment of prey bacteria. Does M. xanthus have some kind of alert system that informs the cell of whether or not Gram (+) bacteria is near? Does this trigger the secretion of proteins? And how does the secretion of these proteins affect the M. xanthus cell? While the article cleared up one question, it left so many unanswered questions for future research to touch on. In the future, it would be incredibly interesting to see studies that explore the existence and/or limitations of this Gram (+)/(-) detection system.
One beneficial aspect of M. xanthus being able to prey on both Gram (+) and (-) is that it could potentially be an antibiotic for many bacteria that are harmful to us. This could be especially useful as it itself is not harmful to humans. Possible avenues for research might include further testing of the range of bacteria that M. xanthus is able to kill. In terms of potential for human treatment, this information might guide the development of medications that use this bacteria to kill harmful bacteria that share similar features with other M. xanthus prey. This could be through further analysis into the content of M. xanthus secretions, or further analysis of the mechanisms of contact predation that we see with Gram (+) bacteria with the potential for isolating certain predatory features to use for killing specific pathogenic bacteria.
Overall, it would be amazing to see exactly what kinds of studies arise from this new information and see how far the boundaries of M. xanthus can be pushed!
About the Authors:
Tahani Ahmed ‘23 is a Biology Major and Art History minor from New Jersey and is pursuing a career as a nurse practitioner after Mount Holyoke. She enjoys being outdoors, swimming, crafting, and being with friends and family.
Myrha-Lissa Chery ‘23 is a Psychology major from Miami, Florida. She enjoys crocheting, being with friends, family, and her cat, and listening to music. She currently works in the Brodie lab on campus studying the effects of temperature on the physiological function of fiddler crabs. After graduation, she hopes to become a physician assistant and work in a holistic healthcare setting.
Alishba Vazquez ‘23.5 is a Neuroscience and Behavior from California. She works in the Camp lab on campus working with a microbe called Bacillus subtilis. She aspires to be a physician assistant after graduation. She enjoys the outdoors, horror movies, and spending time with friends and family.
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